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WO2001048149A1 - Adult bone marrow-origin cell capable of differentiating into heart muscle cell - Google Patents

Adult bone marrow-origin cell capable of differentiating into heart muscle cell Download PDF

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Publication number
WO2001048149A1
WO2001048149A1 PCT/JP2000/001148 JP0001148W WO0148149A1 WO 2001048149 A1 WO2001048149 A1 WO 2001048149A1 JP 0001148 W JP0001148 W JP 0001148W WO 0148149 A1 WO0148149 A1 WO 0148149A1
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seq
amino acid
acid sequence
cell
mef
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PCT/JP2000/001148
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French (fr)
Japanese (ja)
Inventor
Akihiro Umezawa
Jun-Ichi Hata
Keiichi Fukuda
Satoshi Ogawa
Kazuhiro Sakurada
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Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU26950/00A priority Critical patent/AU2695000A/en
Priority to AU10552/01A priority patent/AU1055201A/en
Priority to PCT/JP2000/007741 priority patent/WO2001048150A1/en
Priority to EP00985950A priority patent/EP1254952A4/en
Priority to CA002395950A priority patent/CA2395950A1/en
Priority to AU22281/01A priority patent/AU784618B2/en
Priority to PCT/JP2000/009323 priority patent/WO2001048151A1/en
Priority to US09/749,728 priority patent/US20020142457A1/en
Publication of WO2001048149A1 publication Critical patent/WO2001048149A1/en

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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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Definitions

  • the present invention relates to a method for isolating, purifying, culturing, and inducing differentiation of bone marrow cells having the ability to isolate cardiomyocytes.
  • the present invention also relates to a method for expanding myeloid cells capable of differentiating into cardiomyocytes using various cytokines, transcription factors, and the like, and a method for controlling the division into cardiomyocytes.
  • the present invention further provides a surface antigen specific for bone marrow cells capable of differentiating into cardiomyocytes.
  • Obtaining method obtaining a gene encoding the surface antigen, obtaining an antibody specific to the surface antigen
  • the present invention relates to a method and a method for obtaining proteins and genes involved in proliferation of bone marrow cells capable of differentiating into cardiomyocytes and differentiation into cardiomyocytes.
  • the present invention relates to a therapeutic agent for various heart diseases using bone marrow cells capable of differentiating into cardiomyocytes.
  • cardiomyocytes Before birth, cardiomyocytes actively divide while autonomously beating. However, their division ability is lost at birth, they do not regain cell division ability like hepatocytes, and unlike skeletal muscle cells, they have satellite cells and undifferentiated precursor cells. Absent. Therefore, if cardiomyocytes are necrotic due to myocardial infarction, myocarditis, or aging, in vivo, cell expansion of the remaining cardiomyocytes rather than cell division occurs. Cardiac hypertrophy is initially a physiological adaptation, but it also leads to a decrease in the diastolic function of the heart itself and a decrease in systolic function in conjunction with the fibrosis of the interstitium due to the proliferation of coexisting cardiac fibroblasts, leading to heart failure.
  • heart transplantation is a fundamental treatment for severe heart failure, but heart transplantation is a common medical treatment due to problems such as a shortage of organ donors, difficulty in determining brain death, rejection, and rising medical costs. It is not easy to spread. In fact, heart disease is the third leading cause of death in Japan (Health and Welfare White Paper, Heisei 10), and if we can regenerate lost cardiomyocytes, it will be a major step forward in medical welfare.
  • cell lines that preserve the properties of cardiomyocytes include atrial natriuretic AT-1 cells established from tumors arising in the atrium of transgenic mice produced by recombining the large T antigen of SV40 overnight with the Lemon promoter [Science, 239; 1029-1038 (1988)] ].
  • atrial natriuretic AT-1 cells established from tumors arising in the atrium of transgenic mice produced by recombining the large T antigen of SV40 overnight with the Lemon promoter [Science, 239; 1029-1038 (1988)] ].
  • these cells are not suitable for cell transplantation because they form tumors when transplanted in vivo. Under these circumstances, the following methods were considered to reconstruct the myocardium.
  • the first method is to convert cells other than cardiomyocytes into cardiomyocytes. This was inferred from the fact that introduction of MyoD into fibroblasts could convert them into skeletal muscle cells. Previously, although successful cases have been shown with P19 cells, which are mouse embryonal cancer cells, [Cell Struc. & Func, 21: 101-110 (1996)], successful cases with non-cancer cells Has not been reported.
  • the second method is to give the cardiomyocytes divisional ability again. This is based on the fact that the heart muscle can divide while beating during fetal life. However, no successful cases have been reported so far.
  • the third method is to induce cardiomyocytes from undifferentiated stem cells. Although it has been shown that cardiomyocytes can be induced from embryonic stem cells (ES cells), transplantation of embryonic stem cells themselves into adults has problems such as formation of carcinoma and antigenicity [Nature Biotechnology, 17 , 139-142 (1999)].
  • a method of obtaining undifferentiated myocardial progenitor cells from aborted fetuses and using them for transplantation has also been considered, and it is known that they can function effectively as cardiomyocytes in animal experiments [Science, 264]. , 98-101 (1994)] 0, however, it is difficult to obtain a large amount of cardiac progenitor cells in this way, the applications also to common medical terms of ethical not easy, hematopoiesis in adult bone marrow
  • mesenchymal stem cells in addition to stem cells and vascular stem cells, which include bone cells, chondrocytes, tendon cells, ligament cells, skeletal muscle cells, fat cells, stoma cells, and liver oval cells.
  • a safer and more reliable treatment than the current treatment for heart disease is desired. Therefore, to select bone marrow cells capable of differentiating into cardiomyocytes from bone marrow cells and to control proliferation or sorting of bone marrow cells capable of differentiating into cardiomyocytes, cells using bone marrow-derived cells were used. It is useful for the development of regenerative therapy for myocardium. For this purpose, it is necessary to identify cells capable of dividing bone marrow cells into cardiomyocytes, and to identify cytokines or transcription factors that act on the proliferation or differentiation of the cells. The inventors of the present invention have conducted intensive studies to address the above problems and have obtained the following results.
  • a mouse bone marrow-derived cell line is labeled with a retroviral vector expressing GFP (Green Fluorescent Protein), and by tracking one cell under a fluorescence microscope, it has the ability to differentiate into cardiomyocytes.
  • the cells were found to be pluripotent stem cells capable of inducing differentiation of at least two different types of cells, cardiomyocytes and adipocytes. It was also found that there are myocardial stem cells that are proliferative at the same time and divide only into myocardial cells, and myocardial progenitor cells that have finite cell division ability and differentiate only into myocardial cells.
  • the stem cells can be probabilistically treated by administration of other genomic DNA demethylating agents such as DMSO (dimethyl sulfoxide) as well as 5-azacytidine, which has already been reported under normal culture conditions.
  • DMSO dimethyl sulfoxide
  • 5-azacytidine 5-azacytidine
  • a cytokine such as platelet-derived growth factor (PDGF) or aU-trans retinoic acid, or culturing in a culture dish coated with fibronectin, increased the rate of cardiomyocyte formation.
  • PDGF platelet-derived growth factor
  • FGF- fibroblast growth factor-2
  • the present invention provides the following (1) to (96).
  • the cell according to (1) which is a pluripotent stem cell having at least the ability to differentiate into cardiomyocytes and adipocytes.
  • the cell according to (1) which is a cardiomyocyte that is induced to differentiate only into cardiomyocytes.
  • the factor according to (13) above, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. cell.
  • the factor according to (15), wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors. cell.
  • the transcription factor is Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-
  • Nkx 2. 5 / Csx is Nkx 2. 5 / Csx having the amino acid sequence is represented by SEQ ID NO: 9, the (21), wherein the cell.
  • MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
  • MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17, The cell according to the above (21).
  • MEF-2D is MEF-2D having the amino acid sequence represented by SEQ ID NO: 19.
  • TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
  • TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
  • TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
  • FGF-2 is FGF-2 having the amino acid sequence of SEQ ID NO: 7 or 8.
  • demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
  • demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
  • a method for forming myocardium from bone marrow-derived cells comprising using a factor expressed in a fetal heart development region.
  • the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin, and transcription factors.
  • a method for forming a myocardium from cells derived from bone marrow which comprises using a factor that acts to divide the myocardial cells at the stage of fetal heart development.
  • the factor according to (40), wherein the factor that acts on differentiation into cardiomyocytes during the fetal heart development stage is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors.
  • the PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5, according to (42). the method of.
  • Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
  • MEF-2B are, Ru MEF-2 B der with Amino acid sequence represented by SEQ ID NO: 15, the (46) The method according.
  • MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
  • dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21, The method according to (46) above.
  • TEF-1 is TEF-1 having the amino acid sequence represented by SEQ ID NO: 25.
  • TEF- 3 is a TEF- 3 having an amino acid sequence represented by SEQ ID NO: 27.
  • a cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
  • the factor according to the above (61), wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. Cardiomyogen.
  • a cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
  • the factor described above, wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins, and transcription factors.
  • PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5.
  • the transcription factor is selected from the group consisting of Nkx2.5 / Csx ⁇ GATA4, MEF-2A ⁇ MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5 Selected, above
  • MEF-2A is a represented by MEF-2 A the amino acid sequence of SEQ ID NO: 13, wherein said (69) myocardial former description.
  • MEF-2D is a MEF_ 2 D represented by the amino acid sequence of SEQ ID NO: 19, wherein said (69) myocardial former description.
  • TEF-1 is TEF-1 represented by the amino acid sequence of SEQ ID NO: 25.
  • TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27, (69) The cardiomyogen according to (69).
  • TEF-5 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
  • a method for regenerating a heart destroyed by a heart disease comprising using the cell according to any one of (1) to (34).
  • a therapeutic agent for regenerating heart comprising the cell according to any one of the above (1) to (34) as an active ingredient.
  • a congenital genetic disease which comprises using the cell according to any one of the above (1) to (34), into which a wild-type gene for a mutant gene in a cardiac congenital genetic disease has been introduced.
  • telomere has the amino acid sequence represented by SEQ ID NO: 31.
  • a therapeutic agent for heart disease comprising as an active ingredient the cell according to any one of the above (1) to (34), which has been immortalized by expressing telomerase.
  • telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
  • the bone marrow cells capable of differentiating into cardiomyocytes of the present invention refer to multipotent stem cells, cardiomyocyte stem cells and cardiomyocyte precursor cells isolated from adult bone marrow.
  • Pluripotent stem cells are cells that can induce differentiation of at least two different types of cells, cardiomyocytes and adipocytes.
  • Cardiomyocyte stem cells are cells that are proliferative and have the ability to differentiate only into cardiomyocytes. Induction of myocardial stem cells induces two types of cells, myocardial cells and myocardial stem cells.
  • Cardiac progenitor cells are cells that have a finite cell division ability and have the ability to differentiate only into cardiac myocytes. When differentiation is induced in cardiomyocyte precursor cells, only cardiomyocytes are formed, unlike cardiomyocyte stem cells.
  • the method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from human bone marrow is not particularly limited as long as it is a method that can be safely and efficiently obtained.S.E.Haynesworth et al.
  • the bone marrow cells After collecting the bone marrow cells from the obtained bone marrow fluid by centrifugation at i, ooo xg , the bone marrow cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the bone marrow cells were subjected to 10% FBS (fetal bovine serum) in human MEM (human modified MEM), DMEM
  • a bone marrow cell solution By resuspending the cells in a cell culture medium such as (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium), a bone marrow cell solution can be obtained.
  • a cell culture medium such as (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium)
  • a method for isolating bone marrow cells capable of differentiating into cardiomyocytes from the bone marrow cell solution is to remove other cells mixed in the solution, for example, blood cells, hematopoietic stem cells, vascular stem cells, fibroblasts, and the like.
  • the bone marrow cell fluid was overlaid on percoll having a density of 1.073 g / ml based on the method described in MF Pittenger et al. Science, 284, 143 (1999), and then centrifuged at 1,100 X g for 30 minutes.
  • the cells can be isolated by collecting the cells at the interface.
  • a percoll diluted to 10 X PBS plus 9/10 in bone marrow cell fluid after ⁇ added the same volume, and centrifuged for 30 minutes at 2 0,000 X g, density 1 ⁇ 075 to 1.0
  • a bone marrow cell mixture containing bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
  • the bone marrow cell 'mixture containing bone marrow cells capable of differentiating into cardiomyocytes obtained by the above method is diluted so that only one cell is injected into each well of a 96-well culture plate, and one cell-derived After preparing a large number of clones, the clones are treated using the method described below for inducing cardiomyocytes from bone marrow cells having the ability to differentiate into cardiomyocytes, and clones in which autonomously pulsating cells appear are selected.
  • bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
  • Pluripotent stem cells, myocardial stem cells, and myocardial progenitor cells can be selected by performing single cell marking using a reporter gene such as GFP (Green Fluorescent Protein) described below.
  • the method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from rats and mice is not particularly limited, but can be obtained by the following procedure. Rats or mice are sacrificed by cervical prolapse, and after sufficient disinfection with 70% ethanol, the skin of the femur and the quadriceps are removed. Remove the joint with scissors in the knee joint and remove the muscles behind the femur. Remove the joint with scissors in the hip joint and remove the femur. After removing the muscle attached to the femur with scissors as much as possible, scissors both ends of the femur.
  • FBS fetal calf serum
  • the tip of the needle is inserted into the stump of the femur on the knee joint side.
  • the culture solution in the syringe into the bone marrow the bone marrow cells are pushed out from the hip joint stump.
  • the obtained bone marrow cells are suspended in a culture solution by pipetting.
  • bone marrow cells capable of differentiating into cardiomyocytes can be isolated in the same manner as in the above-mentioned method for isolating bone marrow cells from human bone marrow fluid.
  • Examples of cells isolated by the above method include mouse bone marrow-derived stem cells.
  • BMSC a mouse bone marrow-derived stem cell, was sent to the Ministry of International Trade and Industry by the Ministry of International Trade and Industry's Institute of Biotechnology and Industrial Technology (February 22, 2002) at FERM BP-70 (1-3-3 Tsukuba East, Ibaraki, Japan). Deposited as 43 .
  • the medium used for culturing bone marrow cells capable of differentiating into cardiomyocytes isolated by the method 1 described above is usually a known medium (Tissue Culture Technology Basic Edition, Third Edition, Asakura Shoten 1996).
  • a cell culture medium can be used, and preferably, a cell culture medium such as MEM, DMEM, IMDM, or the like supplemented with 5 to 20% of calories from bovine serum or the like is used.
  • the culturing conditions may be any conditions as long as the cells can be cultivated, but the culturing temperature is preferably 33 to 37 ° C, and the culturing is preferably performed in an incubator filled with 5 to 10% carbon dioxide gas.
  • the bone marrow cells having the ability to divide myocardial cells are attached to a normal plastic culture dish for tissue culture and proliferate.
  • the cells grow on the entire surface of the culture dish, remove the medium and suspend cells by adding trypsin EDTA solution.
  • the suspended cells can be further subcultured by washing with PBS or the cell culture medium, and then diluting 5- to 20-fold with the cell culture medium and adding to a new culture dish.
  • Methods for inducing cardiomyocytes from bone marrow cells capable of differentiating into cardiomyocytes include (1) induction of differentiation by treatment with DNA demethylating agent, (2) factors expressed in the fetal heart development region or fetus. (3) Induction of differentiation by bone marrow cells capable of differentiating into cardiomyocytes or culture supernatant of cardiomyocytes differentiated from the bone marrow cells, etc. Can be mentioned. Simply use these methods By themselves or in combination, cardiomyocytes can be derived from bone marrow cells having the ability to differentiate into cardiomyocytes.
  • the DNA demethylating agent may be any compound that causes demethylation of DNA.
  • DNA demethylating agents include demethylase, an enzyme that specifically inhibits methylation of cytosine residues in GpC sequences in chromosomal DNA, and 5-azacytidine (hereinafter abbreviated as 5-aza-C). And DMSO (dimethyl sulfoxide).
  • demethylase examples include a demethylase having the amino acid sequence of SEQ ID NO: 1 [Nature, 397. 579-583 (1999)]. Specific examples of the induction of differentiation by DNA demethylation treatment are shown below.
  • 5-aza-C is added to a medium containing bone marrow cells capable of differentiating into cardiomyocytes to a concentration between 3 mmol / l and 10 mmol / l, and incubated at the above culture conditions for 24 hours. Bastion. The medium is replaced to remove 5-aza-C, and cultured for another 2-3 weeks to obtain cardiomyocytes.
  • the formed cardiomyocytes are mainly sinus node cells at 2-3 weeks of culture, but can induce differentiation of ventricular cardiomyocytes after 4 weeks of culture.
  • Factors expressed in the fetal heart development region or factors that act on cardiomyocyte differentiation during the fetal heart development stage include cytokines, vitamins, adhesion molecules, transcription factors, and the like.
  • any cytokine may be used as long as it promotes differentiation into bone marrow cells having the ability to divide myocardial cells into cardiomyocytes at the stage of cardiac development.
  • PDGF platelet-derived growth factor
  • bone marrow cells capable of dividing into cardiomyocytes are promoted to be divided into cardiomyocytes at the stage of heart development. It is also possible.
  • cytokines that suppress differentiation into cardiomyocytes include fibroblast growth factor-2 (hereinafter abbreviated as FGF-2), specifically, FGF-2 represented by SEQ ID NO: 7 or 8.
  • FGF-2 fibroblast growth factor-2
  • inhibitors for cytokines that suppress differentiation into cardiomyocytes include substances that inhibit cytokine signaling, such as antibodies that neutralize cytokines and low molecular weight compounds.
  • any vitamin can be used as long as it promotes differentiation into bone marrow cells capable of differentiating into cardiomyocytes, such as retinoic acid, and cardiomyocytes during the developmental stage of the heart. Specifically, and the like retinoic acid 10- 9 M.
  • any adhesion molecule such as fibronectin may be used as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, by culturing bone marrow cells capable of differentiating into the cardiomyocytes in a culture dish coated with fibronectin, filtration into the cardiomyocytes can be promoted.
  • transcription factor examples include homebox-type transcription factor Nkx2.5 / Csx (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: base sequence) and Zinc finger-type transcription factor GATA4 belonging to GATA family (SEQ ID NO: 11: amino acid sequence, SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: Amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence), Basic helix loop dHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: base sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID
  • the above-mentioned transcription factor can induce differentiation into cardiomyocytes by introducing DNA encoding the factor into bone marrow cells capable of differentiating into cardiomyocytes and expressing the DNA.
  • the differentiation into cardiomyocytes can also be induced by adding the factor to a culture solution of bone marrow cells capable of differentiating into cardiomyocytes.
  • the differentiation into cardiomyocytes can be induced by adding the culture supernatant of autonomously beating cardiomyocytes to a bone marrow cell culture medium having the ability to differentiate into cardiomyocytes.
  • a bone marrow-derived cell capable of differentiating into cardiomyocytes can be differentiated into cardiomyocytes by using a cardiomyocyte differentiation-inducing factor (hereinafter referred to as a cardiomyocyte differentiation-inducing factor) obtained by the following method. Can be guided.
  • a cardiomyocyte differentiation-inducing factor hereinafter referred to as a cardiomyocyte differentiation-inducing factor
  • a myocardial differentiation-inducing factor it can be obtained by adding various protease inhibitors to the culture supernatant of autonomously pulsating cells, and combining dialysis, salting out, chromatography and the like.
  • the partial amino acid sequence of the above-described myocardial motility-inducing factor is determined, and a DNA probe designed based on the amino acid sequence is used to prepare the autonomously beating cells.
  • a gene for a myocardial differentiation-inducing factor can be obtained.
  • Cardiac regeneration or heart disease containing bone marrow cells capable of differentiating into cardiomyocytes can be used as therapeutic agents for heart regeneration or heart diseases.
  • heart disease examples include myocardial infarction, ischemic heart disease, congestive heart failure, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, valvular disease and the like.
  • bone marrow cells having the ability to differentiate into cardiomyocytes are contained in high purity, and the bone marrow cells having the ability to differentiate into cardiomyocytes are expanded according to the site and size of the damaged heart.
  • Cells preferably from bone marrow cells capable of differentiating into cardiomyocytes, myocardial endothelial cells (Endocardial endothelial cells), cushion cells (Cushion cells), ventricular-type cardiomyocytes, atrial-type cardiac cells, sinus node cells, etc. Cells capable of inducing differentiation into various cells forming the heart are used.
  • the therapeutic agent is a density gradient centrifugation method described above from the bone marrow fluid of a patient with myocardial infarction, a panning method using an antibody that specifically recognizes cells capable of differentiating into cardiomyocytes described below (J. Immunol. 141 (8), 2797-2800 (1988)] or FACS method [Int. Immunol., 10 (3), 275-283 (1998)], or a gene specific to myeloid cells capable of differentiating into cardiomyocytes
  • a bone marrow capable of differentiating into said cardiomyocytes by a method for constructing a repo-one-line system using It can be produced by purifying cells.
  • the therapeutic agent may be a cell obtained by inducing the differentiation of bone marrow cells capable of differentiating into cardiomyocytes into cardiomyocytes using a cardiomyogen described below, a bone marrow cell obtained from bone marrow of an elderly person, Also included are cells capable of differentiating into cardiomyocytes that have been activated to mitosis using the immortalization method described below.
  • the purity of the therapeutic agent produced by the above method can be assayed by combining the antibody specifically recognizing the cells capable of differentiating into cardiomyocytes with the FACS method.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used.
  • ischemic heart disease As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like
  • eccentric stenosis is subdivided into two types, Type I and Evening II. It is known that the form of stenosis is related to the course and prognosis of angina.Type II eccentric stenosis and multiple wall irregularities are common in unstable angina patients, and there is a high possibility of transition to myocardial infarction . If the blood vessel is completely stenotic, the injected cells may not reach the lesion site.Therefore, the stenosis site must be removed by percutaneous coronary angioplasty (PTCA) or thrombolysis beforehand. It is necessary to restart. Depending on the location of the damaged myocardial cells, the cells to be injected can be differentiated into ventricular and atrial types.
  • PTCA percutaneous coronary angioplasty
  • the catheter insertion method is the Sones method (Medical View, 1993), which is inserted from the right upper arm artery, or the Jundkins method (Medical View, 1993), which is inserted through the femoral artery. 1, MEDICAL VIEW, 1993) can be used.
  • the cardiomyogenic agent of the present invention may be a demethylating agent for chromosomal DNA, a factor expressed in the fetal heart development region, or a factor that acts on cardiomyocyte differentiation during the fetal heart development stage.
  • Bone marrow-derived cells can be induced to differentiate into cardiomyocytes by containing at least one factor as an active ingredient.
  • Myocardial differentiation inducing factors include cytokines, vitamins, adhesion molecules, transcription factors, etc. I can give it.
  • any cytokine can be used as long as it promotes the differentiation of bone marrow cells capable of differentiating into cardiomyocytes and the differentiation into cardiomyocytes at the stage of cardiac development.
  • PDGF 10 to 40 ng / ml of PDGF.
  • PDGF those represented by the amino acid sequence of SEQ ID NO: 3 or 5 are preferably used.
  • any vitamin may be used as the vitamin, as long as it promotes the differentiation of bone marrow cells such as retinoic acid into cardiomyocytes having the ability to divide the myocardial cells into cardiomyocytes at the stage of cardiac development. Specifically, and the like retinoic acid 10- 9 M.
  • the adhesion molecule may be any adhesion molecule, such as fibronectin, as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, differentiation into cardiomyocytes can be promoted by culturing bone marrow cells capable of differentiating into cardiomyocytes in a culture dish coated with fibronectin.
  • the transcription factor Homeobokkusu transcription factor Nkx2.5 / C SX (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: nucleotide sequence), GATA family foremost belongs Zinc finger transcription factor GATA4 (SEQ ID NO: 11: amino acid sequence , SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence) ), Belonging to the basic helix loop helix-type transcription factor.
  • GATA4 Zinc finger transcription factor GATA4
  • MEF-2A belonging to myocyte enhancer factor-2 (MEF
  • DHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: nucleotide sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID NO: 24: nucleotide sequence), TEA- TE belonging to DNA-binding transcription factor family 1 F-1 (SEQ ID NO: 25: amino acid sequence, SEQ ID NO: 26: nucleotide sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28: nucleotide sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence, sequence) No. 30: base sequence).
  • the myocardial agents include those containing a gene for a myocardial differentiation-inducing factor as a main component and those containing a protein that is the main body of a myocardial differentiation-inducing factor.
  • the cardiomyogenic agent of the present invention contains a gene encoding a myocardial differentiation inducing factor as a main component below. The preparation method in this case will be described.
  • a recombinant virus vector Yuichi Plasmid is constructed by inserting a gene DNA fragment or a full-length cDNA of a cardiomyocyte differentiation-inducing factor into the virus vector downstream of Promote One. .
  • the recombinant virus vector-plasmid is introduced into a packaging cell suitable for the virus vector-plasmid.
  • any cell can be used as long as it can supply the protein deficient in recombinant virus vector plasmid which lacks at least one gene encoding a protein necessary for viral packaging. Those can also be used.
  • HEK293 cells derived from human kidney, mouse fibroblast NIH3T3, and the like can be used.
  • Proteins to be supplied by the packaging cells include gag, pol, and env derived from mouse retrovirus in the case of a retrovirus vector, and gag, po env, vpr, and vpu derived from an HIV virus in the case of a lentivirus vector. , Vif, tat, rev, nef, etc., proteins such as E1A and E1B derived from adenovirus in the case of adenovirus vector, Rep (p5, p19, p40), Vp (Cap ) Can be used.
  • a viral vector plasmid that contains a promoter at a position where recombinant viruses can be produced in the above-mentioned packaging cells and a wild-type gene corresponding to a gene causing cardiac congenital genetic disease can be transcribed in cardiomyocytes is used. Used.
  • Viral vector plasmids include MFG [Proc. Natl. Acad. Sci. USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)], LL_CG, CL-CG ⁇ CS-CG, CLG [Journal of Virology, 72, 8150-8157 (1998)], pAdexl [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like are used.
  • any promoter can be used as long as it can be expressed in human tissues.
  • the promoter of the cytomegalovirus (human CMV) IEGmmediate early) gene and the early promoter of SV40 can be used.
  • Retrovirus Promo One, Meta Mouth Choonein Promo One, Heat Shock Protein Promoter, SR Promo One Etc. can be given.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • a cardiomyocyte-specific gene such as the Nkx2.5 / Csx gene, the target gene can be specifically generated in the cardiomyocyte.
  • a recombinant virus vector By introducing the above-mentioned recombinant virus vector plasmid into the above-mentioned packaging cells, a recombinant virus vector can be produced.
  • a method for introducing the viral vector plasmid into the packaging cells include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. Etc. can be given.
  • the above-described recombinant virus vector can be mixed with a base used for a gene therapy agent to produce a cardiomyogenic agent [Nature Genet., 8, 42 (1 "4)].
  • a base used for a gene therapy agent to produce a cardiomyogenic agent
  • Any base can be used as long as it is commonly used for injections, for example, distilled water, salt solutions such as sodium chloride or sodium chloride and a mixture of inorganic salts, mannitol, and lactose.
  • osmotic pressure adjusting agent Using injections as solutions, suspensions, and dispersions, using PH adjusters, vegetable oils such as sesame oil and soybean oil, or auxiliaries such as surfactants such as lecithin or non-ionic surfactants. These injections can also be prepared as preparations for dissolution at the time of use by operations such as pulverization, freeze-drying, etc.
  • the above-mentioned myocardium-forming agent can be used in the form of a solid as it is in the case of a liquid.
  • the myocardium-forming agent of the present invention can be dissolved in the above-mentioned sterilized base immediately before treatment and used for gene therapy if necessary. As described above, a method of local administration using a catheter or the like is used.
  • the above-described recombinant virus vector can be prepared as the above-mentioned cardiomyogen and then administered to a patient after infecting bone marrow cells having the ability to isolate the cardiomyocytes in a test tube. Alternatively, the recombinant viral vector can be administered directly to the affected area of the patient.
  • the cardiomyogenic agent of the present invention contains a cardiac differentiation-inducing factor protein as a main component The law is described.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • the recombinant expression vector of the protein is constructed by inserting the DNA fragment or the full-length cDNA downstream of the promoter in the expression vector.
  • the recombinant expression vector is introduced into a host cell compatible with the expression vector.
  • any capable of expressing the target DNA can be used, and for example, genus Escherichia, genus Serratia, genus Corynebacterium, genus Brevibacterium , Bacteria belonging to the genus Pseudomonas ill genus Bacillus, genus Microno 'icrobacterium, genus Kluyveromyces, genus Saccharomyces f Saccharomyces, genus Shisosaccharomyces omnibus Yeast, animal cells, insect cells and the like belonging to the genus Ron (Trichosporon), the genus Schwanniomyces and the like can be used.
  • Ron Trichosporon
  • those which can be replicated autonomously in the above-mentioned host cells or can be integrated into the chromosome, and which contain a promoter at a position where the gene DNA of the cardiomyocyte differentiation-inducing factor can be transcribed are used.
  • the recombinant expression vector of the cardiomyocyte differentiation-inducing factor is capable of autonomous replication in the bacterium, and at the same time, induces differentiation into a promoter, a ribosome-binding sequence, and a myocardial cell. It is preferably a recombinant expression vector composed of a DNA encoding a possible protein and a transcription termination sequence. A gene that controls a promoter may be included.
  • Expression base Kuta one, for example, pBTrp2, pBTacl, P BTac2 (both Beringa - ⁇ from Mannheim), pKK233 - 2 (Amersham Pharmacia Biotech Co.), (manufactured by Invitrogen Corp.) pSE280, pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO [Japanese Patent Laid-Open No. 58-110600], pYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277] (1989)], pGELl [Proc. Natl. Acad. Sci.
  • the expression vector includes the ribosome binding sequence Shine-Dalgarno.
  • (Dalgarno) sequence and the initiation codon are preferably adjusted to an appropriate distance (for example, 6 to 18 bases).
  • the promoter may be any promoter that can be expressed in the host cell.
  • two sets of P trp are connected in series. Promo one night (P trp x 2), tac promo one
  • Improving the production rate of the target protein by substituting the nucleotide sequence of the portion of the gene encoding the protein of the gene for the cardiomyocyte differentiation-inducing factor gene of the present invention so as to be an optimal codon for expression in the host. Can be done.
  • a transcription termination sequence is not always necessary for the expression of the gene DNA of the cardiac differentiation-inducing factor of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • host cells include microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium, Plevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 ⁇ Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485 Escherichia coli JM109 Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49 N Bacillus subtilis s Bacillus amvloliquefaciens N Brevibacterium am
  • acetoacidophilum ATCC13870 Microbacterium ammoniaphilum ATCC15354 N Pseudomonas sp. D-0110 and the like.
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above-mentioned host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], the protoplast method (JP-A-63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168. Ill (1979).
  • examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
  • Any promoter can be used as long as it can be expressed in yeast.
  • PH05 promoter overnight PGK promoter, GAP promoter overnight, ADH promoter overnight, gal promoter, gal promoter 10 Promoter, Heat Shock Protein Promoter-, MF 1 Promoter, CUP 1 Promoter, etc.
  • the host cells include Saccharomyces f Saccharomyces cerevisiae, Schizosaccharomyces pombe ⁇ ⁇ ⁇ ⁇ ⁇ ... ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • expression vectors include, for example, pcDNAKlnvitrogen), pcDM8 (Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3 -3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103
  • any promoter that can be expressed in animal cells For example, the promoter of the IE (immediate early) gene of the cytomegalovirus (human CMV), the early promoter of the SV40, the promoter of the retrovirus, the metamouth thionine promoter, the heat shock protein promoter, etc. In the evening, SR promoters can be mentioned.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Examples of the host cells include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 [Japanese Patent Application Laid-Open No. 63-299]. .
  • any method capable of introducing DNA into animal cells can be used.
  • the electroporation method [Cytotechnology,, 133 (1990) 3, calcium phosphate Law (JP 2 - 227 075), lipoic Hue transfection method
  • the transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.
  • insect cells When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory, Manual, WH Freeman and Company, New York (1992)], Current ⁇ Protoco — Ruth 'In' Molecular 'Biology Supplement 1-38 (1987-1997),
  • Proteins can be expressed by the method described in Bio / Technology, 6, 47 (1988) and the like.
  • the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is infected to the insect cells to express the protein. be able to.
  • the gene transfer base Kuta one used in the method can be exemplified P VL1392, P VL1393, pBlueBacIII (both manufactured by Invitrogen Corp.).
  • baculovirus for example, an autographa califomica nuclear polyhedro.sis virus or the like, which is a virus infecting night moth insects, can be used.
  • Insect cells include Sf9 and Sf21 which are ovarian cells of Spodoptera frugiperda [Baculovirus Expression Vectors, A La oratory Manual ⁇ WH Freeman and Company, New York, (1992)] ⁇ High 5 which is an ovarian cell of Trichoplusia (Invitrogen Manufactured) can be used.
  • Examples of a method for co-introducing the above-described recombinant gene introduction vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • sugar or sugar chain-added protein When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added protein can be obtained.
  • the transformant having DNA is cultured in a medium to produce and accumulate a protein capable of inducing myocardial differentiation into the cardiomyocyte in the culture, and the protein is collected from the culture to obtain cardiomyocytes.
  • a protein capable of inducing differentiation can be produced.
  • a method for culturing a transformant for producing a protein capable of inducing differentiation into a cardiomyocyte in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, an inorganic substance, etc. which can be utilized by the host. Either a natural medium or a synthetic medium can be used as long as the medium can be cultured efficiently.
  • the carbon source may be any one that can be assimilated by each host, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as ethanol, propanol, etc. can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate Ammonium salts of various inorganic or organic acids such as ammonia and ammonium phosphate, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn starch, casein hydrolyzate, soybean meal, and soybean meal Hydrolysates, various fermentation cells and digests thereof are used. ,
  • potassium potassium phosphate potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • Cultivation is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is
  • the temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.
  • an Indian user may be added to the medium, if necessary.
  • an Indian user may be added to the medium, if necessary.
  • culturing a microorganism transformed with an expression vector using lac promoter overnight use isopropyl-1 /?-D-thiogalactovyranoside (IPTG) or the like, and use trp promoter overnight.
  • IPTG isopropyl-1 /?-D-thiogalactovyranoside
  • trp promoter trp promoter overnight.
  • IAA indoleacrylic acid
  • Culturing is carried out usually pH6 ⁇ 8, 30 ⁇ 40 ° C, 5 % C0 2 present 1 to 7 days under conditions such as lower.
  • Culture media for transformants obtained using insect cells as host cells include commonly used TNM-FH media (Pharmingen), Sf-900II SFM media (Life Technologies), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.
  • Cultivation is usually carried out for 1 to 5 days under conditions such as ⁇ to 7, 25 to 30 ° C.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a normal protein isolation and purification method may be used.
  • a protein capable of inducing shunting into cardiomyocytes is expressed in a dissolved state in the cells
  • the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then sonicated.
  • the cells are disrupted using a crusher, French press, Mentongaulin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), and a cation using a resin such as S-Sepharose FF (manufactured by Amersham Pharmacia Biotech) Exchange mouth chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric point
  • a purified sample can be obtained by using an electrophoresis method such as electrophoresis alone or in combination.
  • the cells When the protein is expressed by forming an insoluble form in the cells, the cells are collected, crushed, and centrifuged to collect the protein insoluble form as a precipitate fraction.
  • the recovered insoluble form of the protein is solubilized with a protein denaturant.
  • the structure of the protein is returned to a normal three-dimensional structure.
  • a purified sample of the protein is obtained by the production method.
  • a derivative such as the protein or a sugar chain-adduct thereof is obtained from the culture supernatant.
  • the culture supernatant is collected from the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • Examples of the protein thus obtained include, for example, proteins having the amino acid sequences represented by SEQ ID NOs: 5, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. I can give it.
  • the protein expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like. .
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like.
  • a protein capable of inducing differentiation into cardiomyocytes can be used to form and use a cardiomyogen in the same manner as in (1) above.
  • diseases that cause heart failure there is a group that causes heart failure due to partial deletion of all necessary proteins due to mutation of a single gene.
  • diseases include familial hypertrophic cardiomyopathy, Fabri disease, long QT syndrome, Marfan syndrome, aortic stenosis, mitochondrial cardiomyopathy, and Duchenne muscular dystrophy.
  • diseases are known to be caused by genetic abnormalities such as myosin, troponin, tropomyosin, voltage-gated Na channel, K channel, fibrin, elastin, mitochondria, and dystrophin [Therapeutics, ⁇ , O 2 -.
  • the antibody of the present invention which recognizes a surface antigen specifically expressed on bone marrow cells capable of differentiating into cardiomyocytes, is capable of differentiation into cardiomyocytes necessary for performing cell therapy for heart diseases such as myocardial infarction. It can be used for purity test and purification of bone marrow cells having the ability.
  • bone marrow cells capable of differentiating into cardiomyocytes of the present invention 3 to 5 ⁇ 10 5 cell S / animal, or a cell membrane fraction l to 10 mg / animal prepared from the cells as an antigen
  • a suitable adjuvant subcutaneously, intravenously or intraperitoneally in non-human mammals such as rabbits, goats, or 3-20 week old rats, mice or hams, [Complete Freund's Adjuvant) or aluminum hydroxide gel, pertussis vaccine, etc.].
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and it is determined whether the serum reacts with the antigen used for immunization by enzyme immunoassay [enzyme immunoassay (ELISA): published by Medical Shoin. I 976 , Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • enzyme immunoassay enzyme immunoassay
  • a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of serum or antibody-producing cells.
  • a polyclonal antibody can be prepared by separating and purifying the serum.
  • a monoclonal antibody is prepared by fusing the antibody-producing cells with myeloma derived from a non-human mammal to produce a hybridoma, and culturing the hybridoma or administering it to an animal to cause the animal to develop ascites carcinoma. It can be prepared by separating and purifying the culture solution or ascites fluid.
  • spleen cells As antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.
  • Myeloma cells include 8-azaguanine-resistant mouse (derived from BALB) myeloma cell line P3-X63Ag8-U1 (P3-U1) strain [Current Topics in Microbiology andlmmunology, 18, 1 (1978)], P3- NSl / 1—Ag41 (NS-1) strain [European J. Immunology, 6, 511 (1976)] ⁇ SP2 / 0-Agl4 (SP-2) strain [Nature, 276, 269 (1978)], P3-X63 -Ag8653 (653) strain [J. Immunology. 123, 1548 (1979)], and mouse-derived cell lines such as P3- ⁇ 3-Ag8 (X6 strain [Nature, 256, 495 (1975)]) are preferably used.
  • P3-X63Ag8-U1 P3-U1 strain [Current Topics in Microbiology andlmmunology, 18, 1 (1978)]
  • Hybridoma cells can be prepared by the following method.
  • Antibody-producing cells and myeloma cells are mixed, suspended in HAT medium (medium containing hypoxanthine, thymidine, and aminopterin in normal medium), and cultured for 7 to 14 days.
  • HAT medium medium containing hypoxanthine, thymidine, and aminopterin in normal medium
  • a portion of the protein is selected from those that react with the antigen but do not react with the protein that does not contain the antigen by enzyme immunoassay or the like.
  • the cells are cloned by the limiting dilution method, and those having a stable and high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridoma cells.
  • Methods for separating and purifying polyclonal or monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or DEAE-Sepharose column, anion exchange column, protein A or G-column or gel filtration column. Chromatography, etc. using these methods alone or in combination.
  • the antibody obtained by the above method and specifically recognizing a surface antigen expressed on bone marrow cells capable of differentiating into cardiomyocytes, the reactivity to sample cells and the hematopoietic stem cells, neural stem cells, etc. By comparing the reactivity with the control cells, it can be easily determined whether or not the sample cells express the above-mentioned specific surface antigen.
  • a subtraction method which is a method for obtaining genes having different expression forms between two samples of different origins, Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988)] and Representational difference analysis [Nucleic Acids Research, 22, 5640-5648 (1994)].
  • subtraction is performed using a cDNA library prepared from bone marrow cells having the ability to divide into cardiomyocytes using mRNA obtained from control cells other than bone marrow cells such as hematopoietic stem cells and neural stem cells.
  • a differential cDNA library enriched for bone marrow cell-specific genes capable of differentiating into cardiomyocytes
  • the ⁇ C DNA sequence randomly from 5 5 side performs the sequence analysis, selecting only those with the secretion signal sequence. By determining the full-length nucleotide sequence of the cDNA thus obtained, it is possible to distinguish whether the protein encoded by the cDNA is a secretory protein or a membrane protein.
  • the signal sequence trap method is a method for selectively screening genes having a secretory signal sequence.
  • a signal sequence trap library was prepared using a vector capable of performing subtraction, and a signal sequence prepared from bone marrow cells having the ability to divide myocardial cells.
  • a preferred method is to perform trap subtraction using mRNA obtained from control cells such as hematopoietic stem cells and neural stem cells.
  • the DNA fragment containing the secretory signal sequence thus obtained can be used as a probe for cloning full-length cDNA.
  • a synthetic peptide is prepared based on the amino acid sequence inferred from the nucleotide sequence, and the synthetic peptide is synthesized.
  • a specific antibody can be obtained by the above method.
  • a membrane protein some of them encode a receptor, and such a receptor is used to regulate the specific proliferation of bone marrow cells capable of differentiating into the cardiomyocytes or the regulation of differentiation into cardiomyocytes. It may be working and can be used to search for ligands for the receptor.
  • a secretory protein it can be used for directly proliferating or differentiating bone marrow cells capable of differentiating into cardiomyocytes.
  • Bone marrow cell growth factor and cardiomyocyte differentiation inducer capable of cardiomyocyte differentiation As a screening method, when culturing bone marrow cells capable of differentiating into cardiomyocytes in a serum-free medium, various substances as specimens are added, and the cells proliferate or differentiate into cardiomyocytes. It can be done by examining whether it is induced.
  • the substance to be the specimen may be any substance such as secreted proteins such as various cytokines and growth factors, membrane-bound proteins such as cell-adhered molecules, tissue extracts, synthetic peptides, synthetic compounds, and microbial culture solutions.
  • the proliferative ability can be examined based on the ability to form the ostium and the uptake of BrdU.
  • the colony forming ability can be examined by seeding the bone marrow cells of the present invention at a low density.
  • BrdU incorporation can be examined by immunostaining using an antibody that specifically recognizes BrdU.
  • Cardiomyocyte differentiation can be evaluated by using autonomic pulsation as an index or by promoting genes specifically expressed in muscle cells and GFP (Gleen fluorescent protein), Luciferase, A method in which the expression of a reporter gene is used as an index by using a reporter cell in which a DNA combined with a reporter gene such as galactosidase is introduced into bone marrow cells capable of differentiating the cardiomyocytes. Is raised.
  • GFP Green fluorescent protein
  • Luciferase Green fluorescent protein
  • TERT gene is a catalytic Sapuyunitto of Teromera Ichize, DNA specifically represented by SEQ ID NO 3 2, the bone marrow Hosohi having minute I spoon ability to cardiomyocytes after introducing a retroviral vector Transfection method, or into bone marrow cells that have the potential to differentiate into cardiomyocytes
  • a method of administering a factor capable of inducing and expressing an endogenous TERT gene to bone marrow cells capable of differentiating into cardiomyocytes or a method of administering a vector containing DNA encoding a factor capable of inducing and expressing TERT gene to bone marrow capable of differentiating into cardiomyocytes
  • Examples of the method include introduction into cells.
  • the factor that induces and expresses the TERT gene is obtained by combining TERT gene promoter and GFP (Green Fluorescent protein), luciferase or galactosidase in bone marrow cells that have the potential to differentiate into cardiomyocytes. Selection can be achieved by introducing the repo overnight system into bone marrow cells capable of differentiating into cardiomyocytes.
  • GFP Green Fluorescent protein
  • Example 1 Obtaining and culturing bone marrow cells capable of differentiating into cardiomyocytes from mouse bone marrow
  • mice Ten 5-week-old C3H / He mice were anesthetized with ether and then killed by cervical dislocation. The mice were placed in a semi-lateral position and disinfected with 70% ethanol. Next, the skin around the femur was incised over a wide area, and the quadriceps femoris over the entire femur was removed with scissors. Light scissors were inserted into the knee joint, the joint was removed, and the muscles behind the femur were resected. A scissor was placed in the hip joint, the joint was removed, and the femur was removed. Muscles attached to the femur were excised with scissors to expose the entire femur.
  • bone marrow-derived primary immortalized cells After culturing under the above conditions for about 4 months and selecting immortalized cells, dilution was used to establish 192 independent single cell-derived cell lines (hereinafter referred to as bone marrow-derived primary immortalized cells). Stocks). After culturing for 24 hours after adding 5-aza-C to each of these independent clone-derived cells to a final concentration of 3 mM, culturing is continued for another 2 weeks by replacing the medium with IMDM medium. A cell producing clone was selected. Primary bone marrow-derived Of the 19 2 immortalized cells, 3 were bone marrow cells capable of differentiating into cardiomyocytes.
  • the percentage of autologous beating cells that non-specifically induce differentiation from pluripotent stem cells is very small.
  • the surrounding area of the self-pulsating cells was collected with a closing syringe, and 5-aza-C was added again to the cultured cells (hereinafter simply referred to as myocardial progenitor cells) and cultured for 24 hours. More autonomously pulsating cells were obtained by further culturing for 2 to 3 weeks instead of IMDM medium.
  • the bone marrow stem cells capable of differentiating into cardiomyocytes exhibit a mononuclear fibroblast-like morphology under proliferating conditions, and hardly express cardiac contractile proteins.
  • 5 - aZaC the morphology changed significantly.
  • RNA was obtained from each of the bone marrow-derived primary immortalized cell lines obtained in Example 1, and possibly cardiomyocytes differentiated from immature stem cells and cardiac progenitor cells, using Trizol Reagents (GIBCO BRL). Next, using the total RNA as a substrate, Supersciptll reverse
  • First strand cDNA was synthesized using transcriptase (GIBCO BRL).
  • Cardiomyocyte-specific genes include ANP, a natriuretic peptide And BNP, myosin heavy chain -MHC and /?-MHC, actin -skeletal actin and /?-Skeletal actin, myosin light chain MLC-2a, MLC-2v, cardiomyocyte-specific transcription factor
  • ANP a natriuretic peptide And BNP
  • myosin heavy chain -MHC and /?-MHC actin -skeletal actin and /?-Skeletal actin
  • myosin light chain MLC-2a MLC-2v
  • cardiomyocyte-specific transcription factor Some Nkx2.5 / Csx, GATA4, TEF-1, MEF-2C, MEF-2D, MEF-2A were used.
  • MEF-2D for amplification of the synthetic DNAs having the nucleotide sequences of SEQ ID Nos. 57 and 58
  • MEF-2A for the amplification of the nucleotide sequences of SEQ ID Nos. 59 and 60 was used.
  • Cardiomyocytes that are induced to differentiate in vivo may contract in fetal, neonatal, or maturation phases, or in atrial or ventricular muscles to create differences in heart rate or energy efficiency of myocardial contraction. There are differences in protein isoforms.
  • natriuretic peptides ANP and BNP
  • myocardial contractile protein the phenotype of bone marrow cells differentiated into cardiomyocytes in the culture system is considered to have the characteristics of fetal ventricular myocytes.
  • the bone marrow cells were separated I spoon into cardiomyocytes in culture systems, Nkx 2. 5 / Csx, GATA4, MEF-2A, MEF-2C, Expression of MEF-2D and TEF-1 genes was observed. Expression of these transcription factors in bone marrow-derived primary immortalized cell lines in proliferation was not observed, but the expression of Nkx2.5 / Csx GATA 4 and MEF-2 C in bone marrow-derived myocardial progenitor cells in proliferation It was observed, with the induction of differentiation into cardiac myocytes, induction of the expression of late MEF-2 a and MEF-2D were observed.
  • the action potential of the bone marrow cells separated into the cardiomyocytes in the culture system was recorded by a glass microelectrode.
  • the action potential is measured at 25 ° C under a Diaphoto-300 stereo microscope (Nikon) by culturing cells in IMDM medium supplemented with 1.49 mM CaCl 2 , 4.23 mM KC, 25 mM HEPES (pH 7.4). did.
  • the glass electrode was filled with 3M KC1 with the electrode resistance set to 15 to 30 ⁇ .
  • the membrane potential was measured in a current clamp mode using MEZ-8300 (manufactured by Nihon Kohden Corporation). The measurement results were recorded on thermal paper using RTA-1100M (manufactured by Nihon Kohden Corporation).
  • sinus node cell type two types differentiated into cardiomyocytes in the culture system were observed: sinus node cell type and ventricular myocyte type.
  • the characteristics of action potentials common to both are: (1) a long action potential duration, (2) a relatively shallow resting potential, and (3) a gradual depolarization of the resting potential seen in pacemaker cells. there were.
  • the action potential In the ventricular myocyte cell type, the action potential exhibited a peak & dome type (having the first phase of action potential).
  • the action potential duration, diastolic membrane potential, and action potential amplitude of the sinus node cell type were similar to those of the sinus node reported in the past.
  • the resting membrane potential tended to be deep and the action potential amplitude tended to be large.
  • the cells of the sinus node cell type were recorded in all the cells for 2 to 3 weeks, but the ventricular myocyte type was observed from about 4 weeks after the induction of differentiation, and gradually increased over time.
  • Mouse bone marrow cells having the ability to divide into cardiomyocytes were seeded at 2 ⁇ 10 4 cells / ml on eOmm culture dish or 60 mm fibronectin-coated dish (Fibronectin-coated dish: Becton Dickinson), and 33 ° C, having conducted a cultured using 5% C0 2 concentration in the incubator unit.
  • the medium was exchanged again, and the same amount as the first group was added to each of the three groups without PDGF alone, both PDGF and retinoic acid, and no addition.
  • the morphology of the cells was observed under a phase-contrast microscope.
  • about 30% of cells in cultured tissue containing only 5-aza-C became myotube-like cells, whereas about 40% when PDGF was added, and about 50% when PDGF and retinoic acid were added simultaneously.
  • Some cells became myotube-like cells.
  • the number of cells that became myotube-like cells increased by about 10% in the three groups with the fibronectin-attached dish compared to the three groups with the culture dish.
  • Example 4 Induction of differentiation of bone marrow-derived stem cells into cardiomyocytes using DMSQ
  • 10 ⁇ M DMSO was added to the obtained bone marrow stem cells capable of differentiating into cardiomyocytes instead of 3 ⁇ M 5 -aza-C, and the cells were cultured for 24 hours. Was replaced with IMDM medium, and the culture was continued for another 6 weeks.
  • beating cardiomyocytes were induced to differentiate, and these cells expressed Nkx2.5 / Csx and GATA4 genes, and exhibited the same properties as when 5-aza-C was added. It was shown to be a cardiomyocyte that had.
  • the results of this analysis indicate that chromosomal DNA demethylation, a common function of 5-aza-C and DMSO, is required for cardiomyocyte differentiation.
  • Example 5 Demonstration that mouse bone marrow cells capable of dividing into cardiomyocytes are multipotent stem cells and cardiomyocyte precursor cells.
  • one cell is labeled by introducing the GFP gene into the virus vector, and then differentiation is induced to determine the type of cells that the labeled cell has differentiated into. It was judged.
  • a retrovirus vector plasmid expressing the GFP gene, GAR3-GFP, and a pCMV-Eco plasmid vector expressing the Ecotropic gene were obtained from Molecular Cionmg, A Laboratory Manual, second Edition, Cold Spring Harbor Laooratory Press (1989), etc. Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Section c. The day before transfection of the DNA, the confluent gag and pol genes were retained. The cells were subcultured to a 10 cm dish at a dilution of 1/5, and cultured using an incubator having a concentration of 37 ° C. and 5% CO 2 .
  • the transfection was conducted as follows.
  • GAR3-GFP retroviral vector one plasmid DNA 1 5 ⁇ g and pCMV-Eco plus Midobekuta one DNA 5 ⁇ g dissolved in addition to 250mM CaCl 2 (pH6.95) 0.5ml, was charged the solution to 15ml tubes 2 X BBS [50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaCl ⁇ 1.5mM Na 2 HP0 4 (pH6.95) ] was added dropwise to 0.5ml static at room temperature for 10 minutes Was placed.
  • BBS 50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaCl ⁇ 1.5mM Na 2 HP0 4 (pH6.95)
  • the DNA solution was added dropwise to 293 in cell culture medium were prepared the day before, were incubated with 37 ° C, '5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured further using a 37 ° C, 5% C0 2 concentration in the incubator unit.
  • IMDM medium 10-10 The solution in IMDM medium 10-10 one 2,
  • Mouse bone marrow cells capable of differentiating into cardiomyocytes to which the virus is introduced are seeded on a 6-well dish at 2 ⁇ 10 4 cells / well on the day before virus influx.
  • diluted solution containing the virus vector For the diluted solution containing the virus vector, add a final concentration of 8 ⁇ g / ml.
  • the Nkx2.5 / Csx or GATA4 gene was introduced using a viral vector, and then differentiation was induced to examine the efficiency of differentiation into cardiomyocytes.
  • Retroviral vector one plasmid P CLNC-Nkx2.5 / Csx and PCLNC- GATA4 and, pCMV-Eco plasmid base click evening one to express Ecotropic gene (Imgenex Fe) 3 ⁇ 4, Molecular Cloning, A Laboratory Manual, Second Edition, Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Cold Spring Harbor Laboratory Press (1989) and the like.
  • pCLNC—Nkx2.5 / Csx or pCLNC—GATA4 15 ⁇ g of retrovirus vector DNA and 5 ⁇ g of pCMV-Eco plasmid vector DNA are added to 0.5 ml of 250 mM CaCl 2 (pH6.95) and dissolved.
  • 2 X BBS 50m BES (N , N- bis (2-hydroxyethl) -2-aminoethanesulfonic acid), 280mM NaCl, 1.5mM Na 2 HP0 4 (pH6.95)] were placed in a tube was dropped into 0.5ml It was left at room temperature for 10 minutes.
  • the DNA solution was added dropwise to 2 ⁇ 3 in cell medium were prepared the day before, were incubated with 37 ° C, 5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured with further 3 7 ° C, 5% C0 2 concentration in the incubator unit.
  • Hexadimethrine bromide (polybrene) (Sigma) was added to the solution containing the virus vector obtained above to a final concentration of 8 ⁇ g / ml, and mouse bone marrow cells capable of differentiating into cardiomyocytes were added. And the culture was performed using an incubator at 33 ° C. and 5% CO 2 concentration. After 5 hours, and replaced with a new MDM medium was cultured 3 ⁇ 4 ⁇ Ryo' with further 3 3 ° C, 5% C_ ⁇ 2 concentration of incubation machine.
  • CS CS was added to a final concentration of 300 g / ml, and culturing was further performed for 3 days. During this time, some cells died and floated. The surviving cells were suspended in trypsin and seeded on new culture dishes.
  • Nkx2.5 / Csx or GATA4 stably transformed cells were induced to differentiate by the method of Example 3 above, and the efficiency of cardiomyocyte differentiation was assayed. As a result, it was found that the forced expression of these transcription factors promoted the efficiency of differentiation into cardiomyocytes.
  • Example 7 Telomerase activity in mouse bone marrow cells capable of differentiating into cardiomyocytes Teguchi-merase activity of mouse bone marrow cells capable of dividing into cardiomyocytes was examined by Telomeric Repeat Amplification Protocol (TRAP) method (Oncor's TRAPeze Telomerase Detection Kit). The measurement of telomerase activity basically followed the attached protocol, and was specifically performed as follows. First, mouse bone marrow cells (approximately 10 6 ) capable of differentiating into cardiomyocytes cultured on a 6 cm diameter culture dish were washed with PBS, added with 1X CHAPS solution, and allowed to stand on ice for 30 minutes. Was placed. Thereafter, the cells in a solution and the co-harvested l.
  • TRAP Telomeric Repeat Amplification Protocol
  • the cell extract of mouse bone marrow cells capable of differentiating into cardiomyocytes obtained under the above conditions was approximately 1 mg / ml.
  • telomere extension reaction was performed according to the protocol.
  • Taq polymerase used was EX Taq polymerase (Takara Shuzo). After the reaction is completed, 1/10 volume of 10X staining solution (0.25% bromophenol blue, 0.25% Xylene cyanol FF, 30% glycerol) is added, and 12.5% polyacrylamide gel (TRAPeze Telomerase Detection Kit protocol) And electrophoresed under a constant voltage of 250 mV. After swimming, the gel was stained with Cyber Green (manufactured by FMC).
  • telomerase activity was detected in a sample having a final concentration of the cell extract of 0.4 to 4 jug / ml.
  • Example 8 Transplantation of mouse bone marrow cells capable of differentiating into cardiomyocytes into the heart.
  • anesthesia was first introduced into C3H / He mice (Charles River Japan, Inc.) using ether and Terumo Anesthesia was maintained by intraperitoneal administration of 30 mg of thiopental using a thermosyringe (1 ml).
  • the limbs of the mouse were fixed to the cork board with tape, and the upper jaw was fixed to the cork board with rubber so that the neck would bend back.
  • electrocardiogram electrodes were inserted into the left and right upper limbs and the right lower limb to monitor the electrocardiogram.
  • the trachea was incised about 1 mm with a microfeather (female), and a Terumo surfro-flash (22G) needle transformed into a J-shape was inserted out of the mouth and taken out of the oral cavity.
  • the outer tube of the flash (G) was inserted into the trachea.
  • a respire overnight (model SN-480-7 manufactured by Shinano Seisakusho) was connected to this outer cylinder, and 100% oxygen was flowed at 1 ml / min.
  • the tidal volume was 1 ml, and the respiration rate was 120 / min. Started.
  • mouse bone marrow cells capable of dividing the transplanted cardiomyocytes were established in the myocardium by extracting the heart of the transplanted mouse, preparing a tissue section, and staining with a GFP-recognizing antibody. .
  • Example 9 Acquisition and culture of bone marrow cells capable of differentiating into cardiomyocytes from rat bone marrow After removing 6 female 5-week-old Wistar rats (Japan SLC Co., Ltd.) from the cervical spine, 70% ethanol was removed. It was disinfected enough. Next, a large incision was made in the skin of the foot, and the femur and tibia were removed while removing the muscle covering the femur and tibia. The removed femur and tibia were transferred to a 10 cm diameter culture dish (manufactured by Iwaki Glass) containing PBS (manufactured by GibcoBRL), and the muscles and joints were completely resected.
  • a 10 cm diameter culture dish manufactured by Iwaki Glass
  • PBS manufactured by GibcoBRL
  • the total number of recovered cells was 2.6 ⁇ 10 9 .
  • 1 ⁇ 10 8 cells were collected from one femur or tibia.
  • the collected cells were diluted to a concentration of 1.3 ⁇ 10 8 cells / ml, and Percoll (manufactured by Amersham Pharmacia Biotech) / D-PBS solution (ml) adjusted to 1.073 g / ml in a 50 ml centrifuge tube After overlaying on top, centrifuged at room temperature at 3,100 rpm; for 30 minutes. After centrifugation, the cells were collected from the interface between the Percoll solution and cell suspension.
  • the bone marrow-derived cells thus fractionated into 3 culture dishes for animal cells with a diameter of 10 cm (Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes) so as to have a size of 2 to 5 x 10 5 cells / cm 2.
  • 10 cm culture dishes The bone marrow-derived cells thus fractionated into 3 culture dishes for animal cells with a diameter of 10 cm (Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes) so as to have a size of 2 to 5 x 10 5 cells / cm 2.
  • 10 cm culture dishes Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes
  • Example 10 Examination of differentiation ability of rat bone marrow-derived cells into cardiomyocytes
  • the confluent rat bone marrow-derived cells were separated again by trypsin EDTA treatment, and the cells were transferred to a 6-well plate (BECTON DICKINSON) in such a manner that 5 x 10 4 cells per 1-well were obtained.
  • the culture dish 6cm diameter coated with Hitofu Eve Lone cutin was reseeded cells to become 1.'3 X 10 5 cells.
  • a bone marrow cell a growth factor, a vitamin, an adhesion molecule, and a method of using the same that are effective for treating a heart disease associated with destruction and degeneration of cardiomyocytes and for searching for a therapeutic agent are provided.
  • SEQ ID NO: 4 1 Description of artificial sequence: Synthetic DNA
  • SEQ ID NO: 46 Description of artificial sequence: Synthetic DNA

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Abstract

Methods for isolating, purifying, culturing and differentiation-inducing bone marrow cells capable of differentiating into heart muscle cells; a method for proliferating bone marrow cells capable of differentiating into heart muscle cells by using various cytokines, transcription factors, etc.; a method for regulating the differentiation into heart muscle cells; a method for obtaining a surface antigen specific to bone marrow cells capable of differentiating into heart muscle cells; a method for obtaining a gene encoding this surface antigen; a method for obtaining an antibody specific to the surface antigen; a method for obtaining a protein and a gene participating in the proliferation and differentiation into heart muscle cells of bone marrow cells capable of differentiating into heart muscle cells; and remedies for various heart diseases with the use of bone marrow cells capable of differentiating into heart muscle cells.

Description

心筋細胞への分化能を有する成体骨髄由来細胞  Adult bone marrow-derived cells capable of differentiating into cardiomyocytes
技術分野 Technical field
本発明は、 心筋細胞への分ィ匕能を有する骨髄細胞の単離、 精製、 培養、 分化誘導法 に関する。 また本発明は、 各種サイトカイン、 転写因子などを用いた、 心筋細胞への 分化能を有する骨髄細胞の増殖方法および心筋細胞への分ィ匕を制御する方法に関す る。本発明はさらに、 心筋細胞への分化能を有する骨髄細胞に特異的な表面抗原の取 明 _  The present invention relates to a method for isolating, purifying, culturing, and inducing differentiation of bone marrow cells having the ability to isolate cardiomyocytes. The present invention also relates to a method for expanding myeloid cells capable of differentiating into cardiomyocytes using various cytokines, transcription factors, and the like, and a method for controlling the division into cardiomyocytes. The present invention further provides a surface antigen specific for bone marrow cells capable of differentiating into cardiomyocytes.
得方法、 該表面抗原をコードする遺伝子の取得方法、 該表面抗原特異的な抗体の取得 田 Obtaining method, obtaining a gene encoding the surface antigen, obtaining an antibody specific to the surface antigen
方法、心筋細胞への分化能を有する骨髄細胞の増殖および心筋細胞への分化に関与す る蛋白質および遺伝子の取得方法に関する。本発明 (ままた、 心筋細胞への分化能を有 する骨髄細胞を用 tヽた各種心臓疾患の治療薬に関する。 The present invention relates to a method and a method for obtaining proteins and genes involved in proliferation of bone marrow cells capable of differentiating into cardiomyocytes and differentiation into cardiomyocytes. The present invention relates to a therapeutic agent for various heart diseases using bone marrow cells capable of differentiating into cardiomyocytes.
背景技術 Background art
心筋細胞は、 出生前は自律拍動しながら活発に細胞分裂を行っている。 しかし、 出 生と同時にその分裂能は喪失し、肝細胞のように再び細胞分裂能を獲得することはな く、 また骨格筋細胞とも異なり衛星細胞といつた未分化な前駆細胞を持つこともない。 従って、 心筋梗塞、 心筋炎または老化等に伴い心筋細胞が壊死すると、 生体内では残 存心筋細胞の細胞分裂ではなく細胞の肥大がおきる。心肥大は初期においては生理的 適応であるが、 また共存する心線維芽細胞の増殖による間質の線維化と相まつて心臓 自体の拡張機能の低下、 さらには収縮機能の低下へと結びつき心不全を呈するように なる。心筋梗塞等による心不全のこれまでの治療は心収縮力の増強、血管拡張薬によ る心臓の圧負荷 ·容量負荷の軽減、利尿薬による血流量の減少等の対症療法を中心に 行われてきた。 これに対し、 心臓移植は重症心不全に対する根本的な治療法であるが、 臓器提供者の不足、 脳死判定の難しさ、 拒絶反応、 医療費の高騰等の問題から心臓移 植が一般的な医療に普及するのは簡単ではない。実際、 心臓病は我が国の死亡原因の 第 3位となっており （厚生白書平成 1 0年）、 失われた心筋細胞を再生することがで きれば医療福祉の大きな前進につながると考えられる。  Before birth, cardiomyocytes actively divide while autonomously beating. However, their division ability is lost at birth, they do not regain cell division ability like hepatocytes, and unlike skeletal muscle cells, they have satellite cells and undifferentiated precursor cells. Absent. Therefore, if cardiomyocytes are necrotic due to myocardial infarction, myocarditis, or aging, in vivo, cell expansion of the remaining cardiomyocytes rather than cell division occurs. Cardiac hypertrophy is initially a physiological adaptation, but it also leads to a decrease in the diastolic function of the heart itself and a decrease in systolic function in conjunction with the fibrosis of the interstitium due to the proliferation of coexisting cardiac fibroblasts, leading to heart failure. Will be presented. Until now, treatment of heart failure due to myocardial infarction has focused on symptomatic treatments such as enhancement of cardiac contractility, reduction of cardiac pressure / volume loading with vasodilators, and reduction of blood flow with diuretics. Was. On the other hand, heart transplantation is a fundamental treatment for severe heart failure, but heart transplantation is a common medical treatment due to problems such as a shortage of organ donors, difficulty in determining brain death, rejection, and rising medical costs. It is not easy to spread. In fact, heart disease is the third leading cause of death in Japan (Health and Welfare White Paper, Heisei 10), and if we can regenerate lost cardiomyocytes, it will be a major step forward in medical welfare.
現在までに、 心筋細胞の性質を保存した細胞株としては、 心房性ナトリウム利尿ホ ルモンのプロモ一夕一に SV40の large T抗原を組み換えて作製したトランスジェニヅ クマウスの心房に生じた腫瘍から株化された AT - 1細胞があげられる [Science, 239; 1029-1038 (1988)]。 しかしながら、 該細胞は in vivoに移植すると腫瘍を形成するため 細胞移植には適さないという問題がある。 そこで、 このような背景のもと、 心筋を再 構築するため以下の方法が考えられた。 To date, cell lines that preserve the properties of cardiomyocytes include atrial natriuretic AT-1 cells established from tumors arising in the atrium of transgenic mice produced by recombining the large T antigen of SV40 overnight with the Lemon promoter [Science, 239; 1029-1038 (1988)] ]. However, there is a problem that these cells are not suitable for cell transplantation because they form tumors when transplanted in vivo. Under these circumstances, the following methods were considered to reconstruct the myocardium.
1つ目の方法は、 心筋細胞以外の細胞を心筋細胞に変換する方法である。 これは、 線維芽細胞に MyoDを導入すると骨格筋細胞に変換できることから類推された。こ： までに、 マウスの胎児性癌細胞である P 1 9細胞での成功例は示されているものの [Cell Struc. &Func, 21: 101-110 (1996)]、 非ガン細胞での成功例は報告されていない。  The first method is to convert cells other than cardiomyocytes into cardiomyocytes. This was inferred from the fact that introduction of MyoD into fibroblasts could convert them into skeletal muscle cells. Previously, although successful cases have been shown with P19 cells, which are mouse embryonal cancer cells, [Cell Struc. & Func, 21: 101-110 (1996)], successful cases with non-cancer cells Has not been reported.
2つ目の方法は、 心筋細胞に再び分裂能を付与する方法である。 これは、 胎児期に 心筋が拍動しながら分裂できる現象に基づいている。 しかしながら、 これまでに成功 例は報告されていない。  The second method is to give the cardiomyocytes divisional ability again. This is based on the fact that the heart muscle can divide while beating during fetal life. However, no successful cases have been reported so far.
. 3つ目の方法は、 未分化な幹細胞から心筋細胞を誘導する方法である。 すでに、 胚 性幹細胞 （ES細胞） から心筋細胞を誘導できることが示されているが、 胚性幹細胞 自身を成体に移植するとカルシノーマを形成すること、抗原性などの問題が存在する [Nature Biotechnology, 17, 139-142 (1999)]。  The third method is to induce cardiomyocytes from undifferentiated stem cells. Although it has been shown that cardiomyocytes can be induced from embryonic stem cells (ES cells), transplantation of embryonic stem cells themselves into adults has problems such as formation of carcinoma and antigenicity [Nature Biotechnology, 17 , 139-142 (1999)].
従って、胚性幹細胞を現実の医療へと応用するためには、 少なくとも心筋前駆細胞 あるいは、 心筋細胞を純粋に精製する技術が不可欠である。抗原性の問題はクローン 化の技術により解決できる可能性は示唆されているが、煩雑な操作を必要とすること から一般的な医療への応用は容易ではない。  Therefore, in order to apply embryonic stem cells to real medicine, at least a technique for purifying myocardial progenitor cells or cardiomyocytes in a pure manner is indispensable. Although it has been suggested that the problem of antigenicity can be solved by cloning technology, it is not easy to apply it to general medical care because it requires complicated operations.
中絶胎児から未分化な細胞である心筋前駆細胞を取得して移植に用いる方法も考 えられており、動物を用いた実験では心筋細胞として有効に機能することが知られて いる [Science, 264, 98-101 (1994)]0 しかしながら、 この方法で大量の心筋前駆細胞を 取得することは困難であり、倫理の観点からも一般的な医療への応用は容易ではない, 成体骨髄には造血系幹細胞および血管幹細胞以外に間葉系幹細胞が存在し、間葉系 幹細胞からは骨細胞、 軟骨細胞、 腱細胞、 靱帯細胞、 骨格筋細胞、 脂肪細胞、 スト口 —マ細胞、 肝臓 oval細胞が分ィ匕誘導できることが報告されている [Science, ， 143- 147 (1999); Science, 284, 1168-1170 (1999)]。 一方、 最近、 マウス成体の骨髄から取得 した細胞から、心筋細胞が分化誘導できることが見い出された Clinical Investigation, !^，10-18 (1999)]。該報告は患者自身から骨髄液を取得して、 in vitroで細胞培養およ び薬剤処理を行った後に、心臓の障害部位へ移植する細胞治療が現実的な医療として 可能になることを示唆している ϋ· Clinical Investigation, 103, 591-592 (1999)]。 しかし ながら、 該報告は、 成体マウスの骨髄から樹立した不死化細胞の一部が心筋細胞に分 化できることを示したものにすぎない。 また、 成体骨髄中の心筋細胞に分化する能力 を有する細胞の特性の同定、 該細胞を増殖する方法、 該細胞から効率的に心筋細胞に 分化誘導する方法については明らかでなかった [J. Clinical Investigation, 103, 591-592 (1999)] o A method of obtaining undifferentiated myocardial progenitor cells from aborted fetuses and using them for transplantation has also been considered, and it is known that they can function effectively as cardiomyocytes in animal experiments [Science, 264]. , 98-101 (1994)] 0, however, it is difficult to obtain a large amount of cardiac progenitor cells in this way, the applications also to common medical terms of ethical not easy, hematopoiesis in adult bone marrow There are mesenchymal stem cells in addition to stem cells and vascular stem cells, which include bone cells, chondrocytes, tendon cells, ligament cells, skeletal muscle cells, fat cells, stoma cells, and liver oval cells. It has been reported that it can be guided [Science, 143-147 (1999); Science, 284, 1168-1170 (1999)]. Meanwhile, recently obtained from adult mouse bone marrow It has been found that cardiomyocytes can be induced to differentiate from the isolated cells. Clinical Investigation,! ^, 10-18 (1999)]. The report suggests that cell therapy, in which bone marrow fluid is obtained from the patient himself and transplanted to a damaged area of the heart after cell culture and drug treatment in vitro, is possible as a realistic medical treatment. Clinical · Clinical Investigation, 103, 591-592 (1999)]. However, the report merely shows that some of the immortalized cells established from the bone marrow of adult mice can differentiate into cardiomyocytes. Furthermore, it was not clear how to identify the characteristics of cells having the ability to differentiate into cardiomyocytes in adult bone marrow, how to proliferate the cells, and how to efficiently induce the cells to differentiate into cardiomyocytes [J. Clinical. Investigation, 103, 591-592 (1999)] o
発明の開示 Disclosure of the invention
現在の心疾患治療より安全かつ確実な治療が望まれている。そこで、骨髄細胞中よ り心筋細胞への分化能を有する骨髄細胞を選別し、心筋細胞への分化能を有する骨髄 細胞の増殖または分ィ匕をコントロールすることは、骨髄由来の細胞を用いた心筋の再 生治療の開発に有用である。 そのために、 骨髄中の細胞から心筋細胞への分ィ匕能を有 する細胞を特定して、該細胞の増殖または分化に働くサイトカインまたは転写因子を 同定することが必要である。 本発明者は上記問題点を閧発すべく鋭意研究し、 以下の結果を得た。すなわち、 マ ウス骨髄由来の細胞株を、 GFP(Green Fluorescent Protein)を発現するレトロウィルス ベクターを用いて標識し、 1つの細胞を蛍光顕微鏡下で追跡することで、 心筋細胞へ の分化能を有する細胞が、心筋細胞および脂肪細胞の少なくとも 2種類の異なる細胞 を分化誘導できる多分化能 (Purulipotent)を持った幹細胞であることを見い出した。 ま た、 同時に増殖性を有し、 心筋細胞だけに分ィ匕する心筋幹細胞、 および有限の細胞分 裂能を有し、 心筋細胞だけに分化する心筋前駆細胞が存在することも見い出した。 さ らに、.該幹細胞は通常の培養条件下ではすでに報告されている 5—ァザシチジンだけ でなく、 DMSO (dimethyl sulfoxide) などの他のゲノム DNAの脱メチル化剤の投与に よっても、 確率的（stochastic) に心筋細胞、脂肪細胞および骨格筋細胞の系列に分化 することを見出し、 ゲノム DNAの脱メチル化が骨髄由来の細胞からの心筋細胞への 分ィ匕誘導に有効であることを明らかにした。 また確率的（stochastic)な分化が、 培養 条件やサイトカインの投与、転写因子の強制発現,などにより心筋細胞、脂肪細胞およ び骨格筋細胞への分化の効率を改変できることを見い出した。すなわち、 血小板由来 増殖因子 (PDGF)等のサイトカインあるいは aU-transレチノイン酸の投与、またはフィ ブロネクチンでコートした培養皿で培養することにより、心筋細胞形成の割合を増加 させること ¾見い出した。 また、 線維芽細胞増殖因子— 2 (FGF- の投与により、 心 筋細胞への分化を抑制できることも見い出した。 A safer and more reliable treatment than the current treatment for heart disease is desired. Therefore, to select bone marrow cells capable of differentiating into cardiomyocytes from bone marrow cells and to control proliferation or sorting of bone marrow cells capable of differentiating into cardiomyocytes, cells using bone marrow-derived cells were used. It is useful for the development of regenerative therapy for myocardium. For this purpose, it is necessary to identify cells capable of dividing bone marrow cells into cardiomyocytes, and to identify cytokines or transcription factors that act on the proliferation or differentiation of the cells. The inventors of the present invention have conducted intensive studies to address the above problems and have obtained the following results. In other words, a mouse bone marrow-derived cell line is labeled with a retroviral vector expressing GFP (Green Fluorescent Protein), and by tracking one cell under a fluorescence microscope, it has the ability to differentiate into cardiomyocytes. The cells were found to be pluripotent stem cells capable of inducing differentiation of at least two different types of cells, cardiomyocytes and adipocytes. It was also found that there are myocardial stem cells that are proliferative at the same time and divide only into myocardial cells, and myocardial progenitor cells that have finite cell division ability and differentiate only into myocardial cells. In addition, the stem cells can be probabilistically treated by administration of other genomic DNA demethylating agents such as DMSO (dimethyl sulfoxide) as well as 5-azacytidine, which has already been reported under normal culture conditions. (Stochastic) to differentiate into cardiomyocyte, adipocyte and skeletal muscle cell lineages, and demethylation of genomic DNA It was clarified that it was effective for shunting guidance. We also found that stochastic differentiation can alter the efficiency of differentiation into cardiomyocytes, adipocytes, and skeletal muscle cells depending on culture conditions, administration of cytokines, and forced expression of transcription factors. That is, it was found that administration of a cytokine such as platelet-derived growth factor (PDGF) or aU-trans retinoic acid, or culturing in a culture dish coated with fibronectin, increased the rate of cardiomyocyte formation. We also found that administration of fibroblast growth factor-2 (FGF-) can suppress differentiation into cardiac myocytes.
すなわち、 本発明は以下の （1) 〜 （96) を提供するものである。  That is, the present invention provides the following (1) to (96).
( 1 ) 骨髄から単離され、 心筋細胞に分化する能力を有する細胞。  (1) Cells isolated from bone marrow and capable of differentiating into cardiomyocytes.
( 2 ) 少なくとも心筋細胞と脂肪細胞に分化する能力を有する多分化能幹細胞であ る上記 （ 1 ) 記載の細胞。  (2) The cell according to (1), which is a pluripotent stem cell having at least the ability to differentiate into cardiomyocytes and adipocytes.
( 3 ) 心筋細胞のみに分化誘導される心筋幹細胞である上記 （ 1 )記載の細胞。 (3) The cell according to (1) above, which is a cardiomyocyte that is induced to differentiate only into cardiomyocytes.
( 4 ) 心筋細胞のみに分化誘導される心筋前駆細胞である上記（ 1 )記載の細胞。(4) The cell according to (1), which is a cardiomyocyte that is induced to differentiate only into cardiomyocytes.
(5) 心室筋細胞に分化する能力を有する、 上記 （1)記載の細胞。 (5) The cell according to (1), which has an ability to differentiate into ventricular myocytes.
( 6 ) 洞結節細胞に分化する能力を有する、 上記 （ 1 )記載の細胞。  (6) The cell according to (1), which has an ability to differentiate into sinus node cells.
(7) 骨髄がほ乳動物由来のものである、 上記 （1)記載の細胞。  (7) The cell according to (1), wherein the bone marrow is derived from a mammal.
(8) ほ乳動物がヒト、 ラヅトおよびマウスから選ばれるものである、 上記 （7) 記載の細胞。  (8) The cell according to (7), wherein the mammal is selected from human, rat, and mouse.
(9) 細胞が、 マウス骨髄由来幹細胞 BMSC (FERM BP-7043) である、 上記 （1) 記載の細胞。  (9) The cell according to (1), wherein the cell is a mouse bone marrow-derived stem cell BMSC (FERM BP-7043).
(10) 染色体 DNAの脱メチル化により心筋細胞に分化する能力を有する、 上記 ( 1 ) 記載の細胞。  (10) The cell according to (1), which has the ability to differentiate into cardiomyocytes by demethylation of chromosomal DNA.
(11) 染色体 DNAの脱メチル化が、 デメチラーゼ、 5—ァザシチジンおよびジ メチルスルフォキシド （DMSO) からなる群から選ばれる少なくとも 1種によるもの であることを特徴とする、 上記 （10)記載の細胞。  (11) The method according to (10), wherein the demethylation of the chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and dimethyl sulfoxide (DMSO). cell.
(12) デメチラ一ゼが、配列番号 1記載で表されるアミノ酸配列を有するデメチ ラ一ゼである、 上記 （11)記載の細胞。  (12) The cell according to (11), wherein the demethylase is a demethylase having an amino acid sequence represented by SEQ ID NO: 1.
(13) 胎児の心臓発生領域で発現している因子により心筋細胞の分ィヒが促進され る上記 （ 1 ) 記載の細胞。 (13) Factors expressed in the fetal heart development region promote cardiomyocyte differentiation The cell according to the above (1).
( 14) 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ夕 ミンおよび転写因子からなる群から選ばれる少なくとも 1種であることを特徴とす る、 上記 （13)記載の細胞。  (14) The factor according to (13) above, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. cell.
(15) 胎児の心臓発生段階において心筋細胞への分化に働く因子により心筋細胞 への分化が促進される、 上記 （1)記載の細胞。  (15) The cell according to (1) above, wherein differentiation into cardiomyocytes is promoted by a factor that acts on cardiomyocyte differentiation at the stage of fetal heart development.
(16) 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、 接着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であるこ とを特徴とする、 上記 （15)記載の細胞。  (16) The factor according to (15), wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors. cell.
(17) サイトカインが血小板由来増殖因子 （PDGF) である、 上記 （ 14) また は （ 16 )記載の細胞。  (17) The cell according to (14) or (16), wherein the cytokine is platelet-derived growth factor (PDGF).
(18) PDGFが配列番号 3または 5で表されるァミノ酸配列を有する PDGFであ る、 上記 （17)記載の細胞。  (18) The cell according to (17) above, wherein the PDGF is PDGF having an amino acid sequence represented by SEQ ID NO: 3 or 5.
(19) 接着分子がフイブロネクチンである、 上記 （14) または （16)記載の 細胞。  (19) The cell according to (14) or (16) above, wherein the adhesion molecule is fibronectin.
(20) ビタミンがレチノイン酸である、 上記（14) または （16)記載の細胞。 (21) 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF-2A、 MEF- 2B、 MEF— 2C、 MEF- (20) The cell according to (14) or (16), wherein the vitamin is retinoic acid. (21) The transcription factor is Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-
2D、 dHAND、 eHAND、 TEF - 1、 TEF-3および TEF- 5からなる群から選ばれるもので ある、 上記 （ 14) または （16)記載の細胞。 The cell according to the above (14) or (16), which is selected from the group consisting of 2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5.
(22) Nkx2.5/Csxが配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csxで ある、 上記 （21)記載の細胞。 (22) Nkx 2. 5 / Csx is Nkx 2. 5 / Csx having the amino acid sequence is represented by SEQ ID NO: 9, the (21), wherein the cell.
(23) GATA が配列番号 11で表されるアミノ酸配列を有する GATA4である、 上記 （ 21 ) 記載の細胞。  (23) The cell according to (21), wherein GATA is GATA4 having an amino acid sequence represented by SEQ ID NO: 11.
(24) MEF-2Aが配列番号 13で表されるァミノ酸配列を有する MEF- 2Aである、 上記 （ 21 ) 記載の細胞。  (24) The cell according to the above (21), wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
(25) MEF-2Bが配列番号 15で表されるァミノ酸配列を有する MEF - 2Bである、 上記 （ 21 ) 記載の細胞。  (25) The cell according to (21), wherein MEF-2B is MEF-2B having an amino acid sequence represented by SEQ ID NO: 15.
(26) MEF- 2Cが配列番号 17で表されるアミノ酸配列を有する MEF-2Cである、 上記 （ 21 ) 記載の細胞。 (26) MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17, The cell according to the above (21).
(27) MEF-2Dが配列番号 19で表されるアミノ酸配列を有する MEF-2Dである、 上記 （ 21 ) 記載の細胞。  (27) The cell according to (21), wherein MEF-2D is MEF-2D having the amino acid sequence represented by SEQ ID NO: 19.
(28) dHANDが配列番号 21で表されるァミノ酸配列を有する dHANDである、 上記 （ 21 )記載の細胞。  (28) The cell according to (21), wherein the dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21.
(29) eHANDが配列番号 23で表されるァミノ酸配列を有する eHANDである、 上記 （ 21 ) 記載の細胞。  (29) The cell according to (21), wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
(30) TEF-1が配列番号 25で表されるァミノ酸配列を有する TEF- 1である、 上記 （ 21 ) 記載の細胞。  (30) The cell according to (21), wherein TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
(31) TEF-3が配列番号 27で表されるァミノ酸配列を有する TEF-3である、 上記 （ 21 )記載の細胞。  (31) The cell according to (21), wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
(32) TEF-5が配列番号 29で表されるァミノ酸配列を有する TEF - 5である、 上記 （ 21 ) 記載の細胞。  (32) The cell according to (21), wherein TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
(33) 線維芽細胞増殖因子— 2 (FGF-2) により心筋細胞への分ィ匕が抑制される、 上記 （ 1 ) 記載の細胞。  (33) The cell according to (1), wherein fibroblast growth factor-2 (FGF-2) suppresses the division into cardiomyocytes.
(34) FGF-2が配列番号 7または 8記載のアミノ酸配列を有する FGF-2である、 上記 （ 33 ) 記載の細胞。  (34) The cell according to (33), wherein FGF-2 is FGF-2 having the amino acid sequence of SEQ ID NO: 7 or 8.
(35) 染色体 DNAの脱メチル化剤を用いて、 骨髄由来の細胞から心筋を形成す る方法。  (35) A method for forming a myocardium from bone marrow-derived cells using a chromosomal DNA demethylating agent.
(36) 染色体 DNAの脱メチル化剤が、 デメチラ一ゼ、 5—ァザシチジンおよび DMSOからなる群から選ばれる少なくとも 1種であることを特徴とする、上記（35) 記載の方法。  (36) The method according to (35), wherein the demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
(37) デメチラ ゼが、配列番号 1記載のアミノ酸配列で表されるデメチラ一ゼ である、 上記 （36) 記載の方法。  (37) The method according to (36) above, wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
(38) 胎児の心臓発生領域で発現している因子を用いることを特徴とする、 骨髄 由来の細胞から心筋を形成する方法。  (38) A method for forming myocardium from bone marrow-derived cells, comprising using a factor expressed in a fetal heart development region.
(39) 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ夕 ミンおよび転写因子からなる群から選ばれる少なくとも 1種であることを特徴とす る、 上記 （38) 記載の方法。 (39) The factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin, and transcription factors. The method according to (38).
(40) 胎児の心臓発生段階において心筋細胞への分ィ匕に働く因子を用いることを 特徴とする、 骨髄由来の細胞から心筋を形成する方法。  (40) A method for forming a myocardium from cells derived from bone marrow, which comprises using a factor that acts to divide the myocardial cells at the stage of fetal heart development.
(41) 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、 接着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であるこ とを特徴とする、 上記 （40) 記載の方法。  (41) The factor according to (40), wherein the factor that acts on differentiation into cardiomyocytes during the fetal heart development stage is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors. Method.
(42) サイ トカインが、 PDGFである、 上記 （39) または （41) 記載の方法 c (43) PDGFが配列番号 3または 5記載のアミノ酸配列で表される PDGFである、 上記 （42)記載の方法。 (42) The method c according to (39) or (41), wherein the cytokine is PDGF. (43) The PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5, according to (42). the method of.
(44) 接着分子がフイブロネクチンである、 上記（39) または （41) 記載の 方法。  (44) The method according to the above (39) or (41), wherein the adhesion molecule is fibronectin.
(45) ビタミンがレチノイン酸である、 上記（39) または （41)記載の方法 c (46) 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF_2A、 MEF- 2B、 MEF-2Cヽ MEF- 2D、 dHAND、 eHAND、 TEF- 1、 TEF-3および TEF - 5からなる群から選ばれる少なく とも 1種であることを特徴する、 上記 （39) または （41) 記載の方法。  (45) The method according to (39) or (41) above, wherein the vitamin is retinoic acid. C (46) The transcription factor is Nkx2.5 / Csx, GATA4, MEF_2A, MEF-2B, MEF-2C ヽ MEF-2D. The method according to (39) or (41), wherein the method is at least one selected from the group consisting of: dHAND, eHAND, TEF-1, TEF-3, and TEF-5.
(47) Nkx2.5/Csxが、 配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csx である、 上記 （46) 記載の方法。  (47) The method according to (46), wherein Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
(48) GATA4が、配列番号 1 1で表されるァミノ酸配列を有する GATA4である、 上記 （ 46 ) 記載の方法。  (48) The method according to the above (46), wherein GATA4 is GATA4 having an amino acid sequence represented by SEQ ID NO: 11.
(49) MEF-2Aが、配列番号 13で表されるァミノ酸配列を有する MEF- 2Aであ る、 上記 （46) 記載の方法。  (49) The method according to (46), wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
(50) MEF-2Bが、 配列番号 15で表されるァミノ酸配列を有する MEF- 2Bであ る、 上記 （46) 記載の方法。 (50) MEF-2B are, Ru MEF-2 B der with Amino acid sequence represented by SEQ ID NO: 15, the (46) The method according.
(51) MEF-2Cが、配列番号 17で表されるァミノ酸配列を有する MEF - 2Cであ る、 上記 （46) 記載の方法。  (51) The method according to (46), wherein MEF-2C is MEF-2C having an amino acid sequence represented by SEQ ID NO: 17.
(52) MEF-2Dが、配列番号 19で表されるァミノ酸配列を有する MEF - 2Dであ る、 上記 （46) 記載の方法。  (52) The method according to (46), wherein MEF-2D is MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
(53) dHANDが、配列番号 21で表されるァミノ酸配列を有する dHANDである、 上記 （46)記載の方法。 (53) dHAND is a dHAND having an amino acid sequence represented by SEQ ID NO: 21, The method according to (46) above.
(54) eHANDが、配列番号 23で表されるァミノ酸配列を有する eHANDである、 上記 （ 46 ) 記載の方法。  (54) The method according to the above (46), wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
(55) TEF - 1が、 配列番号 25で表されるアミノ酸配列を有する TEF - 1である、 上記 （46)記載の方法。  (55) The method according to (46) above, wherein TEF-1 is TEF-1 having the amino acid sequence represented by SEQ ID NO: 25.
(56) TEF - 3が、 配列番号 27で表されるアミノ酸配列を有する TEF -3である、 上記 （46)記載の方法。 (56) The method according to the above (46), wherein the TEF- 3 is a TEF- 3 having an amino acid sequence represented by SEQ ID NO: 27.
(57) EF-5が、 配列番号 29で表されるァミノ酸配列を有する TEF 5である、 上記 （ 46 ) 記載の方^  (57) The method according to the above (46), wherein EF-5 is TEF5 having an amino acid sequence represented by SEQ ID NO: 29.
(58) 染色体 DNAの脱メチル化剤を有効成分として含有することを特徴とする 心筋形成剤。  (58) A cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
(59) 染色体 DNAの脱メチル化剤が、 デメチラ一ゼ、 5一ァザシチジンおよび DMSOからなる群から選ばれる少なくとも 1種である、 上記（58)記載の心筋形成 剤。  (59) The cardiomyogenic agent according to (58), wherein the demethylating agent for chromosomal DNA is at least one member selected from the group consisting of demethylase, 5-azacitidine and DMSO.
(60) デメチラ一ゼが、 配列番号 1記載のアミノ酸配列で表されるデメチラ一ゼ である、 上記 （59)記載の心筋形成剤。  (60) The cardiomyogenic agent according to (59), wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
(61) 胎児の心臓発生領域で発現している因子を有効成分として含有する心筋形 成剤。  (61) A myocardium-forming agent containing, as an active ingredient, a factor expressed in a fetal heart development region.
(62) 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ夕 ミンおよび転写因子からなる群から選ばれる少なくとも 1種であることを特徴とす る、 上記 （ 61 )記載の心筋形成剤。  (62) The factor according to the above (61), wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, bimin and transcription factors. Cardiomyogen.
(63) 胎児の心臓発生段階において心筋細胞への分化に働く因子を有効成分とし て含有することを特徴とする心筋形成剤。  (63) A cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
(64) 胎児の心臓発生段階において心筋細胞への分化に働く因子が、 サイトカイ ン、接着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であ ることを特徴とする、 上記 （63)記載の心筋形成剤。  (64) The factor described above, wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins, and transcription factors. 63) The myocardial agent according to the above.
(65) サイ トカインが、 PDGFである、 上記 （62) または （64)記載の心筋 形成剤。 (66) PDGFが配列番号 3または 5記載のアミノ酸配列で表される、上記（65) 記載の心筋形成剤。 (65) The cardiomyogenic agent according to the above (62) or (64), wherein the cytokine is PDGF. (66) The cardiomyogenic agent according to (65), wherein PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5.
(67) 接着分子がフイブロネクチンである、 上記 （62) または （64)記載の 心筋形成剤。  (67) The cardiomyogenic agent according to (62) or (64), wherein the adhesion molecule is fibronectin.
(68) ビタミンがレチノイン酸である、 上記 （62) または （64) 記載の心筋 形成剤。  (68) The cardiomyogenic agent according to (62) or (64), wherein the vitamin is retinoic acid.
(69) 転写因子が、 Nkx2.5/Csxヽ GATA4、 MEF- 2Aヽ MEF- 2B、 MEF- 2C、 MEF- 2D、 dHAND、 eHAND、 TEF-1、 TEF- 3および TEF- 5からなる群から選ばれる、 上記 (69) The transcription factor is selected from the group consisting of Nkx2.5 / Csx ヽ GATA4, MEF-2A ヽ MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5 Selected, above
(62) または （64) 記載の心筋形成剤。 (62) or (64).
(70) Nkx2.5/Csxが、 配列番号 9記載のァミノ酸配列で表される Nkx2.5/Csxで ある、 上記 （69) 記載の心筋形成剤。  (70) The cardiomyogenic agent according to (69), wherein Nkx2.5 / Csx is Nkx2.5 / Csx represented by the amino acid sequence of SEQ ID NO: 9.
(71) GATA が、配列番号 1 1記載のァミノ酸配列で表される GATA4である、 上記 （ 69 ) 記載の心筋形成剤。  (71) The cardiomyogenic agent according to (69), wherein GATA is GATA4 represented by the amino acid sequence of SEQ ID NO: 11.
(72) MEF-2Aが、 配列番号 13記載のアミノ酸配列で表される MEF- 2Aである、 上記 （ 69 ) 記載の心筋形成剤。 (72) MEF-2A is a represented by MEF-2 A the amino acid sequence of SEQ ID NO: 13, wherein said (69) myocardial former description.
(73) MEF-2Bが、 配列番号 15記載のァミノ酸配列で表される MEF - 2Bである、 上記 （ 69 ) 記載の心筋形成剤。  (73) The cardiomyogenic agent according to (69), wherein MEF-2B is MEF-2B represented by the amino acid sequence of SEQ ID NO: 15.
(74) MEF-2Cが、配列番号 17記載のアミノ酸配列で表される MEF - 2Cである、 上記 （ 69 ) 記載の心筋形成剤。  (74) The cardiomyogenic agent according to (69), wherein MEF-2C is MEF-2C represented by the amino acid sequence of SEQ ID NO: 17.
(75) MEF-2Dが、配列番号 19記載のアミノ酸配列で表される MEF_2Dである、 上記 （ 69 ) 記載の心筋形成剤。 (75) MEF-2D is a MEF_ 2 D represented by the amino acid sequence of SEQ ID NO: 19, wherein said (69) myocardial former description.
(76) dHANDが、配列番号 21記載のアミソ酸配列で表される dHANDである、 上記 （ 69 )記載の心筋形成剤。  (76) The cardiomyogenic agent according to the above (69), wherein the dHAND is a dHAND represented by the amino acid sequence of SEQ ID NO: 21.
(77) eHANDが、配列番号 23記載のァミノ酸配列で表される eHANDである、 上記 （ 69 ) 記載の心筋形成剤。  (77) The cardiomyogenic agent according to the above (69), wherein the eHAND is an eHAND represented by the amino acid sequence of SEQ ID NO: 23.
(78) TEF-1が、 配列番号 25記載のァミノ酸配列で表される TEF- 1である、 上記 （ 69 ) 記載の心筋形成剤。  (78) The cardiomyogen according to (69), wherein TEF-1 is TEF-1 represented by the amino acid sequence of SEQ ID NO: 25.
(79) TEF-3が、 配列番号 27記載のアミノ酸配列で表される TEF - 3である、 上記 （ 69 ) 記載の心筋形成剤。 (79) TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27, (69) The cardiomyogen according to (69).
(80) TEF- 5が、 配列番号 29記載のアミノ酸配列で表される TEF - 5である、 上記 （ 69 ) 記載の心筋形成剤。  (80) The cardiomyogenic agent according to (69), wherein TEF-5 is TEF-5 represented by the amino acid sequence of SEQ ID NO: 29.
(81) 上記 （1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴と する、 心臓疾患により破壊された心臓を再生する方法。  (81) A method for regenerating a heart destroyed by a heart disease, comprising using the cell according to any one of (1) to (34).
(82) 上記 （1)〜 （34)のいずれか 1項に記載の細胞を有効成分とする心臓 再生治療薬。  (82) A therapeutic agent for regenerating heart, comprising the cell according to any one of the above (1) to (34) as an active ingredient.
(83) 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入され た上記 （1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴とする、 先 天性遺伝子疾患での変異遺伝子に対する野生型遺伝子を心筋へ特異的に輸送する方 法。  (83) A congenital genetic disease, which comprises using the cell according to any one of the above (1) to (34), into which a wild-type gene for a mutant gene in a cardiac congenital genetic disease has been introduced. A method for specifically transporting a wild-type gene to a myocardium for a mutant gene in the above.
(84) 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入され た上記 （1)〜 （34)のいずれか 1項に記載の細胞を有効成分として含有する心臓  (84) A heart containing, as an active ingredient, the cell according to any one of the above (1) to (34), into which a wild-type gene for a mutant gene in a congenital heart disease is introduced.
(85) 上記 （1)〜 （34)のいずれか 1項に記載の細胞を免疫原として用いる ことを特徴とする、 該細胞を特異的に認識する抗体を取得する方法。 (85) A method for obtaining an antibody that specifically recognizes the cell according to any one of (1) to (34) above, wherein the cell is used as an immunogen.
(86) 上記（85)記載の方法で取得された抗体を用いることを特徴とする、 ヒ ト骨髄から心筋細胞への分化能を有する成体骨髄由来細胞を単離 ·精製する方法。 (86) A method for isolating and purifying adult bone marrow-derived cells capable of differentiating human bone marrow into cardiomyocytes, comprising using the antibody obtained by the method described in (85).
(87) 上記 （ 1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴と する、 該細胞に特異的な表面抗原を取得する方法。 (87) A method for obtaining a surface antigen specific to a cell according to any one of the above (1) to (34), characterized by using the cell.
(88) 上記 （ 1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴と する、 該細胞を増殖する因子をスクリーニングする方法。  (88) A method for screening for a factor that proliferates the cell, characterized by using the cell according to any one of the above (1) to (34).
(89) 上記 （1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴と する、 該細胞の心筋細胞への分ィヒを誘導する因子をスクリーニングす.る方法。  (89) A method for screening for a factor that induces the differentiation of the cells into cardiomyocytes, comprising using the cells according to any one of the above (1) to (34).
(90) 上記 （ 1)〜 （34)のいずれか 1項に記載の細胞を用いることを特徴と する、 該細胞を不死化する因子をスクリーニングする方法。  (90) A method for screening for a factor that immortalizes a cell, characterized by using the cell according to any one of the above (1) to (34).
(91) 上記 （1)〜 （34)のいずれか 1項に記載の細胞にテロメラ一ゼを発現 させることを特徴とする、 該細胞の不死化方法。 ( 9 2 ) テロメラ一ゼが、 配列番号 3 1記載で表されるアミノ酸配列を有するテロ メラ一ゼである上記 （9 1 ) 記載の方法。 (91) A method for immortalizing a cell according to any one of the above (1) to (34), which comprises expressing telomerase in the cell. (92) The method according to the above (91), wherein the telomerase has the amino acid sequence represented by SEQ ID NO: 31.
( 9 3 ) テロメラ一ゼを発現させることにより、 不死化させた上記（1 )〜（3 4 ) のいずれか 1項に記載の細胞を有効成分として含有する心臓疾患治療薬。  (93) A therapeutic agent for heart disease, comprising as an active ingredient the cell according to any one of the above (1) to (34), which has been immortalized by expressing telomerase.
( 9 4 ) テロメラーゼが、 配列番号 3 1記載で表されるアミノ酸配列を有するテロ メラーゼである上記 （9 3 ) 記載の心臓疾患治療薬。  (94) The therapeutic agent for heart disease according to the above (93), wherein the telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
( 9 5 ) 上記 （ 1 ) 〜 （ 3 4 ) のいずれか 1項に記載の細胞を含んだ培養上清。 ( 9 6 ) 上記 （9 5 )記載の培養上清を用いることを特徴とする、 上記 （1 ) 記載 の細胞を心筋細胞に分化誘導する方法。 本発明の心筋細胞への分化能を有する骨髄細胞とは、成体骨髄から単離された多分 化能幹細胞、 心筋幹細胞および心筋前駆細胞をいう。  (95) A culture supernatant containing the cell according to any one of (1) to (34). (96) A method for inducing differentiation of the cell according to (1) into a cardiomyocyte, comprising using the culture supernatant according to (95). The bone marrow cells capable of differentiating into cardiomyocytes of the present invention refer to multipotent stem cells, cardiomyocyte stem cells and cardiomyocyte precursor cells isolated from adult bone marrow.
多分化能幹細胞とは、心筋細胞および脂肪細胞の少なくとも 2種類の異なる細胞を 分化誘導できる細胞である。心筋幹細胞とは、 増殖性を有し、 心筋細胞だけに分化す る能力を有する細胞である。心筋幹細胞に分ィ匕を誘導すると、 心筋細胞および心筋幹 細胞の 2種類の細胞が形成される。心筋前駆細胞とは、 有限の細胞分裂能を有し、 心 筋細胞だけに分化する能力を有する細胞である。心筋前駆細胞に分化を誘導すると心 筋幹細胞とは異なり心筋細胞のみを形成する。  Pluripotent stem cells are cells that can induce differentiation of at least two different types of cells, cardiomyocytes and adipocytes. Cardiomyocyte stem cells are cells that are proliferative and have the ability to differentiate only into cardiomyocytes. Induction of myocardial stem cells induces two types of cells, myocardial cells and myocardial stem cells. Cardiac progenitor cells are cells that have a finite cell division ability and have the ability to differentiate only into cardiac myocytes. When differentiation is induced in cardiomyocyte precursor cells, only cardiomyocytes are formed, unlike cardiomyocyte stem cells.
以下に、 ヒト、 ラヅト、 マウスなどのほ乳類の成体骨髄から心筋細胞への分化能を 有する骨髄細胞を単離する方法を説明する。  Hereinafter, a method for isolating bone marrow cells capable of differentiating into cardiomyocytes from adult bone marrow of mammals such as human, rat, and mouse will be described.
1 . 心筋細胞への分化能を有する骨髄細胞の単離  1. Isolation of bone marrow cells capable of differentiating into cardiomyocytes
ヒトの骨髄より心筋細胞への分化能を有する骨髄細胞を取得する方法としては、安 全かつ効率的に取得される方法であれば特に限定されないが、 S. E. Haynesworth et al. The method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from human bone marrow is not particularly limited as long as it is a method that can be safely and efficiently obtained.S.E.Haynesworth et al.
Bone, 13, 81 (1992)に記載された方法に基づき行うことができる。 Bone, 13, 81 (1992).
胸骨または腸骨から骨髄穿刺を行う。骨髄穿刺を行う場所の皮膚面を消毒し、局所 麻酔を行う。特に骨膜下を充分に麻酔する。骨髄穿刺針の内筒を抜き、 5000unitsのへ パリンを入れた 10ml注射器を装着して必要量の骨髄液を速やかに吸引する。 平均的 には 10ml〜20mlの骨髄液を吸引する。 骨髄穿刺針を取り外し、 10分間程圧迫止血す る。取得した骨髄液を i，ooo x gの遠心分離により骨髄細胞を回収した後、該骨髄細胞 を PBS (Phosphate Buffered Saline)で洗浄する。 本ステツプを 2回繰り返した後、 該骨 髄細胞を 10%の FBS (牛胎仔血清） を含むひ- MEM (ひ- modified MEM)、 DMEM Perform a bone marrow puncture from the sternum or iliac bone. Disinfect the skin surface at the site of the bone marrow aspiration and perform local anesthesia. In particular, the subperiosteum is sufficiently anesthetized. Remove the inner tube of the bone marrow puncture needle, attach a 10 ml syringe containing 5000 units of heparin, and quickly aspirate the required amount of bone marrow fluid. On average, aspirate 10 to 20 ml of bone marrow fluid. Remove the bone marrow puncture needle and stop bleeding for about 10 minutes You. After collecting the bone marrow cells from the obtained bone marrow fluid by centrifugation at i, ooo xg , the bone marrow cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the bone marrow cells were subjected to 10% FBS (fetal bovine serum) in human MEM (human modified MEM), DMEM
(Dulbecco's modified MEM)あるいは IMDM (Isocove's modified Dulbecco's medium)等の細 胞培養用培地に再浮遊させることにより骨髄細胞液を得ることができる。 By resuspending the cells in a cell culture medium such as (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium), a bone marrow cell solution can be obtained.
該骨髄細胞液から心筋細胞への分化能を有する骨髄細胞を単離する方法としては、 溶液中に混在する他の細胞、 例えば血球系細胞、 造血幹細胞、 血管幹細胞および線維 芽細胞などを除去できれば特に限定されないが、 M. F. Pittenger et al. Science, 284, 143 (1999)に記載された方法に基づき骨髄細胞液を密度 1.073g/mlの percollに重層した後、 1,100 X gで 30分間遠心分離して界面の細胞を回収することにより単離することがで きる。 また、 該骨髄細胞液に 10 X PBSを加えて 9/10に希釈した percollを同容量加え て揮合した後に、 20，000 X gで 30分間遠心分離し、密度 1·075〜1.060の画分を回収す ることにより、該心筋細胞への分化能を有する骨髄細胞を含む骨髄細胞混合物を取得 することができる。 A method for isolating bone marrow cells capable of differentiating into cardiomyocytes from the bone marrow cell solution is to remove other cells mixed in the solution, for example, blood cells, hematopoietic stem cells, vascular stem cells, fibroblasts, and the like. Although not particularly limited, the bone marrow cell fluid was overlaid on percoll having a density of 1.073 g / ml based on the method described in MF Pittenger et al. Science, 284, 143 (1999), and then centrifuged at 1,100 X g for 30 minutes. The cells can be isolated by collecting the cells at the interface. Further, a percoll diluted to 10 X PBS plus 9/10 in bone marrow cell fluid after揮合added the same volume, and centrifuged for 30 minutes at 2 0,000 X g, density 1 · 075 to 1.0 By collecting the 60 fractions, a bone marrow cell mixture containing bone marrow cells capable of differentiating into the cardiomyocytes can be obtained.
上記方法により取得した該心筋細胞への分化能を有する骨髄細胞を含む骨髄細胞 ' 混合物は、 96穴の培養プレートの各穴に 1細胞のみが注入されるように希釈して、 1 細胞由来のクローンを多数調製した後、以下に記載した心筋細胞への分化能を有する 骨髄細胞から心筋細胞を誘導する方法を用いて該クローンを処理し、 自律拍動する細 胞が出現するクローンを選択することにより、該心筋細胞への分化能を有する骨髄細 胞を得ることができる。多分化能幹細胞、 心筋幹細胞および心筋前駆細胞は、後述す る GFP (Gree Fluorescent Protein)などのレポーター遺伝子を用いたシングルセル · マ一キング （single cell marking) を行うことにより選別することができる。  The bone marrow cell 'mixture containing bone marrow cells capable of differentiating into cardiomyocytes obtained by the above method is diluted so that only one cell is injected into each well of a 96-well culture plate, and one cell-derived After preparing a large number of clones, the clones are treated using the method described below for inducing cardiomyocytes from bone marrow cells having the ability to differentiate into cardiomyocytes, and clones in which autonomously pulsating cells appear are selected. Thus, bone marrow cells capable of differentiating into the cardiomyocytes can be obtained. Pluripotent stem cells, myocardial stem cells, and myocardial progenitor cells can be selected by performing single cell marking using a reporter gene such as GFP (Green Fluorescent Protein) described below.
ラットやマウスから心筋細胞への分化能を有する骨髄細胞を取得する方法として は、特に限定されないが以下の手順で取得することができる。 ラットあるいはマウス を頸椎脱曰により致死させ、 70%エタノールで充分消毒した後、 大腿骨の皮膚ならび に大腿四頭筋を切除する。膝関節の部分にハサミをいれて関節をはずし、 大腿骨背面 の筋肉を除去する。股関節の部分にハサミを入れて関節を外し、大腿骨を取り出す。 大腿骨に付着している筋肉をハサミでできるだけ除去した後、大腿骨の両端をハサミ で切断する。 骨の太さに応じた適当なサイズの針を 2.5mlの注射器に装着し、 10%の FBS (牛胎仔血清） を含むひ - MEM、 DMEM、 あるいは IMDM等の細胞培養用培地約 1.5mlを注射器に充填した後、 注射針の先端を大腿骨の膝関節側の断端に差し込む。 注射器内の培養液を骨髄内に注入することで、股関節側の断端から骨髄細胞が押し出 される。得られた骨髄細胞はピペッティングにより培養液中に浮遊させる。該骨髄液 からは、 上記のヒト骨髄液からの骨髄細胞の単離と同様の方法により、 心筋細胞への 分化能を有する骨髄細胞を単離することができる。以上の方法により単離した細胞の 例としては、マウス骨髄由来幹細胞があげられる。マウス骨髄由来幹細胞 BMSCは、 平成 1 2年 2月 2 2日付けで通商産業省工業技術院生命工学工業技術研究所（曰本国 茨城県つくば巿東 1丁目 1番 3号） に FERM BP- 7043として寄託されている。 The method of obtaining bone marrow cells capable of differentiating into cardiomyocytes from rats and mice is not particularly limited, but can be obtained by the following procedure. Rats or mice are sacrificed by cervical prolapse, and after sufficient disinfection with 70% ethanol, the skin of the femur and the quadriceps are removed. Remove the joint with scissors in the knee joint and remove the muscles behind the femur. Remove the joint with scissors in the hip joint and remove the femur. After removing the muscle attached to the femur with scissors as much as possible, scissors both ends of the femur. Disconnect with Attach a needle of an appropriate size according to the thickness of the bone to a 2.5 ml syringe, and add about 1.5 ml of cell culture medium containing 10% FBS (fetal calf serum), such as -MEM, DMEM, or IMDM. After filling the syringe, the tip of the needle is inserted into the stump of the femur on the knee joint side. By injecting the culture solution in the syringe into the bone marrow, the bone marrow cells are pushed out from the hip joint stump. The obtained bone marrow cells are suspended in a culture solution by pipetting. From the bone marrow fluid, bone marrow cells capable of differentiating into cardiomyocytes can be isolated in the same manner as in the above-mentioned method for isolating bone marrow cells from human bone marrow fluid. Examples of cells isolated by the above method include mouse bone marrow-derived stem cells. BMSC, a mouse bone marrow-derived stem cell, was sent to the Ministry of International Trade and Industry by the Ministry of International Trade and Industry's Institute of Biotechnology and Industrial Technology (February 22, 2002) at FERM BP-70 (1-3-3 Tsukuba East, Ibaraki, Japan). Deposited as 43 .
2 . 心筋細胞への分化能を有する骨髄細胞の培養  2. Culture of bone marrow cells capable of differentiating into cardiomyocytes
上記 1の方法により単離した、心筋細胞への分化能を有する骨髄細胞を培養するた めに用いる培地としては、通常公知（組織培養の技術基礎編 第三版、朝倉書店 1996) の組成の細胞培養用培地を用いることができるが、 好ましくは牛等の血清を 5〜20% 添カロした、 - MEM、 DMEMあるいは IMDM等の細胞培養用培地などが用いられる。 培養条件は、細胞が培養可能であればいかなる条件でもよいが、培養温度は 33〜37°C が好ましく、さらに 5〜10%の二酸化炭素ガスで満たした孵卵器で培養することが好ま しい。心筋細胞への分ィ匕能を有する骨髄細胞は、通常の組織培養用のプラスチック製 培養皿に接着して増殖することが好ましい。細胞が培養皿一面に増殖する頃、培地を 除去して、 トリプシン EDTA溶液を加えることで細胞を浮遊させる。 浮遊した細胞 は、 PBSあるいは該細胞培養用の培地で洗浄後、 該細胞培養用の培地で 5倍から 20 倍希釈して新しい培養皿に添加することで、 さらに継代培養することができる。  The medium used for culturing bone marrow cells capable of differentiating into cardiomyocytes isolated by the method 1 described above is usually a known medium (Tissue Culture Technology Basic Edition, Third Edition, Asakura Shoten 1996). A cell culture medium can be used, and preferably, a cell culture medium such as MEM, DMEM, IMDM, or the like supplemented with 5 to 20% of calories from bovine serum or the like is used. The culturing conditions may be any conditions as long as the cells can be cultivated, but the culturing temperature is preferably 33 to 37 ° C, and the culturing is preferably performed in an incubator filled with 5 to 10% carbon dioxide gas. It is preferable that the bone marrow cells having the ability to divide myocardial cells are attached to a normal plastic culture dish for tissue culture and proliferate. When the cells grow on the entire surface of the culture dish, remove the medium and suspend cells by adding trypsin EDTA solution. The suspended cells can be further subcultured by washing with PBS or the cell culture medium, and then diluting 5- to 20-fold with the cell culture medium and adding to a new culture dish.
3 . 心筋細胞への分化能を有する骨髄細胞からの心筋細胞の誘導  3. Induction of cardiomyocytes from bone marrow cells capable of differentiating into cardiomyocytes
心筋細胞への分化能を有する骨髄細胞より心筋細胞を誘導する方法としては、 ( 1 ) DNAの脱メチル化剤処理による分化誘導、 （2 )胎児の心臓発生領域で発現している 因子または胎児の心臓発生段階において心筋細胞への分化に働く因子による分ィ匕誘 導、 （3 )心筋細胞への分化能を有する骨髄細胞または該骨髄細胞から分化した心筋 細胞の培養上清による分化誘導などの方法を挙げることができる。これらの方法を単 独あるいは組み合わせることにより、心筋細胞への分化能を有する骨髄細胞から心筋 細胞を誘導することができる。 Methods for inducing cardiomyocytes from bone marrow cells capable of differentiating into cardiomyocytes include (1) induction of differentiation by treatment with DNA demethylating agent, (2) factors expressed in the fetal heart development region or fetus. (3) Induction of differentiation by bone marrow cells capable of differentiating into cardiomyocytes or culture supernatant of cardiomyocytes differentiated from the bone marrow cells, etc. Can be mentioned. Simply use these methods By themselves or in combination, cardiomyocytes can be derived from bone marrow cells having the ability to differentiate into cardiomyocytes.
DNAの脱メチル化剤としては、 DNAに対して脱メチル化を引き起こす化合物であ ればいかなるものでもよい。 DNAの脱メチル化剤としては、染色体 DNA中の GpC配 列中のシトシン残基のメチル化を特異的に阻害する酵素であるデメチラーゼ、 5-ァザ シチジン （以下 5- aza - Cと略す）、 DMSO (dimethyl sulfoxide) などがあげられる。 デ メチラ一ゼとしては、 配列番号 1記載のアミノ酸配列を有するデメチラーゼ [Nature, 397. 579-583 (1999)]などがあげられる。 DNAの脱メチル化剤処理による分化誘導の具 体例を以下に示す。  The DNA demethylating agent may be any compound that causes demethylation of DNA. DNA demethylating agents include demethylase, an enzyme that specifically inhibits methylation of cytosine residues in GpC sequences in chromosomal DNA, and 5-azacytidine (hereinafter abbreviated as 5-aza-C). And DMSO (dimethyl sulfoxide). Examples of the demethylase include a demethylase having the amino acid sequence of SEQ ID NO: 1 [Nature, 397. 579-583 (1999)]. Specific examples of the induction of differentiation by DNA demethylation treatment are shown below.
3mmol/lから 10mmol/lの間の濃度になるように 5- aza- Cを心筋細胞への分化能を有 する骨髄細胞を含む培地中に添加し、 24時間上記培養条件下でィンキュベーシヨンす る。培地を交換することで 5- aza - Cを除去し、 さらに 2〜 3週間培養することで心筋 細胞を取得することができる。形成される心筋細胞は培養 2〜 3週間目では洞結節細 胞が中心であるが、 培養 4週間目以降心室型心筋細胞を分化誘導することができる。 胎児の心臓発生領域で発現している因子または胎児の心臓発生段階において心筋 細胞への分化に働く因子としては、 サイトカイン、 ビタミン、 接着分子、 転写因子な どをあげることができる。  5-aza-C is added to a medium containing bone marrow cells capable of differentiating into cardiomyocytes to a concentration between 3 mmol / l and 10 mmol / l, and incubated at the above culture conditions for 24 hours. Bastion. The medium is replaced to remove 5-aza-C, and cultured for another 2-3 weeks to obtain cardiomyocytes. The formed cardiomyocytes are mainly sinus node cells at 2-3 weeks of culture, but can induce differentiation of ventricular cardiomyocytes after 4 weeks of culture. Factors expressed in the fetal heart development region or factors that act on cardiomyocyte differentiation during the fetal heart development stage include cytokines, vitamins, adhesion molecules, transcription factors, and the like.
サイトカインとしては、 心筋細胞への分ィ匕能を有する骨髄細胞に、 心臓の発生段階 で心筋細胞への分化を促進するものであればいかなるサイトカインでもよい。  As the cytokine, any cytokine may be used as long as it promotes differentiation into bone marrow cells having the ability to divide myocardial cells into cardiomyocytes at the stage of cardiac development.
具体的には、 10〜40ng/mlの血小板由来増殖因子 （以下、 PDGFと略記する。 ） 、 などをあげることができる。 PDGFとしては、 配列番号 3または 5のアミノ酸配列で 表されるものが好ましく用いられる。  Specifically, 10 to 40 ng / ml of platelet-derived growth factor (hereinafter abbreviated as PDGF), and the like can be mentioned. As PDGF, those represented by the amino acid sequence of SEQ ID NO: 3 or 5 are preferably used.
また、心筋細胞への分化を抑制するサイトカインに対する阻害剤を用いることによ り、 心筋細胞への分ィヒ能を有する骨髄細胞に、 心臓の発生段階で心筋細胞への分ィ匕を 促進することも可能である。  In addition, by using an inhibitor for a cytokine that suppresses differentiation into cardiomyocytes, bone marrow cells capable of dividing into cardiomyocytes are promoted to be divided into cardiomyocytes at the stage of heart development. It is also possible.
心筋細胞への分化を抑制するサイトカインとしては、 線維芽細胞増殖因子— 2 (以 下、 FGF-2と略記する。 ） 、 具体的には、 配列番号 7または 8で表される FGF- 2な どをあげることができる。 心筋細胞への分化を抑 ¾するサイト ンに対する阻害剤としては、サイトカインの 情報伝達を阻害する物質、 例えばサイトカインを中和する抗体、低分子化合物などを あげることができる。 Examples of cytokines that suppress differentiation into cardiomyocytes include fibroblast growth factor-2 (hereinafter abbreviated as FGF-2), specifically, FGF-2 represented by SEQ ID NO: 7 or 8. Can be given. Examples of inhibitors for cytokines that suppress differentiation into cardiomyocytes include substances that inhibit cytokine signaling, such as antibodies that neutralize cytokines and low molecular weight compounds.
ビタミンとしては、 レチノイン酸など心筋細胞への分化能を有する骨髄細胞に、 心 臓の発生段階で心筋細胞への分化を促進するものであればいかなるビタミンでもよ い。 具体的には、 10— 9Mのレチノイン酸などをあげることができる。 As the vitamin, any vitamin can be used as long as it promotes differentiation into bone marrow cells capable of differentiating into cardiomyocytes, such as retinoic acid, and cardiomyocytes during the developmental stage of the heart. Specifically, and the like retinoic acid 10- 9 M.
接着分子としては、 フイブロネクチンなど心臓の発生段階で心臓発生領域で発現し ていればいかなる接着分子でもよい。具体的には、 フイブロネクチンをコートした培 養皿で該心筋細胞への分化能を有する骨髄細胞を培養することにより心筋細胞への 分ィ匕を促進することができる。  As the adhesion molecule, any adhesion molecule such as fibronectin may be used as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, by culturing bone marrow cells capable of differentiating into the cardiomyocytes in a culture dish coated with fibronectin, filtration into the cardiomyocytes can be promoted.
転写因子としては、 ホメォボックス型転写因子 Nkx2.5/Csx (配列番号 9：アミノ酸 配列、 配列番号 10：塩基配列） 、 GATAフアミリーに属する Zinc finger型転写因子 GATA4 (配列番号 11：アミノ酸配列、 配列番号 12：塩基配列） 、 myocyte enhancer factor - 2(MEF- 2)ファミリ一に属する転写因子 MEF- 2A (配列番号 13：ァミノ酸配列、 配列番号 14：塩基配列） 、 MEF- 2B (配列番号 15：アミノ酸配列、 配列番号 16：塩 基配列）、 MEF-2C (配列番号 17：アミノ酸配列、配列番号 18：塩基配列） と MEF - 2D (配列番号 19：アミノ酸配列、 配列番号 20：塩基配列）、 basic helix loop helix型転写 因子に属する dHAND (配列番号 21：ァミノ酸配列、配列番号 22：塩基配列）と eHAND (配列番号 23：ァミノ酸配列、 配列番号 24：塩基配列）、 TEA- DNA結合型転写因子 ファミリ一に属する TEF- 1 (配列番号 25：アミノ酸配列、 配列番号 26：塩基配列）、 TEF-3 (配列番号 27：アミノ酸配列、 配列番号 28：塩基配列） と TEF - 5 (配列番号 29 :アミノ酸配列、 配列番号 30 :塩基配列） などをあげることができる。 Examples of the transcription factor include homebox-type transcription factor Nkx2.5 / Csx (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: base sequence) and Zinc finger-type transcription factor GATA4 belonging to GATA family (SEQ ID NO: 11: amino acid sequence, SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: Amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence), Basic helix loop dHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: base sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID NO: 24: base sequence) belonging to helix-type transcription factor, TEA-DNA binding type TEF- belonging to transcription factor family 1 1 (SEQ ID NO: 25 : amino acid sequence, SEQ ID NO: 26 : base sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28: base sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence, SEQ ID NO: 30: base sequence).
上述した転写因子は、 該因子をコードする DNAを心筋細胞への分化能を有する骨 髄細胞中に導入し、 DNAを発現させることにより心筋細胞への分化を誘導させること ができる。 また、 該因子を心筋細胞への分化能を有する骨髄細胞培養液中に添加する ことによつても、 心筋細胞へ分化誘導させることができる。  The above-mentioned transcription factor can induce differentiation into cardiomyocytes by introducing DNA encoding the factor into bone marrow cells capable of differentiating into cardiomyocytes and expressing the DNA. The differentiation into cardiomyocytes can also be induced by adding the factor to a culture solution of bone marrow cells capable of differentiating into cardiomyocytes.
自律拍動する心筋細胞の培養上清を心筋細胞への分化能を有する骨髄細胞培養液 中に添加することで心筋細胞への分化を誘導させることができる。 また、 以下に示す方法で得られる心筋細胞への分化を誘導する因子（以下、 心筋分 化誘導因子と称する） を用いても、 心筋細胞への分化能を有する骨髄由来細胞を心筋 細胞に分化誘導することができる。 The differentiation into cardiomyocytes can be induced by adding the culture supernatant of autonomously beating cardiomyocytes to a bone marrow cell culture medium having the ability to differentiate into cardiomyocytes. In addition, a bone marrow-derived cell capable of differentiating into cardiomyocytes can be differentiated into cardiomyocytes by using a cardiomyocyte differentiation-inducing factor (hereinafter referred to as a cardiomyocyte differentiation-inducing factor) obtained by the following method. Can be guided.
4 . 心筋分ィ匕誘導因子の取得  4. Acquisition of myocardial diversion inducer
心筋分化誘導因子の取得方法としては、 自律拍動する細胞の培養上清に各種プロテ ァーゼ阻害剤を添加して、 透析、塩析ならびにクロマトグラフィ一などを組み合わせ ることにより取得することができる。  As a method for obtaining a myocardial differentiation-inducing factor, it can be obtained by adding various protease inhibitors to the culture supernatant of autonomously pulsating cells, and combining dialysis, salting out, chromatography and the like.
さらにマイクロシ一ケンサーを用いて、上記の心筋分ィ匕誘導因子の部分ァミノ酸配 列を決定し、 該アミノ酸配列に基づき設計した DNAプロ一ブを用いて該自律拍動す る細胞より作製した cDNAライブラリ一をスクリーニングすることにより、心筋分化 誘導因子の遺伝子を取得することができる。  Further, using a microsequencer, the partial amino acid sequence of the above-described myocardial motility-inducing factor is determined, and a DNA probe designed based on the amino acid sequence is used to prepare the autonomously beating cells. By screening the obtained cDNA library, a gene for a myocardial differentiation-inducing factor can be obtained.
5 . 心筋細胞への分化能を有する骨髄細胞を含む心臓再生治療薬または心臓疾患 本発明の心筋細胞への分化能を有する骨髄細胞は、心臓再生または心臓疾患の治療 薬として用いることができる。  5. Cardiac regeneration or heart disease containing bone marrow cells capable of differentiating into cardiomyocytes The bone marrow cells capable of differentiating into cardiomyocytes of the present invention can be used as therapeutic agents for heart regeneration or heart diseases.
心臓疾患としては、 心筋梗塞、 虚血性心疾患、 うつ血性心不全、 不整脈、 肥大型心 筋症、 拡張型心筋症、 心筋炎、 弁膜症などをあげることができる。  Examples of the heart disease include myocardial infarction, ischemic heart disease, congestive heart failure, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, valvular disease and the like.
心臓再生の治療薬としては、心筋細胞への分化能を有する骨髄細胞を高純度で含み、 心臓の障害部位ならび大きさに応じて、該心筋細胞への分化能を有する骨髄細胞を増 殖させたもの、 好ましくは、 心筋細胞への分化能を有する骨髄細胞から、 心筋内皮細 胞 (Endocardial endothelial cell)、 クッション細胞 (Cushion cell)、 心室型心筋細胞、 心房 型心飭細胞、洞結節細胞等の心臓を形成する様々な細胞へ分化誘導できる細胞が用い られる。  As a therapeutic agent for heart regeneration, bone marrow cells having the ability to differentiate into cardiomyocytes are contained in high purity, and the bone marrow cells having the ability to differentiate into cardiomyocytes are expanded according to the site and size of the damaged heart. Cells, preferably from bone marrow cells capable of differentiating into cardiomyocytes, myocardial endothelial cells (Endocardial endothelial cells), cushion cells (Cushion cells), ventricular-type cardiomyocytes, atrial-type cardiac cells, sinus node cells, etc. Cells capable of inducing differentiation into various cells forming the heart are used.
該治療薬は、 心筋梗塞の患者骨髄液中から上述した密度勾配遠心分離法、後述する 心筋細胞への分化能を有する細胞を特異的に認識する抗体を用いたパニング法 [J. Immunol., 141(8), 2797-2800 (1988)]あるいは FACS法 [Int. Immunol., 10(3), 275-283 (1998)]、 または心筋細胞への分化能を有する骨髄細胞に特異的な遺伝子のプロモー夕 一を用いたレポ一夕一系を構築する方法により該心筋細胞への分化能を有する骨髄 細胞の精製を行うことにより、 製造することができる。 The therapeutic agent is a density gradient centrifugation method described above from the bone marrow fluid of a patient with myocardial infarction, a panning method using an antibody that specifically recognizes cells capable of differentiating into cardiomyocytes described below (J. Immunol. 141 (8), 2797-2800 (1988)] or FACS method [Int. Immunol., 10 (3), 275-283 (1998)], or a gene specific to myeloid cells capable of differentiating into cardiomyocytes A bone marrow capable of differentiating into said cardiomyocytes by a method for constructing a repo-one-line system using It can be produced by purifying cells.
また該治療薬には、 後述する心筋形成剤を用いて、 該心筋細胞への分化能を有する 骨髄細胞を心筋細胞へ分化誘導させた細胞、高齢者の骨髄から取得した骨髄細胞よ、り、 後述する不死化方法を利用して細胞分裂能を賦活させた心筋細胞への分化能を有す る細胞も含まれる。  In addition, the therapeutic agent may be a cell obtained by inducing the differentiation of bone marrow cells capable of differentiating into cardiomyocytes into cardiomyocytes using a cardiomyogen described below, a bone marrow cell obtained from bone marrow of an elderly person, Also included are cells capable of differentiating into cardiomyocytes that have been activated to mitosis using the immortalization method described below.
上記方法で製造した治療薬は、上記心筋細胞への分化能を有する細胞を特異的に認 識する抗体と FACS法を組み合わせることで純度を検定することができる。  The purity of the therapeutic agent produced by the above method can be assayed by combining the antibody specifically recognizing the cells capable of differentiating into cardiomyocytes with the FACS method.
上記の治療薬を障害部位に輸送する方法としては、カテーテルを利用する方法等が 用いられる。以下虚血性心疾患を例に具体的な方法を示す。虚血性心疾患で障害を受 けた心筋細胞は、血管狭窄部位の下流に存在することから、上記の細胞を注入する前 に、 冠動脈造影法 （図説病態内科講座 循環器一 1、 MEDICAL VIEW, 1993) により 血管の狭窄部位を同定しておく必要がある。器質的狭窄病変は狭窄病態に応じて求心 性狭窄、 偏心性狭窄、 多発性壁不整に分類され、 特に偏心性狭窄はタイプ Iおよび夕 イブ IIの 2つのタイプに細分類される。狭窄形態は狭心症の経過、 予後に関連するこ とが知られており、タイプ IIの偏心性狭窄や多発性壁不整は不安定狭心症例に多く、 心筋梗塞に移行する可能性が高い。血管が完全に狭窄している場合には、注入する細 胞が障害部位に到達しない可能性があるので、事前に経皮的冠動脈形成術 (PTCA)ある いは血栓溶解療法などにより狭窄部位を再開することが必要である。障害を受けた心 筋細胞の部位に応じて、注入する細胞を心室型や心房型のように区別することができ る。 カテーテルの挿入法は右上腕動脈より挿入する Sones法 （図説病態内科講座 循 環器一 1、 MEDICAL VIEW, 1 9 9 3 )あるいは大腿動脈より揷入する Jundkins法（図 説病態内科講座 循環器一 1、 MEDICAL VIEW, 1993) を利用することができる。  As a method for transporting the above therapeutic agent to the site of injury, a method using a catheter or the like is used. Hereinafter, a specific method will be described using ischemic heart disease as an example. Cardiomyocytes damaged by ischemic heart disease are located downstream of the site of vascular stenosis. Therefore, coronary angiography (illustrated pathology internal medicine course, cardiology-1, MEDICAL VIEW, 1993) ), It is necessary to identify the stenosis site of the blood vessel. Organic stenotic lesions are classified into afferent stenosis, eccentric stenosis, and multiple wall irregularities according to the stenosis pathology. In particular, eccentric stenosis is subdivided into two types, Type I and Evening II. It is known that the form of stenosis is related to the course and prognosis of angina.Type II eccentric stenosis and multiple wall irregularities are common in unstable angina patients, and there is a high possibility of transition to myocardial infarction . If the blood vessel is completely stenotic, the injected cells may not reach the lesion site.Therefore, the stenosis site must be removed by percutaneous coronary angioplasty (PTCA) or thrombolysis beforehand. It is necessary to restart. Depending on the location of the damaged myocardial cells, the cells to be injected can be differentiated into ventricular and atrial types. The catheter insertion method is the Sones method (Medical View, 1993), which is inserted from the right upper arm artery, or the Jundkins method (Medical View, 1993), which is inserted through the femoral artery. 1, MEDICAL VIEW, 1993) can be used.
6 . 心筋形成剤  6. Cardiomyogen
本発明の心筋形成剤は、 染色体 DNAの脱メチル化剤、 胎児の心臓発生領域で発現 している因子、あるいは胎児の心臓発生段階で心筋細胞への分化に働く因子のいずれ かの心筋分化誘導因子の少なくとも一種類を有効成分として含有し、骨髄由来の細胞 を心筋細胞へ分化誘導させることができる。  The cardiomyogenic agent of the present invention may be a demethylating agent for chromosomal DNA, a factor expressed in the fetal heart development region, or a factor that acts on cardiomyocyte differentiation during the fetal heart development stage. Bone marrow-derived cells can be induced to differentiate into cardiomyocytes by containing at least one factor as an active ingredient.
心筋分化誘導因子としては、 サイトカイン、 ビタミン、 接着分子、 転写因子などを あげることができる。 Myocardial differentiation inducing factors include cytokines, vitamins, adhesion molecules, transcription factors, etc. I can give it.
サイトカインとしては、 心筋細胞への分化能を有する骨髄細胞に、 心臓発生段階で 心筋細胞への分化を促進するものであればいかなるサイトカインでもよい。  As the cytokine, any cytokine can be used as long as it promotes the differentiation of bone marrow cells capable of differentiating into cardiomyocytes and the differentiation into cardiomyocytes at the stage of cardiac development.
具体的には、 10〜40ng/mlの PDGFなどをあげることができる。 PDGFとしては、 配列番号 3または 5のァミノ酸配列で表されるものが好ましく用いられる。  Specific examples include 10 to 40 ng / ml of PDGF. As PDGF, those represented by the amino acid sequence of SEQ ID NO: 3 or 5 are preferably used.
ビタミンとしては、 レチノィン酸など心筋細胞への分ィ匕能を有する骨髄細胞に、 心 臓発生段階で心筋細胞への分化を促進するものであればいかなるビ夕ミンでもよい。 具体的には、 10—9 Mのレチノイン酸などをあげることができる。 Any vitamin may be used as the vitamin, as long as it promotes the differentiation of bone marrow cells such as retinoic acid into cardiomyocytes having the ability to divide the myocardial cells into cardiomyocytes at the stage of cardiac development. Specifically, and the like retinoic acid 10- 9 M.
接着分子としては、 フイブロネクチンなど心臓発生段階で心臓発生領域で発現して いればいかなる接着分子でもよい。具体的には、 フイブロネクチンをコートした培養 皿で該心筋細胞への分化能を有する骨髄細胞を培養することにより心筋細胞への分 化を促進することができる。  The adhesion molecule may be any adhesion molecule, such as fibronectin, as long as it is expressed in the heart development region at the stage of cardiac development. Specifically, differentiation into cardiomyocytes can be promoted by culturing bone marrow cells capable of differentiating into cardiomyocytes in a culture dish coated with fibronectin.
転写因子としては、 ホメォボックス型転写因子 Nkx2.5/CSX (配列番号 9 :アミノ酸 配列、 配列番号 10：塩基配列） 、 GATAファミ リ一に属する Zinc finger型転写因子 GATA4 (配列番号 11：アミノ酸配列、 配列番号 12：塩基配列） 、 myocyte enhancer factor - 2 (MEF - 2)ファミリ一に属する転写因子 MEF- 2A (配列番号 13：アミノ酸配列、 配列番号 14：塩基配列） 、 MEF- 2B (配列番号 15：アミノ酸配列、 配列番号 16：塩 基配列）、 MEF-2C (配列番号 17：アミノ酸配列、配列番号 18：塩基配列） と MEF- 2D (配列番号 19：アミノ酸配列、 配列番号 20：塩基配列）、 basic helix loop helix型転写 因子に属する. dHAND (配列番号 21：ァミノ酸配列、配列番号 22：塩基配列）と eHAND (配列番号 23：ァミノ酸配列、 配列番号 24：塩基配列）、 TEA-DNA結合型転写因子 ファミリ一に属する TEF - 1 (配列番号 25：アミノ酸配列、 配列番号 26：塩基配列）、 TEF-3 (配列番号 27：アミノ酸配列、 配列番号 28 :塩基配列） と TEF-5 (配列番号 29 :アミノ酸配列、 配列番号 30 :塩基配列） などをあげることができる。 The transcription factor, Homeobokkusu transcription factor Nkx2.5 / C SX (SEQ ID NO: 9: amino acid sequence, SEQ ID NO: 10: nucleotide sequence), GATA family foremost belongs Zinc finger transcription factor GATA4 (SEQ ID NO: 11: amino acid sequence , SEQ ID NO: 12: base sequence), transcription factor MEF-2A belonging to myocyte enhancer factor-2 (MEF-2) family 1 (SEQ ID NO: 13: amino acid sequence, SEQ ID NO: 14: base sequence), MEF-2B (SEQ ID NO: 15: amino acid sequence, SEQ ID NO: 16: base sequence), MEF-2C (SEQ ID NO: 17: amino acid sequence, SEQ ID NO: 18: base sequence) and MEF-2D (SEQ ID NO: 19: amino acid sequence, SEQ ID NO: 20: base sequence) ), Belonging to the basic helix loop helix-type transcription factor. DHAND (SEQ ID NO: 21: amino acid sequence, SEQ ID NO: 22: nucleotide sequence) and eHAND (SEQ ID NO: 23: amino acid sequence, SEQ ID NO: 24: nucleotide sequence), TEA- TE belonging to DNA-binding transcription factor family 1 F-1 (SEQ ID NO: 25: amino acid sequence, SEQ ID NO: 26: nucleotide sequence), TEF-3 (SEQ ID NO: 27: amino acid sequence, SEQ ID NO: 28: nucleotide sequence) and TEF-5 (SEQ ID NO: 29: amino acid sequence, sequence) No. 30: base sequence).
該心筋形成剤には心筋分化誘導因子の遺伝子を主成分とするものと、心筋分化誘導 因子の本体である蛋白質を含むものがある。  The myocardial agents include those containing a gene for a myocardial differentiation-inducing factor as a main component and those containing a protein that is the main body of a myocardial differentiation-inducing factor.
( 1 ) 遺伝子を主成分とする心筋形成剤  (1) Gene-based cardiomyogenic agent
以下に本発明の心筋形成剤が心筋分化誘導因子をコードする遺伝子を主成分とする 場合の調製法について述べる。 The cardiomyogenic agent of the present invention contains a gene encoding a myocardial differentiation inducing factor as a main component below. The preparation method in this case will be described.
まず、 心筋分化誘導因子の遺伝子 DNA断片、 あるいは全長 c DNAをウィルスぺク 夕一プラスミ ド内のプロモ一夕一の下流に揷入することにより、組換えウィルスぺク 夕一プラスミ ドを造成する。  First, a recombinant virus vector Yuichi Plasmid is constructed by inserting a gene DNA fragment or a full-length cDNA of a cardiomyocyte differentiation-inducing factor into the virus vector downstream of Promote One. .
該組換えウィルスベクタ一プラスミ ドを、該ウィルスベクタ一プラスミ ドに適合し たパッケージング細胞に導入する。  The recombinant virus vector-plasmid is introduced into a packaging cell suitable for the virus vector-plasmid.
パッケージング細胞としては、 ゥィルスのパヅケージングに必要なタンパク質をコ 一ドする遺伝子の少なくとも 1つを欠損している組換えウィルスぺク夕一プラスミ ドの該欠損する蛋白質を補給できる細胞であればいかなるものも用いることができ る。 例えばヒト腎臓由来の HEK293細胞、 マウス線維芽細胞 NIH3T3などを用いるこ とができる。  As the packaging cell, any cell can be used as long as it can supply the protein deficient in recombinant virus vector plasmid which lacks at least one gene encoding a protein necessary for viral packaging. Those can also be used. For example, HEK293 cells derived from human kidney, mouse fibroblast NIH3T3, and the like can be used.
パッケージング細胞で補給する蛋白質としては、 レトロウィルスベクタ一の場合は マウスレトロウイルス由来の gag、 pol、 envなどの蛋白質、 レンチウィルスベクター の場合は HIVウィルス由来の gag、 poレ env、 vpr、 vpu、 vif、 tat、 rev、 nefなどの蛋白 質、 アデノウイルスベクタ一の場合はアデノウイルス由来の E1A、 E1Bなどの蛋白質、 アデノ随伴ウィルスの場合は Rep(p5，p 19，p40)、 Vp(Cap)などの蛋白質を用いることが できる。  Proteins to be supplied by the packaging cells include gag, pol, and env derived from mouse retrovirus in the case of a retrovirus vector, and gag, po env, vpr, and vpu derived from an HIV virus in the case of a lentivirus vector. , Vif, tat, rev, nef, etc., proteins such as E1A and E1B derived from adenovirus in the case of adenovirus vector, Rep (p5, p19, p40), Vp (Cap ) Can be used.
ウィルスベクタープラスミ ドとしては上記パヅケージング細胞において組換えゥ ィルスが生産でき、心臓先天性遺伝子疾患の原因遺伝子に対する野生型の遺伝子を心 筋細胞で転写できる位置にプロモー夕一を含有しているものが用いられる。  A viral vector plasmid that contains a promoter at a position where recombinant viruses can be produced in the above-mentioned packaging cells and a wild-type gene corresponding to a gene causing cardiac congenital genetic disease can be transcribed in cardiomyocytes is used. Used.
ウィルスベクタ一プラスミ ドとしては MFG [Proc. Natl. Acad. Sci. USA, 92, 6733- 6737 (1995)]、 pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)]， LL_CG、 CL- CGヽ CS - CG、 CLG [Journal of Virology, 72, 8150-8157 (1998)]、 pAdexl [Nucleic Acids Res., 23, 3816-3821 (1995)]等が用いられる。  Viral vector plasmids include MFG [Proc. Natl. Acad. Sci. USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)], LL_CG, CL-CGヽ CS-CG, CLG [Journal of Virology, 72, 8150-8157 (1998)], pAdexl [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like are used.
プロモ一夕一としては、 ヒト組織中で発現できるものであればいずれも用いること ができ、 例えば、 サイ トメガロウィルス （ヒト CMV) の IEGmmediate early)遺伝子の プロモー夕一、 SV40の初期プロモー夕一、 レトロウイルスのプロモ一夕一、 メタ口 チォネインプロモー夕一、 ヒートショック蛋白質プロモーター、 SR プロモー夕一 等をあげることができる。 また、 ヒト CMVの IE遺伝子のェンハンサ一をプロモ一夕 —と共に用いてもよい。また、 Nkx2.5/Csx遺伝子のような心筋細胞特異的な遺伝子の プロモー夕一を用いる.ことで、心筋細胞で特異的に目的の遺伝子を発瑪させることが できる。 Any promoter can be used as long as it can be expressed in human tissues. For example, the promoter of the cytomegalovirus (human CMV) IEGmmediate early) gene and the early promoter of SV40 can be used. , Retrovirus Promo One, Meta Mouth Choonein Promo One, Heat Shock Protein Promoter, SR Promo One Etc. can be given. The enhancer of the IE gene of human CMV may be used together with the promoter. In addition, by using the promoter of a cardiomyocyte-specific gene such as the Nkx2.5 / Csx gene, the target gene can be specifically generated in the cardiomyocyte.
上記組換えウィルスベクタープラスミドを上記パッケ一ジング細胞に導入するこ とで組換えウィルスべク夕一を生産することができる。上記パッケージング細胞への 上記ウィルスベクタープラスミドの導入法としては、例えば、リン酸カルシウム法 [特 開平 2-227075] 、 リポフエクション法 [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] 等をあげることができる。  By introducing the above-mentioned recombinant virus vector plasmid into the above-mentioned packaging cells, a recombinant virus vector can be produced. Examples of a method for introducing the viral vector plasmid into the packaging cells include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. Etc. can be given.
上述した組換えウィルスベクタ一は、遺伝子治療剤に用いる基剤と共に調合して心 筋形成剤を製造することができる [Nature Genet., 8, 42 (1"4)]。遺伝子治療剤に用いる 基剤としては、通常注射剤に用いる基剤であればいかなるものでも用いることができ る。例えば、 蒸留水、 塩ィ匕ナトリゥム又は塩化ナトリゥムと無機塩との混合物等の塩 溶液、 マンニトール、 ラクトース、 デキストラン、 グルコース等の溶液、 グリシン、. アルギニン等のアミノ酸溶液、有機酸溶液又は塩溶液とグルコース溶液との混合溶液 等があげられる。 また常法に従い、 これらの基剤に浸透圧調整剤、 PH調整剤、 ゴマ 油、 ダイズ油等の植物油又はレシチンもしくは非ィォン界面活性剤等の界面活性剤等 の助剤を用いて、 溶液、 懸濁液、 分散液として注射剤を調製してもよい。 これらの注 射剤を、 粉末化、 凍結乾燥等の操作により用時溶解用製剤として調製することもでき る。上記の心筋形成剤は、 液体の場合はそのままで、 固体の場合は治療の直前に必要 により滅菌処理をした上記の基剤に溶解して遺伝子治療に使用することができる。本 発明の心筋形成剤の投与方法は、 患者の治療部位の心筋に吸収されるように、 カテー テル等を用いて局所的に投与する方法等が用いられる。  The above-described recombinant virus vector can be mixed with a base used for a gene therapy agent to produce a cardiomyogenic agent [Nature Genet., 8, 42 (1 "4)". Any base can be used as long as it is commonly used for injections, for example, distilled water, salt solutions such as sodium chloride or sodium chloride and a mixture of inorganic salts, mannitol, and lactose. Dextran, glucose, etc., glycine, amino acid solution such as arginine, organic acid solution or mixed solution of salt solution and glucose solution, etc. In addition, osmotic pressure adjusting agent, Using injections as solutions, suspensions, and dispersions, using PH adjusters, vegetable oils such as sesame oil and soybean oil, or auxiliaries such as surfactants such as lecithin or non-ionic surfactants. These injections can also be prepared as preparations for dissolution at the time of use by operations such as pulverization, freeze-drying, etc. The above-mentioned myocardium-forming agent can be used in the form of a solid as it is in the case of a liquid. In the case of (1), the myocardium-forming agent of the present invention can be dissolved in the above-mentioned sterilized base immediately before treatment and used for gene therapy if necessary. As described above, a method of local administration using a catheter or the like is used.
上述した組換えウィルスベクタ一は試験管内で該心筋細胞への分ィヒ能を有する骨 髄細胞に感染させた後、 上述した心筋形成剤として調製し、 患者に投与することがで きる。 または、 組換えウィルスベクタ一を患者の患部に直接投与することもできる。 ( 2 ) 蛋白質を主成分とする心筋形成剤  The above-described recombinant virus vector can be prepared as the above-mentioned cardiomyogen and then administered to a patient after infecting bone marrow cells having the ability to isolate the cardiomyocytes in a test tube. Alternatively, the recombinant viral vector can be administered directly to the affected area of the patient. (2) Protein-based cardiomyogenic agent
以下に本発明の心筋形成剤が心筋分化誘導因子蛋白質を主成分とする場合の調製 法について述べる。 Preparation when the cardiomyogenic agent of the present invention contains a cardiac differentiation-inducing factor protein as a main component The law is described.
心筋分ィ匕誘導因子蛋白質の完全長 cDNAをもとに、 必要に応じて、 該蛋白質をコ一 ドする部分を含む適当な長さの DNA断片を調製する。  Based on the full-length cDNA of the cardiac inducing factor-inducing protein, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
該 DNA断片、 あるいは完全長 cDNAを発現べクタ一内のプロモーターの下流に揷 入することにより、 該蛋白質の組換発現べクタ一を造成する。  The recombinant expression vector of the protein is constructed by inserting the DNA fragment or the full-length cDNA downstream of the promoter in the expression vector.
該組換発現ベクターを、 該発現ベクターに適合した宿主細胞内に導入する。  The recombinant expression vector is introduced into a host cell compatible with the expression vector.
宿主細胞としては、 目的とする DNAを発現できるものは全て用いることができ、 例えば、 ェシエリヒァ (Escherichia)属、 セラチア (Serratia) 属、 コリネバクテリゥム (Corynebacterium) 属、 ブレビバクテリウム (Brevibacterium)属、 シュ一ドモナス (Pseudomonas) 属ヽ バチルス (Bacillus) 属、 ミクロノ 'クテリゥム ( icrobacterium) 属等に属する細菌、 クルイべ口ミセス (Kluyveromyces) 属、 サヅカロマイセス f Saccharomyces) 属、 シソサッカロマイセス ( Shizosaccharomyces 属、 卜リコスポ ロン (Trichosporon)属、 シヮニォミセス (Schwanniomyces)属等に属する酵母や動物 細胞、 昆虫細胞等を用いることができる。  As the host cell, any capable of expressing the target DNA can be used, and for example, genus Escherichia, genus Serratia, genus Corynebacterium, genus Brevibacterium , Bacteria belonging to the genus Pseudomonas ill genus Bacillus, genus Microno 'icrobacterium, genus Kluyveromyces, genus Saccharomyces f Saccharomyces, genus Shisosaccharomyces omnibus Yeast, animal cells, insect cells and the like belonging to the genus Ron (Trichosporon), the genus Schwanniomyces and the like can be used.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中への 組込みが可能で、 心筋分化誘導因子の遺伝子 DNAを転写できる位置にプロモー夕一 を含有しているものが用いられる。  As the expression vector, those which can be replicated autonomously in the above-mentioned host cells or can be integrated into the chromosome, and which contain a promoter at a position where the gene DNA of the cardiomyocyte differentiation-inducing factor can be transcribed are used.
細菌を宿主細胞として用いる場合は、心筋分化誘導因子の組換え発現べク夕一は該 細菌中で自立複製可能であると同時に、 プロモー夕一、 リボソーム結合配列、 心筋細 胞への分化を誘導できる蛋白質をコードする DNAおよび転写終結配列より構成され た組換え発現ベクターであることが好ましい。プロモーターを制御する遺伝子が含ま れていてもよい。  When a bacterium is used as a host cell, the recombinant expression vector of the cardiomyocyte differentiation-inducing factor is capable of autonomous replication in the bacterium, and at the same time, induces differentiation into a promoter, a ribosome-binding sequence, and a myocardial cell. It is preferably a recombinant expression vector composed of a DNA encoding a possible protein and a transcription termination sequence. A gene that controls a promoter may be included.
発現べクタ一としては、 例えば、 pBTrp2、 pBTacl, PBTac2 (いずれもベーリンガ —マンハイム社より巿販） 、 pKK233 - 2 (Amersham Pharmacia Biotech社製） 、 pSE280 (Invitrogen社製）、 pGEMEX-1 (Promega社製）、 pQE- 8 (QIAGEN社製）、 pKYPIO [特開昭 58-110600] 、 p YP200 [Agricultural Biological Chemistry, 48, 669 (1984)] 、 pLSAl [Agric. Biol. Chem., 53, 277 (1989)] 、 pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)] 、 pBluescript II S (-) (Stratagene社製) 、 pGEX (Amersham Pharmacia Biotech 社製） 、 pET-3 (Novagen社製）、 pTerm2(USP4686191、 USP4939094、 USP5160735)、 pSupexヽ pUB110、 pTP5、 pC194、 pEG400 [J. BacterioL, 172， 2392 (1990)]等を例示す ることができる。 Expression base Kuta one, for example, pBTrp2, pBTacl, P BTac2 (both Beringa -巿販from Mannheim), pKK233 - 2 (Amersham Pharmacia Biotech Co.), (manufactured by Invitrogen Corp.) pSE280, pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO [Japanese Patent Laid-Open No. 58-110600], pYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277] (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II S (-) (Stratagene), pGEX (Amersham Pharmacia Biotech) PET-3 (Novagen), pTerm2 (USP4686191, USP4939094, USP5160735), pSupexuppUB110, pTP5, pC194, pEG400 [J. BacterioL, 172, 2392 (1990)] it can.
発現べクタ一としては、 リボソーム結合配列であるシャイン一ダルガノ （Shine- The expression vector includes the ribosome binding sequence Shine-Dalgarno.
Dalgarno) 配列と開始コドンとの間を適当な距離 （例えば 6〜1 8塩基） に調節した ものを用いることが好ましい。 (Dalgarno) sequence and the initiation codon are preferably adjusted to an appropriate distance (for example, 6 to 18 bases).
プロモ—夕—としては、宿主細胞中で発現できるものであればいかなるものでもよ い。 例えば、 trpプロモ一夕一 （P trp) 、 lacプロモーター （P lac) 、 PLプロモー夕The promoter may be any promoter that can be expressed in the host cell. For example, trp promoter Isseki one (P trp), lac promoter (P lac), P L promoter evening
―、 PRプロモーター、 T7プロモ一夕一等の大腸菌やファージ等に由来するプロモ一 夕一、 SPOlプロモー夕一、 SP02プロモー夕一、 penPプロモーター等をあげること ができる。 また P trpを 2つ直列させたプロモ一夕一 （P trp x 2 ) 、 tacプロモ一夕-, Promo one evening one derived from the P R promoter, T7 promoter Isseki one like Escherichia coli or phage, etc., spol promoter evening one, SP02 promoter evening one, can be mentioned penP promoter. In addition, two sets of P trp are connected in series. Promo one night (P trp x 2), tac promo one
―、 letlプロモ一夕一 [Gene, 44， 29 (1986) ] 、 lacT7プロモー夕一のように人為的に 設計改変されたプロモーター等も用いることができる。 -Promoters designed and modified artificially, such as letl Promo Overnight [Gene, 44, 29 (1986)] and lacT7 Promo Overnight, can also be used.
本発明の心筋分化誘導因子の遺伝子 DNAの蛋白質をコードする部分の塩基配列を、 宿主の発現に最適なコドンとなるように、 塩基を置換することにより、 目的とする蛋 白質の生産率を向上させることができる。  Improving the production rate of the target protein by substituting the nucleotide sequence of the portion of the gene encoding the protein of the gene for the cardiomyocyte differentiation-inducing factor gene of the present invention so as to be an optimal codon for expression in the host. Can be done.
本発明の心筋分化誘導因子の遺伝子 DNAの発現には転写終結配列は必ずしも必要 ではないが、 好適には構造遺伝子直下に転写終結配列を配置することが望ましい。 宿主細胞としては、 ェシエリヒア属、 セラチア属、 コリネバクテリウム属、 プレビ バクテリウム属、 シユードモナス属、 バチルス属、 ミクロバクテリゥム属等に属する 微生物、 例えば、 Escherichia coli XL1-Blue、 Escherichia coli XL2- Blue、 Escherichia coli DH1ヽ Escherichia coli MC1000、 Escherichia coli KY3276、 Escherichia coli W1485 Escherichia coli JM109 Escherichia coli HB101、 Escherichia coli No.49、 Escherichia coli W3110、 Escherichia coli NY49N Bacillus subtiliss Bacillus amvloliquefaciensN Brevibacterium ammoniagenesヽ Brevibacterium immariophilum ATCC 14068、 Brevibacterium sa'ccharolyticum ATCC14066、 Corynebacterium glutamicum ATCC13032、 Corynebacterium glutamicum ATCC14067、 Corynebacterium glutamicum ATCC13869、 Corynebacterium A transcription termination sequence is not always necessary for the expression of the gene DNA of the cardiac differentiation-inducing factor of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene. Examples of host cells include microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium, Plevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1ヽEscherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485 Escherichia coli JM109 Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49 N Bacillus subtilis s Bacillus amvloliquefaciens N Brevibacterium ammoniagenesヽBrevibacterium immariophilum ATCC 14068, Brevibacterium sa'ccharolyticum ATCC14066, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium
acetoacidophilum ATCC13870、 Microbacterium ammoniaphilum ATCC15354N Pseudomonas sp. D- 0110等をあげることができる。 acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354 N Pseudomonas sp. D-0110 and the like.
組換えベクターの導入方法としては、 上記宿主細胞へ DNAを導入する方法であれ ばいずれも用いることができ、 例えば、 カルシウムイオンを用いる方法 [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)] 、 プロトプラスト法 （特開昭 63-248394) 、 または Gene, 17, 107 (1982)や Molecular & General Genetics, 168. I l l (1979)に記載の方法等を あげることができる。  Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above-mentioned host cells.For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], the protoplast method (JP-A-63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168. Ill (1979).
酵母を宿主細胞として用いる場合には、 発現べクタ一として、 例えば、 YEpl3 (ATCC37115) 、 YEp24 (ATCC37051) 、 YCp50 (ATCC37419) 、 pHS19、 pHS15等 を例示することができる。  When yeast is used as a host cell, examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
プロモ一夕一としては、酵母中で発現できるものであればいかなるものでもよく、 例えば、 PH05プロ乇一夕一、 PGKプロモーター、 GAPプロモ一夕一、 ADHプロモ 一夕一、 gal lプロモーター、 gal 10プロモー夕一、 ヒートショック蛋白質プロモ一夕 ―、 MFひ 1プロモーター、 CUP 1プロモー夕一等をあげることができる。  Any promoter can be used as long as it can be expressed in yeast. For example, PH05 promoter overnight, PGK promoter, GAP promoter overnight, ADH promoter overnight, gal promoter, gal promoter 10 Promoter, Heat Shock Protein Promoter-, MF 1 Promoter, CUP 1 Promoter, etc.
宿主細胞としては、 サヅカロミセス 'セレビシェ f Saccharomyces cerevisiae) 、 シ ソサヅカロミセス ·ボンべ (Schizosaccharomyces pombe ヽ クリユイべ口ミセス .ラ クチス i luyveromyces lactis")、 トリコス不ロン 'プルランス f Trichosporon pullulans) シュヮニォミセス ·アルビウス (Schwanniomyces alluvius)等をあげることができる。 組換えベクターの導入方法としては、 酵母に DNAを導入する方法であればいずれ も用いることができ、 例えば、 エレクトロボレ一シヨン法 [Methods in Enzymol., 194, 182 (1990)] 、 スフエロプラスト法 [Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)]、 酢 酸リチウム法 []. BacterioL, 153, 163 (1983)、 Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)] 等をあげることができる。  The host cells include Saccharomyces f Saccharomyces cerevisiae, Schizosaccharomyces pombe ヽ ク リ ユ... ク ク ク 、, ト リ ラ ン ス ラ ン ス プ ル ラ ン ス プ ル プ ル ラ ン ス ラ ン ス プ ル プ ル ラ ン ス プ ルAny method can be used as a method for introducing a recombinant vector, as long as it is a method for introducing DNA into yeast. For example, an electro-boration method [Methods in Enzymol., 194, 182] Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method []. BacterioL, 153, 163 (1983), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)].
動物細胞を宿主細胞として用いる場合には、 発現べクタ一として、 例えば、 pcDNAKlnvitrogen社製)、 pcDM8 (Invitrogen社製） 、 pAGE107 [特開平 3-22979； Cytotechnology, 3, 133 (1990)]、 pAS3- 3 (特開平 2 - 227075)、 pCDM8 [Nature, 329, 840 (1987)]、 pcDNAI/Amp ( Invitrogen社製）、 pREP4( Invitrogen社製）、 pAGE103 |j. Biochem" 101, 1307 (1987)] 、 pAGE210等を例示することができる。  When animal cells are used as host cells, expression vectors include, for example, pcDNAKlnvitrogen), pcDM8 (Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3 -3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 | j. Biochem "101, 1307 (1987) ], PAGE210 and the like.
プロモーターとしては、動物細胞中で発現できるものであればいずれも用いること ができ、 例えば、 サイ トメガロウィルス （ヒト CMV) の IE(immediate early)遺伝子の プロモーター、 SV40の初期プロモ一夕一、 レトロウイルスのプロモ一夕一、 メタ口 チォネインプロモーター、 ヒートショック蛋白質プロモ一夕一、 SR プロモーター 等をあげることができる。 また、 ヒト CMVの IE遺伝子のェンハンサ一をプロモ一夕 —と共に用いてもよい。 Use any promoter that can be expressed in animal cells. For example, the promoter of the IE (immediate early) gene of the cytomegalovirus (human CMV), the early promoter of the SV40, the promoter of the retrovirus, the metamouth thionine promoter, the heat shock protein promoter, etc. In the evening, SR promoters can be mentioned. The enhancer of the IE gene of human CMV may be used together with the promoter.
宿主細胞としては、 ヒトの細胞であるナマルバ （Namalwa) 細胞、 サルの細胞であ る COS細胞、 チャイニーズ 'ハムスターの細胞である CHO細胞、 HBT5637 [特開昭 63-299] 等をあげることができる。  Examples of the host cells include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 [Japanese Patent Application Laid-Open No. 63-299]. .
,組換えべク夕一の導入法としては、 動物細胞に DNAを導入できるいかなる方法も 用いることができ、 例えば、 エレクトロポ一レ一シヨン法 [Cytotechnology, ， 133 (1990)3 、 リン酸カルシゥム法 （特開平 2 -227075) 、 リポフエクション法 As a method for introducing a recombinant vector, any method capable of introducing DNA into animal cells can be used. For example, the electroporation method [Cytotechnology,, 133 (1990) 3, calcium phosphate Law (JP 2 - 227 075), lipoic Hue transfection method
[Proc.Natl.Acad.Sci., USA, 84, 7413 (1987)、 Virology, 52, 456 (1973)] 等を用いること ができる。 形質転換体の取得および培養は、 特開平 2- 227075号公報あるいは特開平 2-257891号公報に記載されている方法に準じて行なうことができる。  [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987), Virology, 52, 456 (1973)] and the like can be used. The transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.
昆虫細胞を宿主として用いる場合には、 例えばバキュロウィルス ·ェクスプレツシ ョン ·ベクターズ, ァ ·ラホラトリー 'マニュアル [Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)]、 カレント ■プロトコ —ルズ 'イン 'モレキュラー 'バイオロジー サプルメント 1-38(1987-1997)、  When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory, Manual, WH Freeman and Company, New York (1992)], Current ■ Protoco — Ruth 'In' Molecular 'Biology Supplement 1-38 (1987-1997),
Bio/Technology, 6, 47 (1988)等に記載された方法によって、蛋白質を発現することがで ぎる。 Proteins can be expressed by the method described in Bio / Technology, 6, 47 (1988) and the like.
即ち、組換え遺伝子導入べクタ一およびバキュロウィルスを昆虫細胞に共導入して 昆虫細胞培養上清中に組換えウィルスを得た後、 さらに組換えウィルスを昆虫細胞に 感染させ、 蛋白質を発現させることができる。  That is, the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is infected to the insect cells to express the protein. be able to.
該方法において用いられる遺伝子導入べクタ一としては、 例えば、 PVL1392、 PVL1393、 pBlueBacIII (ともに Invitrogen社製） 等をあげることができる。 The gene transfer base Kuta one used in the method, for example, can be exemplified P VL1392, P VL1393, pBlueBacIII (both manufactured by Invitrogen Corp.).
バキュロウィルスとしては、 例えば、 夜盗蛾科昆虫に感染するウィルスであるァゥ トグラファ 'カリフオル二力 'ヌクレァ一 ·ポリへドロシス 'ウィルス (Autographa califomica nuclear polyhedro.sis virus等を用いることができる。 昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞である Sf9、 Sf21 [Baculovirus Expression Vectors, A Laり oratory Manual^ W.H. Freeman and Company, New York, (1992)]ヽ Trichoplusia の卵巣細胞である High 5 (Invitrogen社製） 等を用いることができる。 組換えゥィルスを調製するための、昆虫細胞への上記組換え遺伝子導入べク夕一と 上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシウム法 [特開平 2-227075]、 リポフエクシヨン法 [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]等をあ げることができる。 As the baculovirus, for example, an autographa califomica nuclear polyhedro.sis virus or the like, which is a virus infecting night moth insects, can be used. Insect cells include Sf9 and Sf21 which are ovarian cells of Spodoptera frugiperda [Baculovirus Expression Vectors, A La oratory Manual ^ WH Freeman and Company, New York, (1992)] ヽ High 5 which is an ovarian cell of Trichoplusia (Invitrogen Manufactured) can be used. Examples of a method for co-introducing the above-described recombinant gene introduction vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
遺伝子の発現方法としては、 直接発現以外に、 モレキュラー ·クロ一ニング 第 2 /ΪΚ. [Molecularし lomng, A Laooratory Manual, Second Edition, Cold Spring Harbor  In addition to direct expression, the methods of gene expression include Molecular Cloning Part II / II. [Molecular and lomng, A Laooratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press (1989)] に記載されている方法等に準じて、 分泌生産、 融合蛋白質発 現等を行うことができる。 Laboratory Press (1989)], secretory production, fusion protein expression and the like can be performed.
酵母、 動物細胞または昆虫細胞により発現させた場合には、 糖あるいは糖鎖が付加 された蛋白質を得ることができる。  When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added protein can be obtained.
心筋細胞への分化を誘導できる蛋白質をコードする DNAを組み込んだ組換え体  Recombinant incorporating DNA encoding a protein capable of inducing differentiation into cardiomyocytes
DNAを保有する形質転換体を培地に培養し、培養物中に心筋細胞への分ィヒを誘導でき る蛋白質を生成蓄積させ、 該培養物より該蛋白質を採取することにより、 心筋細胞へ の分化を誘導できる蛋白質を製造することができる。 The transformant having DNA is cultured in a medium to produce and accumulate a protein capable of inducing myocardial differentiation into the cardiomyocyte in the culture, and the protein is collected from the culture to obtain cardiomyocytes. A protein capable of inducing differentiation can be produced.
心筋細胞への分化を誘導できる蛋白質製造用の形質転換体を培地に培養する方法 は、 宿主の培養に用いられる通常の方法に従って行うことができる。  A method for culturing a transformant for producing a protein capable of inducing differentiation into a cardiomyocyte in a medium can be performed according to a usual method used for culturing a host.
大腸菌等の原核生物あるいは酵母等の真核生物を宿主として得られた形質転換体 を培養する培地としては、 該宿主が資化し得る炭素源、 窒素源、 無機物等を含有し、 形質転換体の培養を効率的に行える培地であれば天然培地、合成培地のいずれでもよ い。  A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, an inorganic substance, etc. which can be utilized by the host. Either a natural medium or a synthetic medium can be used as long as the medium can be cultured efficiently.
炭素源としては、 それぞれの宿主が資化し得るものであればよく、 グルコース、 フ ラクトース、 スクロース、 これらを含有する糖蜜、 デンプンあるいはデンプン加水分 解物等の炭水化物、 酢酸、 プロビオン酸等の有機酸、 エタノール、 プロパノールなど のアルコ一ル類を用レ、ることができる。  The carbon source may be any one that can be assimilated by each host, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as ethanol, propanol, etc. can be used.
窒素源としては、 アンモニア、 塩化アンモニゥム、 硫酸アンモニゥム、 酢酸アンモ 二ゥム、 リン酸アンモニゥム等の各種無機酸若しくは有機酸のアンモニゥム塩、 その 他含窒素化合物、 並びに、 ペプトン、 肉エキス、 酵母エキス、 コーンスチ一プリカ一、 カゼイン加水分解物、大豆粕および大豆粕加水分解物、 各種発酵菌体およびその消化 物等が用いられる。 ， Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate Ammonium salts of various inorganic or organic acids such as ammonia and ammonium phosphate, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn starch, casein hydrolyzate, soybean meal, and soybean meal Hydrolysates, various fermentation cells and digests thereof are used. ,
無機物としては、 リン酸第一カリウム、 リン酸第二カリウム、 リン酸マグネシウム、 硫酸マグネシウム、 塩化ナトリウム、 硫酸第一鉄、 硫酸マンガン、 硫酸銅、 炭酸カル シゥム等を用いることができる。  As the inorganic substance, potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
培養は、 振盪培養または深部通気攪抻培養などの好気的条件下で行う。培養温度は Cultivation is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is
15〜40°Cがよく、 培養時間は、 通常 16時間〜 7日間である。 培養中 pHは、 3·0〜9·0 に保持する。 p Hの調整は、 無機あるいは有機の酸、 アルカリ溶液、 尿素、 炭酸カル シゥム、 アンモニアなどを用いて行う。 The temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days. During the cultivation, maintain the pH between 3.0 and 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
また培養中必要に応じて、 アンピシリンゃテトラサイクリン等の抗生物質を培地に 添加してもよい。  If necessary, an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.
プロモー夕一として誘導性のプロモー夕一を用いた発現ベクターで形質転換した 微生物を培養するときには、必要に応じてィンデユーザーを培地に添加してもよい。 例えば、 lacプロモ一夕一を用いた発現べクタ一で形質転換した微生物を培養すると きにはイソプロピル一/?— D—チォガラクトビラノシド （IPTG) 等を、 trpプロモ一 夕一を用いた発現ベクターで形質転換した微生物を培養するときにはインド一ルァ クリル酸 （IAA) 等を培地に添加してもよい。  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an Indian user may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using lac promoter overnight, use isopropyl-1 /?-D-thiogalactovyranoside (IPTG) or the like, and use trp promoter overnight. When culturing a microorganism transformed with the expression vector, indoleacrylic acid (IAA) or the like may be added to the medium.
動物細胞を宿主細胞として得られた形質転換体を培養する培地としては、一般に使 用されている RPMI1640培地 [The Journal of the American Medical Association, 199, 519 (1967)]、 Eagleの MEM培地 [Science, 122, 501 (1952)] 、 ダルべヅコ改変 MEM培地 As a medium for culturing transformants obtained using animal cells as host cells, commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science , 122, 501 (1952)], Dalbeco modified MEM medium
[Virology, 8, 396 (1959)] 、 1 9 9培地 [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)]またはこれら培地に牛胎児血清等を添カロした培地等を用いるこ とができる。 [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], or use a medium supplemented with calf fetal serum etc. Can be.
培養は、 通常 pH6〜8、 30〜40°C、 5%C02存在下等の条件下で 1〜7日間行う。 Culturing is carried out usually pH6~ 8, 30~40 ° C, 5 % C0 2 present 1 to 7 days under conditions such as lower.
また、 培養中必要に応じて、 カナマイシン、 ペニシリン等の抗生物質を培地に添カロ してもよい。 昆虫細胞を宿主細胞として得られた形質転換体を培養する培地としては、一般に使 用されている TNM- FH培地（Pharmingen社製）、 Sf- 900 II SFM培地（Life Technologies 社製)、 ExCell400、 ExCell405 (いずれも JRH Biosciences社製）、 Grace's Insect Medium [Grace, T.C.C., Nature, 195, 788 (1962)]等を用いることができる。 If necessary, an antibiotic such as kanamycin or penicillin may be added to the medium during the culture. Culture media for transformants obtained using insect cells as host cells include commonly used TNM-FH media (Pharmingen), Sf-900II SFM media (Life Technologies), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.
培養は、 通常 ρΗδ〜7、 25〜30°C等の条件下で、 1〜5日間行う。 Cultivation is usually carried out for 1 to 5 days under conditions such as ρΗδ to 7, 25 to 30 ° C.
また、 培養中必要に応じて、 ゲン夕マイシン等の抗生物質を培地に添加してもよい。 上述の形質転換体の培養物から、心筋細胞への分化を誘導できる蛋白質を単離精製 するには、 通常の蛋白質の単離、 精製法を用いればよい。  If necessary, an antibiotic such as genyumycin may be added to the medium during the culture. In order to isolate and purify a protein capable of inducing differentiation into cardiomyocytes from a culture of the above-mentioned transformant, a normal protein isolation and purification method may be used.
例えば、 心筋細胞への分ィ匕を誘導できる蛋白質が、 細胞内に溶解状態で発現した場 合には、 培養終了後、 細胞を遠心分離により回収し水系緩衝液にけん濁後、 超音波破 砕機、 フレンチプレス、 マントンガウリンホモゲナイザー、 ダイノミル等により細胞 を破碎し、 無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られた 上清から、 通常の蛋白質の単離精製法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱 塩法、有機溶媒による沈殿法、ジェチルァミノエ^ル（DEAE)—セファロース、 DIAION HPA-75 (三菱化学社製） 等レジンを用いた陰イオン交換クロマトグラフィー法、 S - Sepharose FF(Amersham Pharmacia Biotech社製)等のレジンを用いた陽イオン交換ク口 マトグラフィ一法、 プチルセファロース、 フエ二ルセファロース等のレジンを用いた 疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、 ァフィ二ティ一クロマト グラフィ一法、 クロマトフォーカシング法、 等電点電気泳動等の電気泳動法等の手法 を単独あるいは組み合わせて用い、 精製標品を得ることができる。  For example, if a protein capable of inducing shunting into cardiomyocytes is expressed in a dissolved state in the cells, the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then sonicated. The cells are disrupted using a crusher, French press, Mentongaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), and a cation using a resin such as S-Sepharose FF (manufactured by Amersham Pharmacia Biotech) Exchange mouth chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric point A purified sample can be obtained by using an electrophoresis method such as electrophoresis alone or in combination.
また、 該蛋白質が細胞内に不溶体を形成して発現した場合は、 細胞を回収後破砕し、 遠心分離することにより、 沈殿画分として蛋白質の不溶体を回収する。  When the protein is expressed by forming an insoluble form in the cells, the cells are collected, crushed, and centrifuged to collect the protein insoluble form as a precipitate fraction.
回収した該蛋白質の不溶体を蛋白質変性剤で可溶化する。  The recovered insoluble form of the protein is solubilized with a protein denaturant.
該可溶化液を、 希釈あるいは透析により、該可溶化液中の蛋白質変性剤の濃度を下 げることにより、 該蛋白質の構造を正常な立体構造に戻した後、 上記と同様の単離精 製法により該蛋白質の精製標品を得る。  After reducing the concentration of the protein denaturing agent in the lysate by diluting or dialysis the lysate, the structure of the protein is returned to a normal three-dimensional structure. A purified sample of the protein is obtained by the production method.
心筋細胞への分化を誘導できる蛋白質あるいはその糖修飾体等の誘導体が細胞外 に分泌された場合には、 培養上清から、 該蛋白質あるいはその糖鎖付加体等の誘導体 を回収することができる。即ち、培養物から遠心分離等の手法により培養上清を回収 し、 該培養上清から、 上記と同様の単離精製法を用いることにより、 精製標品を得る ことができる。 When a protein capable of inducing differentiation into cardiomyocytes or a derivative thereof such as a sugar-modified product is secreted extracellularly, a derivative such as the protein or a sugar chain-adduct thereof is obtained from the culture supernatant. Can be recovered. That is, the culture supernatant is collected from the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
このようにして取得される蛋白質として、 例えば、 配列番号 5、 6、 10、 12、 14、 16、 18、 20、 22、 24、 26、 28および 30で表されるアミノ酸配列を有する蛋白質等をあげ ることができる。  Examples of the protein thus obtained include, for example, proteins having the amino acid sequences represented by SEQ ID NOs: 5, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. I can give it.
また、 上記方法により発現させた蛋白質を、 Fmoc法 (フルォレニルメチルォキシカ ルポニル法)、 tBoc法 (t-プチルォキシカルボニル法)等の化学合成法によっても製造す ることができる。 また、米国 Advanced ChemTech社製、 Perkin-Elmer社製、 Amersham Pharmacia Biotech社製、米国 Protein Technology Instrument社製、米国 Synthecell-Vega 社製、 米国 PerSeptive社製、 島津製作所社製等のペプチド合成機を利用して合成する こともできる。  Further, the protein expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl) method, the tBoc method (t-butyloxycarbonyl method), and the like. . Utilize a peptide synthesizer manufactured by Advanced ChemTech, U.S.A., Perkin-Elmer, Amersham Pharmacia Biotech, Protein Technology Instrument, U.S.A., Synthecell-Vega, U.S.A., PerSeptive, U.S.A., Shimadzu, etc. Can also be combined.
心筋細胞への分化を誘導できる蛋白質ほ、 上記（1 ) と同様にして心筋形成剤を形 成し使用することができる。  A protein capable of inducing differentiation into cardiomyocytes can be used to form and use a cardiomyogen in the same manner as in (1) above.
7 . 先天性遺伝子疾患の治療への利用  7. Use for the treatment of congenital genetic diseases
心不全を来す疾患の中には、 一部であるが単一遺伝子の変異により、 本来必要な蛋 白質が全て欠損するために心不全を来す一群がある。 このような疾患としては、 家族 性肥大型心筋症、 Fabri病、 QT延長症候群、 マルファン症候群、 大動脈弁狭窄症、 ミ トコンドリァ心筋症、 Duchenne型筋ジストロフィ一症等があげられる。 これらの疾 患は、 ミオシン、 トロポニン、 トロポミオシン、 電位依存性 Naチャンネル、 Kチヤ ンネル、 フイブリン、 エラスティン、 ミ トコンドリア、 ジストロフィンなどの遺伝子 異常が原因であることが知られている [治療学，^， O2- 1306(1"6)] 。 すなわち、 こ れら患者より本発明の心筋細胞への分化能を有する骨髄細胞を取得し、正常な遺伝子 を導入して心臓に移植することで上記疾患を治療することができる。正常な遺伝子は、 上記 6 ( 1 )で記載した遺伝子治療用のベクターを用いることで本発明の心筋細胞へ の分化能を有する骨髄細胞に導入することができる。 Among the diseases that cause heart failure, there is a group that causes heart failure due to partial deletion of all necessary proteins due to mutation of a single gene. Such diseases include familial hypertrophic cardiomyopathy, Fabri disease, long QT syndrome, Marfan syndrome, aortic stenosis, mitochondrial cardiomyopathy, and Duchenne muscular dystrophy. These diseases are known to be caused by genetic abnormalities such as myosin, troponin, tropomyosin, voltage-gated Na channel, K channel, fibrin, elastin, mitochondria, and dystrophin [Therapeutics, ^, O 2 -. 1 3 0 6 (1 "6)] that is, to get the bone marrow cells capable of differentiating into cardiomyocytes of the present invention from these patients, be implanted into a heart by introducing normal genes The normal gene can be introduced into bone marrow cells capable of differentiating into cardiomyocytes of the present invention by using the vector for gene therapy described in 6 (1) above. it can.
8. 心筋細胞への分化能を有する骨髄細胞特異的な表面抗原を特異的に認識する抗 体の取得 以下に、本発明の心筋細胞への分化能を有する骨髄細胞で発現している表面抗原を 特異的に認識する抗体の調製法について述べる。 8. Acquisition of antibodies that specifically recognize bone marrow cell-specific surface antigens that have the ability to differentiate into cardiomyocytes Hereinafter, a method for preparing an antibody that specifically recognizes a surface antigen expressed on bone marrow cells capable of differentiating into cardiomyocytes according to the present invention will be described.
本発明の心筋細胞への分化能を有する骨髄細胞で特異的に発現している表面抗原 を認識する抗体は、心筋梗塞などの心臓病の細胞治療を実施する上で必要な心筋細胞 への分化能を有する骨髄細胞の純度検定や精製に用いることができる。  The antibody of the present invention, which recognizes a surface antigen specifically expressed on bone marrow cells capable of differentiating into cardiomyocytes, is capable of differentiation into cardiomyocytes necessary for performing cell therapy for heart diseases such as myocardial infarction. It can be used for purity test and purification of bone marrow cells having the ability.
該抗体を取得する方法として、本発明の心筋細胞への分化能を有する骨髄細胞 3〜5 X 105cellS/匹、あるいは該細胞から調製した細胞膜画分 l〜10mg/匹程を抗原として、 ゥサギ、 ャギまたは 3~20週令のラット、 マウスもしくはハムス夕一等の非ヒトほ乳 動物の皮下、 静脈内または腹腔内に、 適当なアジュバント [例え^、 フロインドの完 全アジュバント（Complete Freund's Adjuvant)または、 水酸化アルミ ゥムゲル、 百日 咳菌ワクチンなど]とともに投与する。 As a method for obtaining the antibody, bone marrow cells capable of differentiating into cardiomyocytes of the present invention 3 to 5 × 10 5 cell S / animal, or a cell membrane fraction l to 10 mg / animal prepared from the cells as an antigen A suitable adjuvant subcutaneously, intravenously or intraperitoneally in non-human mammals such as rabbits, goats, or 3-20 week old rats, mice or hams, [Complete Freund's Adjuvant) or aluminum hydroxide gel, pertussis vaccine, etc.].
該抗原の投与は、 1回目の投与の後 1〜2週間おき 3〜10回行う。 各投与後、 3 〜 7日目に眼底静脈叢より採血し、該血清が免疫に用いた抗原と反応するか否かを酵 素免疫測定法 [酵素免疫測定法 （ELISA法） ：医学書院刊 I976年、 Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988] などで調べる。 The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and it is determined whether the serum reacts with the antigen used for immunization by enzyme immunoassay [enzyme immunoassay (ELISA): published by Medical Shoin. I 976 , Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
免疫に用いた抗原に対し、 その血清が充分な抗体価を示した非ヒトほ乳動物を、 血 清または抗体産生細胞の供給源とする。  A non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of serum or antibody-producing cells.
ポリクロ一ナル抗体は、 該血清を分離、精製することにより調製することができる。 モノクローナル抗体は、該抗体産生細胞と非ヒトほ乳動物由来の骨髄腫細 とを融 合させてハイプリドーマを作製し、 該ハイプリドーマを培養するか、 動物に投与して 該動物を腹水癌化させ、 該培養液または腹'水を分離、精製することにより調製するこ とができる。  A polyclonal antibody can be prepared by separating and purifying the serum. A monoclonal antibody is prepared by fusing the antibody-producing cells with myeloma derived from a non-human mammal to produce a hybridoma, and culturing the hybridoma or administering it to an animal to cause the animal to develop ascites carcinoma. It can be prepared by separating and purifying the culture solution or ascites fluid.
抗体産生細胞としては、 脾細胞、 リンパ節、 末梢血中の抗体産生細胞、 特に脾細胞 が好適に用いられる。  As antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.
骨髄腫細胞としては、 8—ァザグァニン耐性マウス （BALBん由来） 骨髄腫細胞株 である P3 - X63Ag8— U1(P3— U1)株 [Current Topics in Microbiology andlmmunology, 18， 1 (1978)] 、P3— NSl/1— Ag41(NS - 1)株 [European J. Immunology, 6, 511 (1976)] ヽ SP2/0- Agl4(SP- 2)株 [Nature, 276, 269 (1978)] 、 P3 - X63 - Ag8653(653)株 [J. Immunology. 123, 1548 (1979)] 、 P3- Χδ3- Ag8(X6 株 [Nature, 256, 495 (1975)] 等、 マウス由来の株化細 胞が好適に用いられる。 Myeloma cells include 8-azaguanine-resistant mouse (derived from BALB) myeloma cell line P3-X63Ag8-U1 (P3-U1) strain [Current Topics in Microbiology andlmmunology, 18, 1 (1978)], P3- NSl / 1—Ag41 (NS-1) strain [European J. Immunology, 6, 511 (1976)] ヽ SP2 / 0-Agl4 (SP-2) strain [Nature, 276, 269 (1978)], P3-X63 -Ag8653 (653) strain [J. Immunology. 123, 1548 (1979)], and mouse-derived cell lines such as P3-Δδ3-Ag8 (X6 strain [Nature, 256, 495 (1975)]) are preferably used.
ハイプリドーマ細胞は、 以下の方法により作製できる。  Hybridoma cells can be prepared by the following method.
抗体産生細胞と骨髄腫細胞を混合し、 H A T培地（正常培地にヒポキサンチン、 チ ミジンおよびアミノプテリンを加えた培地） に懸濁したのち、 7〜1 4日間培養する c 培養後、 培養上清の一部をとり酵素免疫測定法などにより、 抗原に反応し、 抗原を含 まない蛋白質には反応しないものを選択する。ついで、 限界希釈法によりクロ一ニン グを行い、酵素免疫測定法により安定して高い抗体価の認められたものをモノクロ一 ナル抗体産生ハイプリド一マ細胞として選択する。  Antibody-producing cells and myeloma cells are mixed, suspended in HAT medium (medium containing hypoxanthine, thymidine, and aminopterin in normal medium), and cultured for 7 to 14 days. A portion of the protein is selected from those that react with the antigen but do not react with the protein that does not contain the antigen by enzyme immunoassay or the like. Then, the cells are cloned by the limiting dilution method, and those having a stable and high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridoma cells.
ポリクロ一ナル抗体またはモノクローナル抗体を分離、精製する方法としては、 遠 心分離、 硫安沈殿、 力プリル酸沈殿、 または DEAE—セファロ一スカラム、 陰イオン 交換カラム、プロテイン Aまたは G—カラムあるいはゲル濾過カラム等を用いるクロ マトグラフィ一等を、 単独または組み合わせて処理する方法があげられる。  Methods for separating and purifying polyclonal or monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or DEAE-Sepharose column, anion exchange column, protein A or G-column or gel filtration column. Chromatography, etc. using these methods alone or in combination.
上記方法で取得した、該心筋細胞への分化能を有する骨髄細胞で発現している表面 抗原を特異的に認識する抗体を用いて、検体細胞に対する反応性と造血系幹細胞、 神 経系幹細胞などの対照となる細胞に対する反応性とを比較することで、検体細胞が上 記特異的表面抗原を発現しているかどうかを容易に検定することができる。  Using the antibody obtained by the above method and specifically recognizing a surface antigen expressed on bone marrow cells capable of differentiating into cardiomyocytes, the reactivity to sample cells and the hematopoietic stem cells, neural stem cells, etc. By comparing the reactivity with the control cells, it can be easily determined whether or not the sample cells express the above-mentioned specific surface antigen.
9 . 心筋細胞への分化能を有する骨髄細胞で発現している表面抗原および該表面 抗原をコ一ドする遺伝子の取得  9. Acquisition of surface antigens expressed on bone marrow cells capable of differentiating into cardiomyocytes and genes encoding the surface antigens
該心筋細胞への分化能を有する骨髄細胞で特異的に発現している表面抗原遺伝子 の取得方法としては、二つの異なる由来のサンプル間で異なる発現形態 取る遺伝子 を取得する方法であるサブトラクション法 [Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988)]や Representational difference analysis [Nucleic Acids Research, 22, 5640-5648 (1994)]による方法をあげることができる。  As a method for obtaining a surface antigen gene specifically expressed in bone marrow cells capable of differentiating into cardiomyocytes, a subtraction method, which is a method for obtaining genes having different expression forms between two samples of different origins, Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988)] and Representational difference analysis [Nucleic Acids Research, 22, 5640-5648 (1994)].
まず、心筋細胞への分ィ匕能を有する骨髄細胞より作製した cDNAライブラリ一を、 造血系幹細胞や神経系幹細胞などの骨髄細胞以外の対照細胞より取得した mRNAを 用いてサブトラクシヨンを行う。心筋細胞への分化能を有する骨髄細胞特異的な遺伝 子を濃縮した差分化 cDNAライブラリ一を調製した後、該差分化 cDNAライプラリ一 の揷入CDNA配列を5 5側よりランダムに塩基配列解析を行い、 分泌シグナル配列を 持つものだけを選択する。このようにして得られた cDNAの全長塩基配列を決定する ことにより、該 cDNAがコードする蛋白質が分泌蛋白質か膜蛋白質かを区別すること ができる。 First, subtraction is performed using a cDNA library prepared from bone marrow cells having the ability to divide into cardiomyocytes using mRNA obtained from control cells other than bone marrow cells such as hematopoietic stem cells and neural stem cells. After preparing a differential cDNA library enriched for bone marrow cell-specific genes capable of differentiating into cardiomyocytes, the differential cDNA library was prepared. The揷入C DNA sequence randomly from 5 5 side performs the sequence analysis, selecting only those with the secretion signal sequence. By determining the full-length nucleotide sequence of the cDNA thus obtained, it is possible to distinguish whether the protein encoded by the cDNA is a secretory protein or a membrane protein.
上記の方法において、 ランダム配列解析の代わりに、 シグナルシーケンストラヅプ 法も用いることもできる [Science, 261, 600-603 (1993); Nature Biotechnology, 17, 487-490 (1999)]。 シグナルシーケンストラップ法とは、 分泌シグナル配列をもつ遺伝 子を選択的にスクリーニングする方法である。  In the above method, a signal sequence trap method can be used instead of the random sequence analysis [Science, 261, 600-603 (1993); Nature Biotechnology, 17, 487-490 (1999)]. The signal sequence trap method is a method for selectively screening genes having a secretory signal sequence.
効率よく特異的な表面抗原を取得するためには、 シグナルシーケンストラップライ ブラリーをサブトラクシヨンが行えるベクタ一を用いて作製し、心筋細胞への分ィ匕能 を有する骨髄細胞から作製したシグナルシーケンストラップライプラリーを造血系 幹細胞や神経系幹細胞などの対照となる細胞より取得した mRNAを用いてサブトラ クションを行う方法が望ましい。このようにして取得された分泌シグナル配列を含む DNA断片は全長 cDNAをクロ一ン化するためのプローブとして用いることができる。 全長 cDNAはその全長塩基配列を解析することで、該 cDNAがコードする蛋白質が 分泌蛋白質か膜蛋白質かを区別することができる。  In order to efficiently obtain specific surface antigens, a signal sequence trap library was prepared using a vector capable of performing subtraction, and a signal sequence prepared from bone marrow cells having the ability to divide myocardial cells. A preferred method is to perform trap subtraction using mRNA obtained from control cells such as hematopoietic stem cells and neural stem cells. The DNA fragment containing the secretory signal sequence thus obtained can be used as a probe for cloning full-length cDNA. By analyzing the full-length nucleotide sequence of the full-length cDNA, it is possible to distinguish whether the protein encoded by the cDNA is a secretory protein or a membrane protein.
ランダム配列解析あるいはシグナルシーケンストラップ法を用いた場合でも、得ら れたクローンが膜蛋白質をコードする場合は、塩基配列から類推されるァミノ酸配列 に基づき合成べプチドを作製し、該合成べプチドを抗原として上記方法により特異的 な抗体を取得することができる。  Even when random sequence analysis or signal sequence trapping is used, if the obtained clone encodes a membrane protein, a synthetic peptide is prepared based on the amino acid sequence inferred from the nucleotide sequence, and the synthetic peptide is synthesized. Using the above as an antigen, a specific antibody can be obtained by the above method.
また、 膜蛋白質の場合は、 受容体をコードしているものがあり、 このような受容体 は該心筋細胞への分化能を有する骨髄細胞の特異的な増殖または心筋細胞への分化 の調節に働いている可能性があり、当該受容体のリガンドの探索に用いることができ る。分泌蛋白質の場合は、 直接心筋細胞への分化能を有する骨髄細胞を増殖あるいは 分化させるために用いることができる。  In the case of a membrane protein, some of them encode a receptor, and such a receptor is used to regulate the specific proliferation of bone marrow cells capable of differentiating into the cardiomyocytes or the regulation of differentiation into cardiomyocytes. It may be working and can be used to search for ligands for the receptor. In the case of a secretory protein, it can be used for directly proliferating or differentiating bone marrow cells capable of differentiating into cardiomyocytes.
1 0 .心筋細胞への分化能のある骨髄細胞の増殖因子および心筋細胞への分化誘導 因子のスクリーニング  10. Screening of growth factor of bone marrow cells capable of differentiating into cardiomyocytes and differentiation inducing factor into cardiomyocytes
心筋細胞への分化能のある骨髄細胞の増殖因子および心筋細胞への分化誘導因子 のスクリーニング方法としては、心筋細胞への分化能を有する骨髄細胞を無血清培地 中で培養させる際に、 検体である種々の物質を添加させ、 該細胞が増殖するか、 また は心筋細胞へ分化誘導されるかで調べることにより行うことができる。 Bone marrow cell growth factor and cardiomyocyte differentiation inducer capable of cardiomyocyte differentiation As a screening method, when culturing bone marrow cells capable of differentiating into cardiomyocytes in a serum-free medium, various substances as specimens are added, and the cells proliferate or differentiate into cardiomyocytes. It can be done by examining whether it is induced.
検体となる物質としては、 各種サイトカインゃ増殖因子などの分泌蛋白質、 細胞接 着分子などの膜結合蛋白質、 組織抽出液、 合成ペプチド、 合成化合物、 微生物培養液 等などいかなるものでもよい。  The substance to be the specimen may be any substance such as secreted proteins such as various cytokines and growth factors, membrane-bound proteins such as cell-adhered molecules, tissue extracts, synthetic peptides, synthetic compounds, and microbial culture solutions.
増殖能力はコ口二一形成能や BrdUの取り込みなどで調べることができる。  The proliferative ability can be examined based on the ability to form the ostium and the uptake of BrdU.
コロニ一形成能は、本発明の骨髄細胞を低密度で播種することにより調べることが できる。  The colony forming ability can be examined by seeding the bone marrow cells of the present invention at a low density.
BrdUの取り込みは、 BrdUを特異的に認識する抗体を用いた免疫染色により調べる ことができる。  BrdU incorporation can be examined by immunostaining using an antibody that specifically recognizes BrdU.
心筋細胞への分化を評価する方法としては、 自律拍動を指標にするかまたは筋細胞 で特異的に発現する遺伝子のプロモ一夕一と GFP(Gleen fluorescent protein) 、 ルシフ エラ一ゼ、ベー夕一ガラクトシダ一ゼなどのレポ一夕一遺伝子とを組み合わせたべク 夕一 DNAを該心筋細胞への分化能を有する骨髄細胞に導入したレポー夕一細胞を用 いてレポーター遺伝子の発現を指標にする方法があげられる。  Cardiomyocyte differentiation can be evaluated by using autonomic pulsation as an index or by promoting genes specifically expressed in muscle cells and GFP (Gleen fluorescent protein), Luciferase, A method in which the expression of a reporter gene is used as an index by using a reporter cell in which a DNA combined with a reporter gene such as galactosidase is introduced into bone marrow cells capable of differentiating the cardiomyocytes. Is raised.
レポ一夕一系の構築には cardiac troponin I(cTNI)のプロモ一夕一を用いる方法があ げられる [J. Biological Chemistry, 273, 25371 - 25380 (1998)]。  One method of constructing the repo overnight system is to use the cardiac troponin I (cTNI) promoter overnight [J. Biological Chemistry, 273, 25371-25380 (1998)].
1 1 . 心筋細胞への分化能のある骨髄細胞の不死化  1 1. Immortalization of bone marrow cells capable of differentiating into cardiomyocytes
心臓疾患の患者、 特に高齢者に本発明の治療薬を投与する場合、 本発明の心筋細胞 への分化能を有する骨髄細胞をガン化させずに細胞分裂の回数を増やすことが望ま しい。  When administering the therapeutic agent of the present invention to a patient with a heart disease, particularly an elderly person, it is desirable to increase the number of cell divisions without causing the bone marrow cells capable of differentiating into cardiomyocytes of the present invention to become cancerous.
骨髄細胞をガン化させずに細胞分裂の回数を増やす方法としては、テロメラーゼを 本発明の心筋細胞への分化能を有する骨髄細胞に発現させる方法をあげることがで ぎる。  As a method of increasing the number of cell divisions without causing bone marrow cells to become cancerous, a method of expressing telomerase in bone marrow cells capable of differentiating into cardiomyocytes of the present invention can be mentioned.
例えば、 テロメラ一ゼの触媒サプュニットである TERT遺伝子、 具体的には配列番 号 32で表される DNAを、 レトロウイルスベクターに導入した後に心筋細胞への分ィ匕 能を有する骨髄細碑に導入する方法、 または心筋細胞への分化能を有する骨髄細胞に 内在する TERT遺伝子を誘導発現させる因子を心筋細胞への分化能を有する骨髄細胞 に投与する方法、 あるいは TERT遺伝子を誘導発現させる因子をコードする DNAを 含むベクターを心筋細胞への分化能を有する骨髄細胞に導入する方法などをあげる ことができる。 For example, TERT gene is a catalytic Sapuyunitto of Teromera Ichize, DNA specifically represented by SEQ ID NO 3 2, the bone marrow Hosohi having minute I spoon ability to cardiomyocytes after introducing a retroviral vector Transfection method, or into bone marrow cells that have the potential to differentiate into cardiomyocytes A method of administering a factor capable of inducing and expressing an endogenous TERT gene to bone marrow cells capable of differentiating into cardiomyocytes, or a method of administering a vector containing DNA encoding a factor capable of inducing and expressing TERT gene to bone marrow capable of differentiating into cardiomyocytes Examples of the method include introduction into cells.
このような TERT遺伝子を誘導発現させる因子は、 心筋細胞への分化能を有する骨 髄細胞に TERT遺伝子プロモ一夕一と GFP(Green Fluorescent protein), ルシフェラー ゼ、あるいはべ一夕一ガラクトシダーゼを組み合わせたレポ一夕一系を心筋細胞への 分化能を有する骨髄細胞に導入することで選別することができる。  The factor that induces and expresses the TERT gene is obtained by combining TERT gene promoter and GFP (Green Fluorescent protein), luciferase or galactosidase in bone marrow cells that have the potential to differentiate into cardiomyocytes. Selection can be achieved by introducing the repo overnight system into bone marrow cells capable of differentiating into cardiomyocytes.
以下に実施例をあげて、 本発明を具体的に示す。  Hereinafter, the present invention will be specifically described with reference to examples.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
実施例 1 . マウス骨髄からの心筋細胞への分化能を有する骨髄細胞の取得と培養 Example 1. Obtaining and culturing bone marrow cells capable of differentiating into cardiomyocytes from mouse bone marrow
5週齢の C3H/Heマウス 10匹をエーテルを用いて麻酔し、 そのうえで頸椎脱臼に より致死させた。 マウスを半側臥位にして、 70%エタノールを充分かけ消毒した。 次に大腿骨周辺の皮膚を広い範囲にわたり切開し、大腿骨全面の大腿四頭筋をはさ みで切除した。膝関節の部分に軽くはさみを入れ、 関節を外し、 さらに大腿骨背面の 筋肉を切除した。股関節の部分にはさみを入れ関節を外し、 大腿骨を取り出した。大 腿骨に付着している筋肉をはさみで切除し、 大腿骨全体を露出させた。大腿骨の両端 をはさみで切断後、 テルモ製 23Gの針を装着した 2.5ml注射器に 20%FCSを含有する IMDM培地を約 l.5ml入れ、 注射針の先端を大腿骨の膝関節側の断端に差し込み、 試 験管の中に培養液を吹き出すことで、骨髄細胞を押し出した。取得した細胞は、 20%FCS、 100mg/ml penicillin^ 250ng/ml streptomycin^ 85mg/ml amphotericin 含有 "5る IMDM培 地中で 33°Cで、 5% C02濃度の孵卵機を用いて培養を行った。 継代を続けることで、 細胞は間葉系の細胞へと均一化し、 造血系の細胞は消失した。 Ten 5-week-old C3H / He mice were anesthetized with ether and then killed by cervical dislocation. The mice were placed in a semi-lateral position and disinfected with 70% ethanol. Next, the skin around the femur was incised over a wide area, and the quadriceps femoris over the entire femur was removed with scissors. Light scissors were inserted into the knee joint, the joint was removed, and the muscles behind the femur were resected. A scissor was placed in the hip joint, the joint was removed, and the femur was removed. Muscles attached to the femur were excised with scissors to expose the entire femur. After cutting both ends of the femur with scissors, about the IMDM medium containing 20% FCS to 2. 5 ml syringe fitted with a needle manufactured by Terumo 23G l. 5 ml were placed, the tip of the injection needle femoral knee Bone marrow cells were extruded by inserting them into the side stumps and blowing the culture into the test tubes. Acquired cells, in 2 0% FCS, 100mg / ml penicillin ^ 250ng / ml streptomycin ^ 85mg / ml amphotericin -containing "5 Ru IMDM culture ground at 33 ° C, with 5% C0 2 concentration in the incubator machine culture By continuing the passage, the cells were homogenized into mesenchymal cells, and the hematopoietic cells disappeared.
約 4ヶ月上記条件で培養を行い、 不死化した細胞を選択した後、 希釈により 192種 類の独立した単一細胞 (single cell)由来の細胞株を樹立した （以下、 骨髄由来初代不死 化細胞株と称する） 。 これら独立のクローン由来の細胞にそれぞれに 3mMの終濃度 になるように 5 - aza-Cを添加し 24時間培養した後、 培地を IMDM培地に代えてさら に 2週間培養することで拍動する細胞を産生するクローンを選択した。骨髄由来初代 不死化細胞 192個のうち、 心筋細胞への分化能を有する骨髄細胞は 3個であった。該 心筋細胞への分化能を有する骨髄細胞は、心筋細胞以外に脂肪細胞を分化誘導したこ とからいずれも多分化能幹細胞であることが示された。多分化能幹細胞から、 非特異 的に分化誘導してくる自己拍動細胞の割合は非常に少ない。 After culturing under the above conditions for about 4 months and selecting immortalized cells, dilution was used to establish 192 independent single cell-derived cell lines (hereinafter referred to as bone marrow-derived primary immortalized cells). Stocks). After culturing for 24 hours after adding 5-aza-C to each of these independent clone-derived cells to a final concentration of 3 mM, culturing is continued for another 2 weeks by replacing the medium with IMDM medium. A cell producing clone was selected. Primary bone marrow-derived Of the 19 2 immortalized cells, 3 were bone marrow cells capable of differentiating into cardiomyocytes. All of the bone marrow cells capable of differentiating into cardiomyocytes induced pluripotent cells in addition to cardiomyocytes, indicating that they were all multipotent stem cells. The percentage of autologous beating cells that non-specifically induce differentiation from pluripotent stem cells is very small.
しかし、 この自己拍動する細胞周辺をクロ一ニングシリンジで採取し、 培養した細 胞 （以下、 単に心筋前駆細胞と称する） に、 再び 5-aza-Cを添加し 24時間培養した 後、 培地を IMDM培地に代えてさらに 2〜3週間培養することでより多くの自律拍動 する細胞を取得した。該心筋細胞への分化能を有する骨髄幹細胞は、増殖条件下では、 単核の線維芽細胞様の形態を呈し、 心筋収縮蛋白質はほとんど発現していない。 しか し 5 - aZa-Cにより最終分化を誘導すると形態は著しく変化した。 However, the surrounding area of the self-pulsating cells was collected with a closing syringe, and 5-aza-C was added again to the cultured cells (hereinafter simply referred to as myocardial progenitor cells) and cultured for 24 hours. More autonomously pulsating cells were obtained by further culturing for 2 to 3 weeks instead of IMDM medium. The bone marrow stem cells capable of differentiating into cardiomyocytes exhibit a mononuclear fibroblast-like morphology under proliferating conditions, and hardly express cardiac contractile proteins. However, when terminal differentiation was induced by 5 - aZaC , the morphology changed significantly.
分化誘導 1週間目頃より、一部の細胞は細胞質が大きくなり円形あるいは棒状を呈 し、後に自律拍動を開始する細胞となるが、 この時点では自律拍動を行うことは少な かった。分化誘導後 2週間になると、 自己拍動を開始した。 この自 3拍動した細胞は 互いに連結しあい、 縦に連結して筋管細胞様となった。 3週間以後には多くの細胞が 舉に 1列にならび、 同期して収縮した。分ィ匕後 4週間以後には培養皿の上の直接連結 される細胞は、すべて同期して収縮し心筋組織様になった。マウスの心臓は、毎分 300 〜400回程度の心拍数で収縮するが、 これに対してマウス成体骨髄由来の細胞より分 化した心筋細胞は、培養条件下において毎分 120〜250回の速さで規則的に収縮した。  Differentiation induction From about the first week, some cells have increased cytoplasm and have a round or rod-like shape, and later become cells that start autonomous pulsation. At this point, however, seldom autonomous pulsation was performed. Two weeks after induction of differentiation, self-pulsation started. The three beats of the cells connected to each other, and connected vertically to form myotube cells. After three weeks, many cells lined up in a row, and contracted synchronously. After 4 weeks, the directly connected cells on the culture dish all contracted synchronously and became like myocardial tissue. The mouse heart contracts at a heart rate of about 300 to 400 beats per minute, whereas cardiomyocytes differentiated from cells derived from adult mouse bone marrow produce a rapid rate of 120 to 250 beats per minute under culture conditions. It shrunk regularly.
実施例 2 . マウス骨髄細胞から誘導される心筋細胞の特性  Example 2. Characteristics of cardiomyocytes derived from mouse bone marrow cells
骨髄由来細胞から形成される自律拍動する心筋様細胞が、実際に心筋細胞の性質を 保有しているかどうかの解析を行った。  We analyzed whether autonomously beating cardiomyocytes formed from bone marrow-derived cells actually possess the properties of cardiomyocytes.
実施例 1で取得した、 骨髄由来初代不死化細胞株、 多分ィヒ能幹細胞および心筋前駆 細胞から分化誘導した心筋細胞から、 それぞれ Trizol Reagents(GIBCO BRL社製)を用 いて全 RNAを取得した。 次に、 該全 RNAを基質として Supersciptll reverse  Total RNA was obtained from each of the bone marrow-derived primary immortalized cell lines obtained in Example 1, and possibly cardiomyocytes differentiated from immature stem cells and cardiac progenitor cells, using Trizol Reagents (GIBCO BRL). Next, using the total RNA as a substrate, Supersciptll reverse
transcriptase(GIBCO BRL社製)を用いて First strand cDNAを合成した。 First strand cDNA was synthesized using transcriptase (GIBCO BRL).
次に、 心筋細胞特異的な遺伝子の発現を検討するために、該 First strand cDNAを基 質として、配列番号 33〜58に示した塩基配列を有する合成 DNAを用いて定量的 PCR を行った。 心筋細胞特異的な遺伝子としては、 ナトリウム利尿ペプチドである ANP および BNP、ミオシン重鎖である -MHCおよび/? - MHC、ァクチンであるひ -skeletal actinおよび/? - skeletal actin、 ミオシン軽鎖である MLC- 2a、 MLC- 2v、 心筋細胞特異 的転写因子である Nkx2.5/Csx、 GATA4、 TEF- 1、 MEF- 2C、 MEF - 2D、 MEF- 2Aを用 いた。 Next, in order to examine the expression of a cardiomyocyte-specific gene, quantitative PCR was performed using the first strand cDNA as a base and a synthetic DNA having the nucleotide sequence shown in SEQ ID NOS: 33 to 58. Cardiomyocyte-specific genes include ANP, a natriuretic peptide And BNP, myosin heavy chain -MHC and /?-MHC, actin -skeletal actin and /?-Skeletal actin, myosin light chain MLC-2a, MLC-2v, cardiomyocyte-specific transcription factor Some Nkx2.5 / Csx, GATA4, TEF-1, MEF-2C, MEF-2D, MEF-2A were used.
ANPの増幅には配列番号 33、 34の塩基配列を有する合成 DNAを、 BNPの増幅には 配列番号 35、 36の塩基配列を有する合成 DNAを、 ひ- MHCの増幅には配列番号 37、 38の塩基配列を有する合成 DNAを、 MHCの増幅には配列番号 39、 40の塩基配列 を有する合成 DNAを、 ひ- skeletal actinの増幅には配列番号 41、 42の塩基配列を有す る合成 DNAを、 ? -skeletal actinの増幅には配列番号 43、 44の塩基配列を有する合成 DNAを、 MLC- 2aの増幅には配列番号 45、 46の塩基配列を有する合成 DNAを、 MLC- 2v の増幅には配列番号 47、 48の塩基配列を有する合成 DNAを、 Nkx2.5/Csxの増幅には 配列番号 49、 50の塩基配列を有する合成 DNAを、 GATA4の増幅には配列番号 51、 52の塩基配列を有する合成 DNAを、 TEF- 1の増幅には配列番号 53、 54の塩基配列を 有する合成 DNAを、 MEF- 2Cの増幅には配列番号 55、 56の塩基配列を有する合成 DNA を、 MEF- 2Dの増幅には配列番号 57、 58の塩基配列を有する合成 DNAも、 MEF-2A の増幅には配列番号 59、 60の塩基配列を有する合成 DNAを用いた。  For the amplification of ANP, a synthetic DNA having the nucleotide sequence of SEQ ID NOs: 33 and 34, for the amplification of BNP, a synthetic DNA having the nucleotide sequences of SEQ ID NOS: 35 and 36, and for the amplification of MHC, SEQ ID NOs: 37 and 38 Synthetic DNA having the nucleotide sequence of SEQ ID NO: 39 or 40 for amplification of MHC, and synthetic DNA having the nucleotide sequence of SEQ ID NO: 41 or 42 for amplification of human skeletal actin For the amplification of? -Skeletal actin, synthetic DNA having the base sequence of SEQ ID NO: 43 or 44, for the amplification of MLC-2a, the synthetic DNA having the base sequence of SEQ ID NO: 45 or 46, and for the amplification of MLC-2v The synthetic DNA having the nucleotide sequence of SEQ ID NO: 47 or 48, the synthetic DNA having the nucleotide sequence of SEQ ID NO: 49 or 50 for amplification of Nkx2.5 / Csx, and the amplification of GATA4 by the DNA of SEQ ID NO: 51 or 52 Synthetic DNA having the nucleotide sequence of SEQ ID NOS: 53 and 54 for amplification of TEF-1 and amplification of MEF-2C for amplification of TEF-1 Synthetic DNAs having the nucleotide sequences of Nos. 55 and 56, MEF-2D for amplification of the synthetic DNAs having the nucleotide sequences of SEQ ID Nos. 57 and 58, and MEF-2A for the amplification of the nucleotide sequences of SEQ ID Nos. 59 and 60 Was used.
生体内で分化誘導する心筋細胞は、心筋収縮の心拍数またはエネルギー効率に違い を持たせるために、 胎児期、 新生児期あるいは成熟期によって、 または心房筋あるい は心室筋の相違によって、 心筋収縮蛋白質のァイソフォームに違いがある。  Cardiomyocytes that are induced to differentiate in vivo may contract in fetal, neonatal, or maturation phases, or in atrial or ventricular muscles to create differences in heart rate or energy efficiency of myocardial contraction. There are differences in protein isoforms.
培養系で心筋細胞に分化した骨髄細胞の場合、 アイソフォームの発現様式は ーァ クチンの場合は骨格筋型のほうが心筋型より多く発現し、 ミオシン重鎖の場合は/?型 のほうがひ型よりも多く発現していた。 ミオシン軽鎖では 2 V型が発現しているのに 対し、 2 a型の発現は観察されなかった。  In the case of bone marrow cells differentiated into cardiomyocytes in the culture system, the expression form of isoforms in actin is higher in skeletal muscle than in cardiac muscle, and in myosin heavy chain, the /? Was more frequently expressed. In myosin light chain, type 2V was expressed, whereas type 2a was not expressed.
また、 培養系で心筋細胞に分ィ匕した骨髄細胞の分化誘導後には、 ナトリウム利尿べ プチドである A N Pおよび B N Pの発現が見られた。以上の心筋収縮蛋白質の発現様 式より判断すると、培養系で心筋細胞に分化した骨髄細胞の表現型は胎児型心室筋細 胞の形質を有すると考えられる。  In addition, expression of natriuretic peptides, ANP and BNP, was observed after differentiation induction of bone marrow cells separated from cardiomyocytes in the culture system. Judging from the above expression of myocardial contractile protein, the phenotype of bone marrow cells differentiated into cardiomyocytes in the culture system is considered to have the characteristics of fetal ventricular myocytes.
培養系で心筋細胞に分ィ匕した骨髄細胞では、 Nkx2.5/Csx、 GATA4, MEF-2A, MEF-2C, MEF-2D, TEF- 1遺伝子の発現が観察された。 増殖中の骨髄由来初代不死化細胞株で はこれらの転写因子の発現は認められなかったが、増殖中の骨髄由来心筋前駆細胞で は Nkx2.5/Csx GATA4および MEF- 2Cの発現が観察され、 心筋細胞への分化誘導に 伴い、 遅れて MEF- 2Aおよび MEF- 2Dの発現誘導が観察された。 The bone marrow cells were separated I spoon into cardiomyocytes in culture systems, Nkx 2. 5 / Csx, GATA4, MEF-2A, MEF-2C, Expression of MEF-2D and TEF-1 genes was observed. Expression of these transcription factors in bone marrow-derived primary immortalized cell lines in proliferation was not observed, but the expression of Nkx2.5 / Csx GATA 4 and MEF-2 C in bone marrow-derived myocardial progenitor cells in proliferation It was observed, with the induction of differentiation into cardiac myocytes, induction of the expression of late MEF-2 a and MEF-2D were observed.
次に、 ガラス微少電極により、培養系で心筋細胞に分ィ匕した骨髄細胞の活動電位を 記録した。 活動電位は、 細胞を 1.49mM CaCl2、 4.23mM KCレ 25mM HEPES(pH7.4)を 添加した IMDM培地中で培養し、 Diaphoto- 300実体顕微鏡 (ニコン社製)下、 温度 25°C で測定した。ガラス電極は電極抵抗を 15〜30 Ωに設定して 3M KC1を充填した。膜電 位の測定は MEZ- 8300 (日本光電社製） を用いて電流クランプモードで行った。 測定 結果は RTA- 1100M (日本光電社製） を用いて熱感紙に記録した。 その結果、 培養系 で心筋細胞に分化した骨髄細胞は、洞結節細胞型と心室筋細胞型の 2種類が観察され た。両者に共通する活動電位の特徴は、 ①活動電位持続時間が長いこと、 ②比較的浅 い静止期電位を持つこと、③ペースメーカー細胞にみられる静止期電位の緩やかな脱 分極が認められることであった。 また、 心室筋細胞型では活動電位は Peak&Dome型 (活動電位第 1相を持つ） を呈した。洞結節細胞型の活動電位持続時間、 拡張期膜電 位、活動電位振幅は従来ゥサギゃラヅトで報告されている洞結節の活動電位と近似し ていた。心室筋細胞型ではこれに比べて、 静止期膜電位は深く、 活動電位振幅は大き い傾向を示した。分化誘導後、 2〜3週間の細胞はすべて洞結節細胞型が記録されたが、 分化誘導後 4週間頃より心室筋細胞型が観察され時間経過とともに次第に増加した。 Next, the action potential of the bone marrow cells separated into the cardiomyocytes in the culture system was recorded by a glass microelectrode. The action potential is measured at 25 ° C under a Diaphoto-300 stereo microscope (Nikon) by culturing cells in IMDM medium supplemented with 1.49 mM CaCl 2 , 4.23 mM KC, 25 mM HEPES (pH 7.4). did. The glass electrode was filled with 3M KC1 with the electrode resistance set to 15 to 30Ω. The membrane potential was measured in a current clamp mode using MEZ-8300 (manufactured by Nihon Kohden Corporation). The measurement results were recorded on thermal paper using RTA-1100M (manufactured by Nihon Kohden Corporation). As a result, two types of bone marrow cells differentiated into cardiomyocytes in the culture system were observed: sinus node cell type and ventricular myocyte type. The characteristics of action potentials common to both are: (1) a long action potential duration, (2) a relatively shallow resting potential, and (3) a gradual depolarization of the resting potential seen in pacemaker cells. there were. In the ventricular myocyte cell type, the action potential exhibited a peak & dome type (having the first phase of action potential). The action potential duration, diastolic membrane potential, and action potential amplitude of the sinus node cell type were similar to those of the sinus node reported in the past. In the ventricular myocyte cell type, the resting membrane potential tended to be deep and the action potential amplitude tended to be large. After induction of differentiation, the cells of the sinus node cell type were recorded in all the cells for 2 to 3 weeks, but the ventricular myocyte type was observed from about 4 weeks after the induction of differentiation, and gradually increased over time.
実施例 3 . サイトカインを用いた心筋細胞への分ィ匕の促進  Example 3. Promotion of shunting to cardiomyocytes using cytokines
心筋細胞への分化能を有するマウス骨髄細胞の心筋分化誘導率を増加させるため、 5- aza - Cで分化誘導をおこなう際に、 各種サイトカインを添加して誘導率が増加する かどうか角军析をおこなった。  In order to increase the induction rate of myocardial differentiation in mouse bone marrow cells that have the ability to differentiate into cardiomyocytes, it was analyzed whether addition of various cytokines would increase the induction rate when inducing differentiation with 5-aza-C. Was done.
心筋細胞への分ィ匕能を有するマウス骨髄細胞を 2 X 104細胞/ mlとなるように eOmm 培養デイツシュあるいは 60mmフイブロネクチン付着デイツシュ (fibronectin- coated dish:Becton Dickinson社製） に蒔き、 33°C、 5%C02濃度の孵卵機を用いて培養を行つ た。 Mouse bone marrow cells having the ability to divide into cardiomyocytes were seeded at 2 × 10 4 cells / ml on eOmm culture dish or 60 mm fibronectin-coated dish (Fibronectin-coated dish: Becton Dickinson), and 33 ° C, having conducted a cultured using 5% C0 2 concentration in the incubator unit.
翌日、 該培養液に 5 - aza-Cを終濃度 3 z Mとなるよう添加した上で、 更に、 PDGF のみ、 PDGFとレチノイン酸の両方、添カロなし、の 3種類の群で培地中に添カロした（終 濃度は PDGFは 10ng/ml、 レチノィン酸は 10— 9M) 。 On the next day, 5-aza-C was added to the culture solution to a final concentration of 3 zM. Only, or both PDGF and retinoic acid, without added Caro was added Caro in the medium in three groups of (final concentration PDGF is 10 ng / ml, Rechinoin acid 10- 9 M).
それから更に 2日後、 4日後にも培地交換した後、 PDGFのみ、 PDGFとレチノィ ン酸の両方、 添加なしの 3群にそれぞれ最初と同量を添加した。  After 2 or 4 days, the medium was exchanged again, and the same amount as the first group was added to each of the three groups without PDGF alone, both PDGF and retinoic acid, and no addition.
薬剤を加えてから 4週間後、 細胞の形態を位相差顕微鏡下で観察した。 その結果、 5-aza-Cのみを添加した培養デイツシュでは約 3割の細胞が筋管様細胞となるのに対 し、 PDGFを添加すると約 4割、 PDGFとレチノイン酸を同時に添加すると約 5割の 細胞が筋管様細胞となった。 また、 フイブロネクチン付着ディッシュの 3群では、 培 養ディッシュの 3群に比べて、 筋管様細胞になる細胞数が約 1割程度ずつ増加した。  Four weeks after adding the drug, the morphology of the cells was observed under a phase-contrast microscope. As a result, about 30% of cells in cultured tissue containing only 5-aza-C became myotube-like cells, whereas about 40% when PDGF was added, and about 50% when PDGF and retinoic acid were added simultaneously. Some cells became myotube-like cells. In addition, the number of cells that became myotube-like cells increased by about 10% in the three groups with the fibronectin-attached dish compared to the three groups with the culture dish.
実施例 4 . DMSQを用いた骨髄由来幹細胞からの心筋細胞への分化誘導  Example 4. Induction of differentiation of bone marrow-derived stem cells into cardiomyocytes using DMSQ
実施例 1に示した方法により、 取得した心筋細胞への分化能のある骨髄幹細胞に 3 〃 Mの 5- aza- Cの代わりに 10〃 Mの DMSOを添加し 2 4時間培養した後、 培地を IMDM培地に代えて、 さらに 6週間培養を続けた。 According to the method described in Example 1, 10 μM DMSO was added to the obtained bone marrow stem cells capable of differentiating into cardiomyocytes instead of 3 μM 5 -aza-C, and the cells were cultured for 24 hours. Was replaced with IMDM medium, and the culture was continued for another 6 weeks.
その結果、 拍動する心筋細胞が分化誘導されることを見出し、 これらの細胞には Nkx2.5/Csxおよび GATA4遺伝子が発現しており、 5 - aza - Cを添加したときと同様の 性質を有した心筋細胞であることが示された。 この解析結果は、 5- aza - Cと DMSOの 共通の機能である染色体 DNAの脱メチル化が心筋細胞の分化に必要であることを示 している。  As a result, they found that beating cardiomyocytes were induced to differentiate, and these cells expressed Nkx2.5 / Csx and GATA4 genes, and exhibited the same properties as when 5-aza-C was added. It was shown to be a cardiomyocyte that had. The results of this analysis indicate that chromosomal DNA demethylation, a common function of 5-aza-C and DMSO, is required for cardiomyocyte differentiation.
実施例 5 .心筋細胞への分ィ匕能を有するマウス骨髄細胞が多分化能を有する幹細胞 および心筋前駆細胞であることの証明 .  Example 5: Demonstration that mouse bone marrow cells capable of dividing into cardiomyocytes are multipotent stem cells and cardiomyocyte precursor cells.
マウス骨髄細胞から分化誘導する拍動細胞が心筋細胞の性質を保有していること は示されたが、 心筋細胞への分化能を有するマウス骨髄細胞に、 心筋前駆細胞が存在 しているのか、 もつと未分化で心筋細胞以外の、例えば脂肪細胞などに分ィ匕可能な幹 細胞が存在するかを調べるため、 シングルセル ·マ一キング（Single cell marking)の実 験を行った。  It was shown that the pulsatile cells induced to differentiate from mouse bone marrow cells possess the properties of cardiomyocytes, but do cardiomyocyte progenitors exist in mouse bone marrow cells that have the ability to differentiate into cardiomyocytes? In order to investigate whether undifferentiated stem cells other than cardiomyocytes, such as adipocytes, exist, a single cell marking experiment was performed.
具体的には、 分化誘導を行う前に、 ある 1つの細胞に GFP遺伝子をウィルスべク 夕一を導入して標識し、その後分化誘導させて標識した細胞がどのような細胞に分化 したかで判断した。 まず、 GFP遺伝子を発現させるレトロウイルスベクタ一プラスミド GAR3-GFPお よび、 Ecotropic遺伝子を発現させる pCMV- Ecoプラスミドベクタ一を、 Molecular Cionmg, A Laboratory Manual, second Edition, Cold Spring Harbor Laooratory Press (1989) 等に記載のアル力リ中和法および PEG沈殿法を用いて、 純度の高い DNAを取得した c この DNAをトランスフエクシヨンさせる前日に、 コンフルェン卜になった、 gagお よび pol遺伝子を保有する 293細胞を 1/5希釈で 10cmディヅシュに継代し、一晚 37°C；、 5%C02濃度の孵卵機を用いて培養をおこなった。 Specifically, before inducing differentiation, one cell is labeled by introducing the GFP gene into the virus vector, and then differentiation is induced to determine the type of cells that the labeled cell has differentiated into. It was judged. First, a retrovirus vector plasmid expressing the GFP gene, GAR3-GFP, and a pCMV-Eco plasmid vector expressing the Ecotropic gene were obtained from Molecular Cionmg, A Laboratory Manual, second Edition, Cold Spring Harbor Laooratory Press (1989), etc. Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Section c. The day before transfection of the DNA, the confluent gag and pol genes were retained. The cells were subcultured to a 10 cm dish at a dilution of 1/5, and cultured using an incubator having a concentration of 37 ° C. and 5% CO 2 .
トランスフエクシヨンは以下の通り 行った。  The transfection was conducted as follows.
GAR3-GFPレトロウイルスベクタ一プラスミド DNA 1 5〃 gと pCMV- Ecoプラス ミドベクタ一 DNA 5〃 gを 250mM CaCl2 (pH6.95) 0.5mlに加えて溶解させ、 その溶液 を 15mlのチューブに入れた 2 X BBS [50mM BES(N，N- bis(2- hydroxyethl)- 2 - aminoethanesulfonic acid)、 280mM NaClヽ 1.5mM Na2HP04(pH6.95)] 0.5mlに滴下して 10 分間室温で静置させた。 その後、 この DNA溶液を、 前日に用意した 293細胞培地中 に滴下させ、 37°C、 ' 5%C02濃度の孵卵機を用いて培養を行った。 翌日、 培地を交換し、 更に 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 GAR3-GFP retroviral vector one plasmid DNA 1 5〃 g and pCMV-Eco plus Midobekuta one DNA 5〃 g dissolved in addition to 250mM CaCl 2 (pH6.95) 0.5ml, was charged the solution to 15ml tubes 2 X BBS [50mM BES (N , N- bis (2- hydroxyethl) - 2 - aminoethanesulfonic acid), 280mM NaClヽ 1.5mM Na 2 HP0 4 (pH6.95) ] was added dropwise to 0.5ml static at room temperature for 10 minutes Was placed. Thereafter, the DNA solution was added dropwise to 293 in cell culture medium were prepared the day before, were incubated with 37 ° C, '5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured further using a 37 ° C, 5% C0 2 concentration in the incubator unit.
培地を交換して 2日後に、培養上清を 0.45〃 mのフィル夕一（Millipore社製）でろ 過し、 ウィルスベクターを含む溶液を回収した。 この溶液を IMDM培地で 10— 10一2、Two days after the medium was replaced, the culture supernatant was filtered through a 0.45 μm filter (Millipore) to recover a solution containing the virus vector. The solution in IMDM medium 10-10 one 2,
10一3、 10—4ヽ 10— 5に希釈した >。 10 one 3, was diluted to 10-4ヽ10- 5>.
ウィルスぺク夕一を導入される側の心筋細胞への分化能を有するマウス骨髄細胞 は、ウィルスをインフエクシヨンさせる前日に 2 X 104細胞/ゥエルとなるように 6ゥ エル ·ディヅシュに蒔いた。 Mouse bone marrow cells capable of differentiating into cardiomyocytes to which the virus is introduced are seeded on a 6-well dish at 2 × 10 4 cells / well on the day before virus influx. Was.
希釈した、 ウィルスベクタ一を含む溶液には、 終濃度 8〃g/mlとなるように、  For the diluted solution containing the virus vector, add a final concentration of 8 μg / ml.
Hexadimethrine bromide(polybrene) ( S i gm a社製） を添加し、 心筋細胞への分化能 を有するマウス骨髄細胞の培養上清 2 m lをウィルス液 2mlと置換し、 33°C, 5%C02 濃度の孵卵機を用いて培養をおこなった。 5時間後、培養上清を新しい IMDM培地に 交換し、 更に 33° 5%C02濃度の孵卵機を用いて培養を行った。 Was added Hexadimethrine bromide (polybrene) (S i gm a Corporation), the culture supernatant a 2 ml murine bone marrow cells with ability to differentiate into cardiomyocytes and replaced with virus solution 2ml, 33 ° C, 5% C0 2 Culture was carried out using an incubator having a concentration. After 5 hours, replacing the culture supernatant to a new IMDM medium were incubated with further 33 ° 5% C0 2 concentration in the incubator unit.
2日間培養を行った後、 蛍光顕微鏡下で GFPを発現している細胞を観察し、 細胞 1000個あたり 1つの GFP陽性細胞があるような細胞群を得た。 ' 該細胞を 8 X 103細胞/ディヅシュとなるよう、 35mmガラスベースディヅシュ （旭 テクノグラス社製） に蒔き、 33°C、5%C02濃度の孵卵機を用いて培養を行った。 After culturing for 2 days, cells expressing GFP were observed under a fluorescence microscope, and a cell group having one GFP-positive cell per 1000 cells was obtained. 'The cells so as to be 8 X 10 3 cells / Didzushu were plated 35 mm glass base di Uz Gerhard (manufactured by Asahi Techno Glass), and cultured using a 33 ° C, 5% C0 2 concentration incubator machine Was.
翌日、 5- aza - C (Sigma社製） 、 PDGF-BB (Peprotech社製） 、 all transレチノイン 酸 （Sigma社製） をそれぞれ終濃度 3〃 M、 10ng/ml, 10—gMとなるよう添カ卩し、 添カロ .して 2日後および 4日後には、培地交換を行うとともに、再度 PDGF— B B (以降 PDGF と略す） 、 all transレチノイン酸を上述と同じ濃度で添加した。 The next day, 5-aza-C (Sigma), PDGF-BB (Peprotech), and all trans retinoic acid (Sigma) were adjusted to a final concentration of 3 M, 10 ng / ml, and 10— g M, respectively. Two days and four days after the addition, the medium was replaced, and PDGF-BB (hereinafter abbreviated as PDGF) and all trans retinoic acid were added again at the same concentrations as described above.
4週間後、 蛍光顕微鏡で GFP陽性細胞がどのように分化したかを観察すると、 心 筋細胞のみが GFP陽性になっている細胞集団、 心筋細胞と未分化幹細胞が GFP陽性 になっている細胞集団、 ならびに心筋細胞、 脂肪細胞および未分ィ匕幹細胞の 3者が GFP陽性になっている細胞集団の 3種類の細胞集団が見られた。すなわち、多分化能 幹細胞から心筋幹細胞さらに心筋前駆細胞へと確率的（stochastic)に誘導してくるこ とが明らかとなった。 またこの結果は、 心筋細胞への分化能を有するマウス骨髄細胞 には多分化能をもつ幹細胞が存在することを示している。  Four weeks later, observation of how the GFP-positive cells differentiated by fluorescence microscopy revealed that only the myocardial cells were GFP-positive, and that myocardial cells and undifferentiated stem cells were GFP-positive , And cardiomyocytes, adipocytes and undivided stem cells were found to be GFP-positive. In other words, it was clarified that pluripotent stem cells are induced stochastically into cardiac stem cells and cardiac progenitor cells. This result also indicates that pluripotent stem cells exist in mouse bone marrow cells capable of differentiating into cardiomyocytes.
実施例 6 . 転写因子の強制発現による心筋細胞分化の促進  Example 6. Promotion of cardiomyocyte differentiation by forced expression of transcription factor
マウス心筋細胞への分化能を有する骨髄細胞に心筋細胞分化に関係する転写因子 を強制的に発現させることによる心筋細胞への分ィヒに与える影響を角牟析した。  The effect of forcibly expressing a transcription factor associated with cardiomyocyte differentiation on bone marrow cells capable of differentiating into mouse cardiomyocytes was examined for the effect on cardiomyocyte differentiation.
具体的には、 分化誘導を行う前に、 Nkx2.5/Csxまたは GATA4遺伝子をウィルスべ クタ一を用いて導入して、その後分化誘導させて心筋細胞への分化の効率を検討した まず、 Nkx2.5/Csxと GATA4の遺伝子を発現させる目的で、 レトロウイルスベクタ 一プラスミド pCLNCX (Imgenex社)に Nkx2.5/Csxと GATA4をそれぞれ組み込み、 PCLNC-Nkx2.5/Csxと pCLNC- GATA4を調製した。レトロウィルスベクタ一プラスミ ド PCLNC-Nkx2.5/Csxと pCLNC- GATA4および、 Ecotropic遺伝子を発現させる pCMV-Ecoプラスミドべク夕一 (Imgenex不ェ) ¾、 Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)等に記載のアル力リ中和法お よび PEG沈殿法を用いて、 純度の高い DNAを取得した。 Specifically, prior to inducing differentiation, the Nkx2.5 / Csx or GATA4 gene was introduced using a viral vector, and then differentiation was induced to examine the efficiency of differentiation into cardiomyocytes. in .5 / Csx and purpose of expressing a gene of GATA4, the retroviral vector one plasmid pCLNCX (Imgenex Corp.) incorporation Nkx2.5 / Csx and GATA4 respectively, prepared P CLNC-Nkx2.5 / Csx and PCLNC- GATA4 did. Retroviral vector one plasmid P CLNC-Nkx2.5 / Csx and PCLNC- GATA4 and, pCMV-Eco plasmid base click evening one to express Ecotropic gene (Imgenex Fe) ¾, Molecular Cloning, A Laboratory Manual, Second Edition, Highly pure DNA was obtained using the neutralization method and the PEG precipitation method described in Cold Spring Harbor Laboratory Press (1989) and the like.
これらの DNAをトランスフエクシヨンさせる前日に、 コンフルェントになった、 gagおよび pol遺伝子を保有する 293細胞を I/5希釈で 10cmディヅシュに継代し、一 晚 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 トランスフヱクシヨンは以下の通りにおこなった。 These DNA the day prior to transflector Ekushi Yong became Konfuruento, two 93 cells carrying the gag and pol genes and passaged 10cm Didzushu in I / 5 dilution, One晚37 ° C, 5% C0 2 concentration Was cultured using an incubator. The transfection was performed as follows.
pCLNC— Nkx2.5/Csxあるいは pCLNC— GATA4レトロウィルスベクタ一プラスミド DNA15〃gと pCMV - Ecoプラスミドベクタ一 DNA 5〃 gを 250mMCaCl2 (pH6.95) 0.5ml に加えて溶解させ、 その溶液を 15mlのチューブに入れた 2 X BBS [50m BES(N，N- bis(2-hydroxyethl)-2-aminoethanesulfonic acid), 280mM NaCl、 1.5mM Na2HP04(pH6.95)] 0.5mlに滴下して 10分間室温で静置させた。 その後、 この DNA溶液を、 前日に用意 した 2Θ3細胞培地中に滴下させ、 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 翌日、 培地を交換し、 更に 37°C、 5%C02濃度の孵卵機を用いて培養を行った。 pCLNC—Nkx2.5 / Csx or pCLNC—GATA4 15 〃g of retrovirus vector DNA and 5 〃g of pCMV-Eco plasmid vector DNA are added to 0.5 ml of 250 mM CaCl 2 (pH6.95) and dissolved. 2 X BBS [50m BES (N , N- bis (2-hydroxyethl) -2-aminoethanesulfonic acid), 280mM NaCl, 1.5mM Na 2 HP0 4 (pH6.95)] were placed in a tube was dropped into 0.5ml It was left at room temperature for 10 minutes. Thereafter, the DNA solution was added dropwise to 2Θ3 in cell medium were prepared the day before, were incubated with 37 ° C, 5% C0 2 concentration in the incubator unit. The next day, the medium was changed, were cultured with further 3 7 ° C, 5% C0 2 concentration in the incubator unit.
培地を交換して 2日後に、培養上清を 0,45〃 mのフィル夕一（Millipore社製）でろ 過し、 ウィルスベクタ一を含む溶液を回収した。 - ウィルスベクタ一を導入される側の心筋細胞への分化能を有するマウス骨髄細胞 は、ウィルスをインフエクシヨンさせる前日に 2 X 104細胞/ゥエルとなるように 6ゥ エル ·ディヅシュに蒔いておいた。 2 days later the medium was changed to a culture supernatant 0, 45 〃 fill evening (manufactured by Millipore Co.) one m Delo spent to recover a solution containing viral vectors scratch. -Mouse bone marrow cells capable of differentiating into the cardiomyocyte to which the virus vector is introduced are seeded on a 6 ゥ dish at 2 × 10 4 cells / ゥ on the day before virus infection. Oita.
上記で取得したウィルスベクターを含む溶液に、 終濃度 8〃g/mlとなるように、 Hexadimethrine bromide (polybrene) (Sigma社製） を添加し、 心筋細胞への分化能を有す るマウス骨髄細胞の培地と置換し、 33°C、 5%C02濃度の孵卵機を用いて培養を行った。 5時間後、新しい MDM培地に交換し、更に 33°C、 5%C〇2濃度の孵卵機を用いて培養 ¾ί了った。 Hexadimethrine bromide (polybrene) (Sigma) was added to the solution containing the virus vector obtained above to a final concentration of 8 μg / ml, and mouse bone marrow cells capable of differentiating into cardiomyocytes were added. And the culture was performed using an incubator at 33 ° C. and 5% CO 2 concentration. After 5 hours, and replaced with a new MDM medium was cultured ¾ί Ryo' with further 3 3 ° C, 5% C_〇 2 concentration of incubation machine.
2日間培養を行った後、 C Sを終濃度 300 g/mlになるように添加し、 さらに 3 日間培養した。 この間に一部の細胞は死滅して浮遊した。生き残った細胞をトリプシ ンで浮遊させ、 新しい培養皿に播種した。  After culturing for 2 days, CS was added to a final concentration of 300 g / ml, and culturing was further performed for 3 days. During this time, some cells died and floated. The surviving cells were suspended in trypsin and seeded on new culture dishes.
このようにして、取得した Nkx2.5/Csxあるいは GATA4の安定形質転換細胞につい て、 上記実施例 3の方法により分化誘導を行い、 心筋細胞への分化の効率を検定した。 その結果、 これら転写因子の強制発現により、 心筋細胞への分化の効率が促進された ことが分かった。  The thus obtained Nkx2.5 / Csx or GATA4 stably transformed cells were induced to differentiate by the method of Example 3 above, and the efficiency of cardiomyocyte differentiation was assayed. As a result, it was found that the forced expression of these transcription factors promoted the efficiency of differentiation into cardiomyocytes.
実施例 7 . 心筋細胞への分化能を有するマウス骨髄細胞でのテロメラ一ゼ活性 心筋細胞への分ィ匕能を有するマウス骨髄細胞のテ口メラーゼ活性は Telomeric Repeat Amplification Protocol(TRAP)法により検討した ( Oncor社製 TRAPeze Telomerase Detection Kit) 。 テロメラーゼ活性の測定は原則的に添付されていたプロ トコールに従ったが、 具体的には以下の通りに行った。 まず、 6cm径の培養皿上で培 養した心筋細胞への分化能を有するマウス骨髄細胞 （およそ 106個） を PBSで洗浄し た後、 の 1 X CHAPS液を加え、 氷上で 30分間静置した。 その後、 溶液と共 に細胞を l.5ml容遠沈管に回収し、 14，000rpmで 20分間遠心分離 (4°C、 HITACHI社製 himacCF15)し、 上清を細胞抽出液として回収した。 Protein assay (BioRad社製） を用 いて蛋白質含有量を測定したところ、上記条件で取得した心筋細胞への分化能を有す るマウス骨髄細胞の細胞抽出液はおよそ lmg/mlであった。 Example 7. Telomerase activity in mouse bone marrow cells capable of differentiating into cardiomyocytes Teguchi-merase activity of mouse bone marrow cells capable of dividing into cardiomyocytes was examined by Telomeric Repeat Amplification Protocol (TRAP) method (Oncor's TRAPeze Telomerase Detection Kit). The measurement of telomerase activity basically followed the attached protocol, and was specifically performed as follows. First, mouse bone marrow cells (approximately 10 6 ) capable of differentiating into cardiomyocytes cultured on a 6 cm diameter culture dish were washed with PBS, added with 1X CHAPS solution, and allowed to stand on ice for 30 minutes. Was placed. Thereafter, the cells in a solution and the co-harvested l. 5 ml volume centrifuge tubes, centrifuged for 20 minutes at 14,000rpm (4 ° C, HITACHI Co. HimacCF15), and the supernatant was collected as a cell extract. When the protein content was measured using a protein assay (manufactured by BioRad), the cell extract of mouse bone marrow cells capable of differentiating into cardiomyocytes obtained under the above conditions was approximately 1 mg / ml.
次にこの細胞抽出液を用いて、 プロトコールに従ってテロメァ伸長反応及び PCR 増幅を行った。 Taqポリメラーゼは EX Taq polymerase (宝酒造製） を用いた。反応終 了後の試料は 10 X染色液 （0.25%bromophenol blue, 0.25%Xylene cyanol FF, 30% glycerol) を 1/10量添加し、 12.5%ポリアクリルアミドゲル (TRAPeze Telomerase Detection Kit のプロトコールに記載されている通り調製） に載せ、 250mV定電圧下で泳動した。泳 動後、 ゲルをサイバーグリーン （FMC社製） で染色し、 蛍光色素分析装置、  Next, using this cell extract, telomere extension reaction and PCR amplification were performed according to the protocol. Taq polymerase used was EX Taq polymerase (Takara Shuzo). After the reaction is completed, 1/10 volume of 10X staining solution (0.25% bromophenol blue, 0.25% Xylene cyanol FF, 30% glycerol) is added, and 12.5% polyacrylamide gel (TRAPeze Telomerase Detection Kit protocol) And electrophoresed under a constant voltage of 250 mV. After swimming, the gel was stained with Cyber Green (manufactured by FMC).
FluoroImager(Molecular Dynamics社製)を用 、て解析した。 その結果、 細胞抽出液の終 濃度が 0.4〜4 ju g/mlの試料でテロメラーゼ活性が検出された。 Analysis was performed using FluoroImager (Molecular Dynamics). As a result, telomerase activity was detected in a sample having a final concentration of the cell extract of 0.4 to 4 jug / ml.
実施例 8 . 心筋細胞への分化能を有するマウス骨髄細胞の心臓への移植 .  Example 8. Transplantation of mouse bone marrow cells capable of differentiating into cardiomyocytes into the heart.
心筋細胞への分化能を有するマウス骨髄細胞をマウス心臓に移植するために、 まず C3H/Heマウス （日本チャールズリバ一社製） に、 ェ一テルを用いて麻酔の導入を行 い、 テルモ製のテルモシリンジ （1ml) を用いてチォペンタール 30mgの腹腔内投与 することで麻酔の維持を行った。マウスの四肢をテープでコルク板に固定し、 さらに 首が反り返るように上顎をゴムでコルク板に固定した。この時点 、左右の上肢及び右 下肢に心電図電極を刺入し心電図のモニタリングを行った。 続いて、 メ一ョ剪刀 (N〇NAKA RIKAKI CO.，LTD NK- 174 - 14)で頸部を気管にそって 1 cmほど切開し、 白十 字社のベビー綿棒で甲状腺を左右に剥離をし、 気管周囲の筋肉をマイクロ剪刀 (NONAKA RIKAKI CO., LTD NY- 334- 08)で切開し気管を露出した。 ついでマイクロフ ェザー（メス）で気管を 1mmほど切開しここから J型に変型させたテルモ製サーフロ —フラッシュ （22G) の針を挿入し口腔から外に出し、 この針をガイドにサ一フロー フラッシュ （ G) の外筒を気管内に挿入した。 この外筒にレスピレ一夕 （シナノ製 作所製の MODEL SN-480-7)をつなぎ 100パーセント酸素を 1ml/分で流し、一回換気 量は lml、 呼吸回数は 120/分で人工呼吸を開始した。 このときにガイド針を揷入した 穴からエアーがもれるので気管周囲の皮膚をモスキ一ト鉗子 (NONAKA RIKA I CO.，LTD) を用いて気管をおおうようにして閉鎖した。 つぎに、 胸骨柄より頸部に向 かい 2cmほどメーョ剪刀で切開、ついで胸骨を 2cmほど胸骨柄から類部に向かい切開 をした。 出血をバイポーラの電気メスで止血し、 テルモ製のテルモシリンジ （1ml) にジーエルサイエンス社製の30Gの針 （メタルハプ交換針 N730) をつけて心尖部に 実施例 1で得た心筋細胞への分化能を有するマウス骨髄細胞を PBSに浮遊した液体 を 0.1ml注入した。ついで ETHICON社製の 4-0 ETHIBOND X761を用いて胸骨の閉鎖、 皮膚の閉鎖を行い、 同じ針糸で頸部の皮膚の閉鎖をした。 自発呼吸の出現を確認しレ スピレー夕をはずしィンファントウォーマ一を 37°Cに加熱しこの中で覚醒を待った。 なお本実験の操作は DESIGN FOR VISON 4.5 X SURGICAL TELESCOPESを用いて行 つた。 In order to transplant mouse bone marrow cells capable of differentiating into cardiomyocytes into the mouse heart, anesthesia was first introduced into C3H / He mice (Charles River Japan, Inc.) using ether and Terumo Anesthesia was maintained by intraperitoneal administration of 30 mg of thiopental using a thermosyringe (1 ml). The limbs of the mouse were fixed to the cork board with tape, and the upper jaw was fixed to the cork board with rubber so that the neck would bend back. At this time, electrocardiogram electrodes were inserted into the left and right upper limbs and the right lower limb to monitor the electrocardiogram. Next, a 1 cm incision was made along the trachea along the trachea with a macho scissor (N〇NAKA RIKAKI CO., LTD NK-174-14), and the thyroid gland was exfoliated to the left and right with a white cotton swab. Then, the muscle around the trachea was incised with a micro-scissor (NONAKA RIKAKI CO., LTD NY-334-08) to expose the trachea. Then, the trachea was incised about 1 mm with a microfeather (female), and a Terumo surfro-flash (22G) needle transformed into a J-shape was inserted out of the mouth and taken out of the oral cavity. The outer tube of the flash (G) was inserted into the trachea. A respire overnight (model SN-480-7 manufactured by Shinano Seisakusho) was connected to this outer cylinder, and 100% oxygen was flowed at 1 ml / min. The tidal volume was 1 ml, and the respiration rate was 120 / min. Started. At this time, since air leaked from the hole into which the guide needle was inserted, the skin around the trachea was closed using mosquito forceps (NONAKA RIKA I CO., LTD) to cover the trachea. Next, an incision was made with a Mayo scissor about 2 cm from the sternum handle toward the neck, and then an incision was made about 2 cm from the sternum handle toward the analogous part. The bleeding hemostasis bipolar electrocautery, Terumo Terumo syringe (1 ml) to GL Sciences Inc. of 3 0G needle cardiomyocytes obtained in (Metaruhapu exchange needle N 73 0) with the apex in Example 1 0.1 ml of a liquid in which mouse bone marrow cells capable of differentiating into cells were suspended in PBS was injected. Then, the sternum and the skin were closed using 4-0 ETHIBOND X761 manufactured by ETHICON, and the neck skin was closed with the same needle thread. After confirming the appearance of spontaneous breathing, the respiratory evening was removed and the infant warmer was heated to 37 ° C and awakened in this. The operation of this experiment was performed using DESIGN FOR VISON 4.5 X SURGICAL TELESCOPES.
移植した心筋細胞への分ィ匕能を有するマウス骨髄細胞が心筋に定着したことは、被 移植マウスの心臓を摘出後、組織切片を作製し、 GFPを認識する抗体で染色すること により確認した。  It was confirmed that mouse bone marrow cells capable of dividing the transplanted cardiomyocytes were established in the myocardium by extracting the heart of the transplanted mouse, preparing a tissue section, and staining with a GFP-recognizing antibody. .
実施例 9 . ラット骨髄からの心筋細胞への分化能を有する骨髄細胞の取得と培養 5週齢の Wistar rat (日本 SLC株式会社)雌 6匹を頸椎脱日した後、 70%エタノ一ルを 充分かけ消毒した。次に足部の皮膚を広範囲に渡り切開し、 大腿骨や脛骨を覆う筋肉 を切除しながら、 大腿骨と脛骨を取り出した。 取り出した大腿骨と脛骨は PBS (GibcoBRL社製） の入った 10cm径培養皿 (岩城硝子社製)に移し、 筋肉及び関節を完 全に切除した。 続いてこれらの骨の両端をハサミで切り、 20G注射針を付けた 10ml 用注射器（テルモ社製） を用いて、 培養液（D_PBS、 GibclBRL社製） の水流で骨髄中 の内容物を押し出した。取得した細胞塊はさらに注射器を通して一様になるようにほ ぐした。このようにして得た細胞浮遊液は 50ml容遠沈管（BECTON DICKINSON社製） に回収し、 l，500rpmで 10分間遠心分離し （TOMY社製低速遠心機） 、 沈殿した細胞 を Smlの D- PBS中に懸濁した。改良ノィバウエル型血球計算盤にて細胞数を計測した ところ、 回収した細胞は合計 2.6 X 109個であった。大腿骨または脛骨 1本当たりから 1 X 108個の細胞を回収したことになる。 回収した細胞は lml当たり 1.3 X 108個の濃度 になるよう希釈し、 50ml容遠沈管に入った 1.073g/mlに調製された Percoll(Amersham Pharmacia Biotech社製)/ D- PBS液（ ml)上に ¾nl重層した後、室温で 3，100rpmで；30 分間遠心分離した。遠心分離後、 Percoll液と細胞浮遊液との界面より細胞を回収し、 . D- PBSで 4倍に希釈した後、 2300rpmで 10分間遠心分離し、 分画した細胞集団を回 収した。 回収した細胞は 20%FCS、 100 j g/ml penicillin, 250 ng/ml streptomycin, 85 I g/ml amphotericin (GibcoBRL社製） を含む IMDM培地 （GibcoBRL社製） に懸濁し た。 この時点で再度細胞数を計測したところ、 回収した骨髄由来細胞は合計 4J X 107 個あり、 処理前の細胞の約 2%相当を回収したことになる。 このようにして分画した 骨髄由来細胞は 2〜5 X 105個 /cm2になるように 10cm径の動物細胞用の培養皿 （岩城 硝子社製、以下 10cm培養皿と略す） 3枚に撒き、 C02培養器（夕バイ社製）にて 33°C、 5%C02濃度で培養を開始した。培地は ¾時間後、 72時間後にそれぞれ半分交換した。 その 3〜4日後に培地を半分交換した。 I5日経過し、コロニーが密集してきたので、 細胞をトリプシン EDTA処理ではがし、 2/3は 4mlの保存液 （10%DMSO、 50%の骨髄 由来細胞培養上清、 4096の未使用上記培地） に懸濁し、 2ml容チューブ（住友べ一クラ ィト社製） に 1本当たり lml分注して凍結保存し、 残り I/3は 10cm培養皿 2枚に蒔 き直し継代した。 Example 9. Acquisition and culture of bone marrow cells capable of differentiating into cardiomyocytes from rat bone marrow After removing 6 female 5-week-old Wistar rats (Japan SLC Co., Ltd.) from the cervical spine, 70% ethanol was removed. It was disinfected enough. Next, a large incision was made in the skin of the foot, and the femur and tibia were removed while removing the muscle covering the femur and tibia. The removed femur and tibia were transferred to a 10 cm diameter culture dish (manufactured by Iwaki Glass) containing PBS (manufactured by GibcoBRL), and the muscles and joints were completely resected. Subsequently, both ends of these bones were cut with scissors, and the contents of the bone marrow were extruded with a stream of culture solution (D_PBS, GibclBRL) using a 10 ml syringe (Termo) with a 20G injection needle. . The obtained cell mass was further loosened through a syringe. Thus obtained cell suspension was collected in 5 0 ml volumes centrifuge tube (BECTON DICKINSON Co., Ltd.), l, 5 were centrifuged for 10 minutes at rpm (TOMY Co. slow centrifuge), the precipitated cells of Sml It was suspended in D-PBS. Cell count was measured with a modified Neubauer hemocytometer. However, the total number of recovered cells was 2.6 × 10 9 . This means that 1 × 10 8 cells were collected from one femur or tibia. The collected cells were diluted to a concentration of 1.3 × 10 8 cells / ml, and Percoll (manufactured by Amersham Pharmacia Biotech) / D-PBS solution (ml) adjusted to 1.073 g / ml in a 50 ml centrifuge tube After overlaying on top, centrifuged at room temperature at 3,100 rpm; for 30 minutes. After centrifugation, the cells were collected from the interface between the Percoll solution and cell suspension. After diluted 4-fold with D-PBS, 23 were centrifuged 10 minutes at rpm, was fractionated cell populations times Carabid. The collected cells were suspended in an IMDM medium (GibcoBRL) containing 20% FCS, 100 jg / ml penicillin, 250 ng / ml streptomycin, and 85 Ig / ml amphotericin (GibcoBRL). At this time, the number of cells was counted again. As a result, a total of 4J × 10 7 bone marrow-derived cells were recovered, which means that about 2% of the cells before the treatment were recovered. The bone marrow-derived cells thus fractionated into 3 culture dishes for animal cells with a diameter of 10 cm (Iwaki Glass, hereinafter abbreviated as 10 cm culture dishes) so as to have a size of 2 to 5 x 10 5 cells / cm 2. plated, and culturing was started at 3 3 ° C, 5% C0 2 concentration in C0 2 incubator (evening by Co.). The medium was changed half after ¾ hour and after 72 hours. Three to four days later, the medium was changed half. Elapsed I 5 days, because colonies have dense, the cells were detached with trypsin EDTA treatment, 2/3 storage solution (10% DMSO, 50% of bone marrow-derived cell culture supernatant 4 ml, unused above 4096 The medium was suspended in a 2 ml tube (manufactured by Sumitomo Beikurato Co., Ltd.), and the mixture was cryopreserved, and the remaining I / 3 was replated on two 10 cm culture dishes and subcultured.
実施例 1 0 . ラット骨髄由来細胞の心筋細胞への分化能の検討  Example 10: Examination of differentiation ability of rat bone marrow-derived cells into cardiomyocytes
上記で継代したラット骨髄由来細胞は密集したところを再度トリプシン EDTA処 理ではがし、 6ゥエルプレート （BECTON DICKINSON社製） には 1ゥエル当たり 5 X 104個になるように、 ま feヒトフイブロネクチンをコートした 6cm径の培養皿 (BECTON DICKINSON社製 Biocoat)には 1.'3 X 105個になるように細胞を蒔き直した。 1日後に5-ァザシチジン （Sigma社製、 終濃度 10〃M) のみを加えたものと、 5-ァザ シチジン、 PDGF-BB (Pepro Tech EC LTD.社製、 終濃度 10ng/ml)、 aU-transレチノ イン酸 （RA、 Sigma社製、 終濃度 10— 9Μ)を加えた二種類の異なる培養条件培養を行い、 2日間培養した後に培地を交換した（後者の場合は培地交換時に再度 PDGF、 all-trans レチノイン酸を加え、 2日後と 4日後にさらに加えた）。その 3〜4日後に、培地を交 換し、 3週間培養した。その結果 5-ァザシチジン、 PDGF - BB、 レチノイン酸を加えた もので筋管様細胞の分化が観察された。 ' ' 産業上の利用可能性 The confluent rat bone marrow-derived cells were separated again by trypsin EDTA treatment, and the cells were transferred to a 6-well plate (BECTON DICKINSON) in such a manner that 5 x 10 4 cells per 1-well were obtained. the culture dish 6cm diameter coated with Hitofu Eve Lone cutin (BECTON DICKINSON Co. Biocoat) was reseeded cells to become 1.'3 X 10 5 cells. One day later, only 5 -azacitidine (Sigma, final concentration 10 M) was added, and 5 -azacitidine, PDGF-BB (Pepro Tech EC LTD., Final concentration 10 ng / ml), aU -trans retinoic acid (RA, Sigma Co., final concentration 10- 9 Micromax) to perform two different culture conditions culture added, the medium was changed after 2 days of culture (in the latter case again during medium replacement PDGF, all-trans retinoic acid was added, and 2 and 4 days later). Three to four days later, change the medium. And cultured for 3 weeks. As a result, differentiation of myotube-like cells was observed with 5-azacitidine, PDGF-BB, and retinoic acid. '''' Industrial applicability
本発明によれば、心筋細胞の破壊ならびに変性を伴う心疾患の治療ならびに治療薬 の探索に有効な骨髄細胞、 増殖因子、 ビタミン、 接着分子、 及びこれらの利用法が提 供される。  According to the present invention, a bone marrow cell, a growth factor, a vitamin, an adhesion molecule, and a method of using the same that are effective for treating a heart disease associated with destruction and degeneration of cardiomyocytes and for searching for a therapeutic agent are provided.
「配列表フリーテキスト」  "Sequence List Free Text"
配列番号 3 3一人工配列の説明:合成 DNA SEQ ID NO: 33 Description of artificial sequence: Synthetic DNA
配列番号 3 4一人工配列の説明:合成 DNA SEQ ID NO: 34 Description of artificial sequence: Synthetic DNA
配列番号 3 5一人工配列の説明:合成 DNA SEQ ID NO: 35 Description of artificial sequence: Synthetic DNA
配列番号 3 6—人工配列の説明:合成 DNA SEQ ID NO: 36—Description of artificial sequence: Synthetic DNA
配列番号 3 7一人工配列の説明:合成 DNA SEQ ID NO: 37 Description of artificial sequence: Synthetic DNA
配列番号 3 8一人工配列の説明:合成 DNA SEQ ID NO: 38 Description of artificial sequence: Synthetic DNA
配列番号 3 9—人工配列の説明:合成 DNA SEQ ID NO: 39—Description of artificial sequence: Synthetic DNA
配列番号 4 0一人工配列の説明:合成 DNA SEQ ID NO: 40 Description of artificial sequence: Synthetic DNA
配列番号 4 1一人工配列の説明:合成 DNA SEQ ID NO: 4 1 Description of artificial sequence: Synthetic DNA
配列番号 4 2一人工配列の説明:合成 DNA SEQ ID NO: 42 Description of artificial sequence: Synthetic DNA
配列番号 4 3一人工配列の説明:合成 DNA SEQ ID NO: 43 Description of artificial sequence: Synthetic DNA
配列番号 4 4一人工配列の説明:合成 DNA SEQ ID NO: 4 Description of artificial sequence: Synthetic DNA
配列番号 4 5一人工配列の説明:合成 DNA SEQ ID NO: 45 Description of artificial sequence: Synthetic DNA
配列番号 4 6一人工配列の説明:合成 DNA SEQ ID NO: 46 Description of artificial sequence: Synthetic DNA
配列番号 4 7—人工配列の説明:合成 DNA SEQ ID NO: 4 7—Description of artificial sequence: synthetic DNA
配列番号 4 8 -人工配列の説明:合成 DNA SEQ ID NO: 48-Description of Artificial Sequence: Synthetic DNA
配列番号 4 9一人工配列の説明:合成 DNA SEQ ID NO: 49 Description of artificial sequence: Synthetic DNA
配列番号 5 0 -人工配列の説明:合成 DNA SEQ ID NO: 50-Description of artificial sequence: Synthetic DNA
配列番号 5 1一人工配列の説明:合成 DNA SEQ ID NO: 5 Description of artificial sequence: Synthetic DNA
配列番号 5 2 -人工配列の説明:合成 DNA SEQ ID NO: 52 Description of artificial sequence: Synthetic DNA
配列番号 5 3 -人工配列の説明:合成 DNA ' 配列番号 5 4—人工配列の説明:合成 DNA 配列番号 5 5—人工配列の説明:合成 DNA 配列番号 5 6一人工配列の説明:合成 DNA 配列番号 5 7—人工配列の説明:合成 DNA 配列番号 5 8—人工配列の説明:合成 DNA 配列番号 5 9一人工配列の説明:合成 DNA 配列番号 6 0—人工配列の説明:合成 DNA SEQ ID NO: 53-Description of Artificial Sequence: Synthetic DNA ' SEQ ID No. 5 4—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 5—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 6 — Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 7—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 5 8—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 5 9—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 60—Description of Artificial Sequence: Synthetic DNA

Claims

請求の範囲 The scope of the claims
1. 骨髄から単離され、 心筋細胞に分ィヒする能力を有する細胞。  1. Cells that are isolated from bone marrow and have the ability to divide into cardiomyocytes.
2. 少なくとも心筋細胞と脂肪細胞に分化する能力を有する多分化能幹細胞である、 請求項 1記載の細胞。  2. The cell according to claim 1, which is a pluripotent stem cell capable of differentiating at least into cardiomyocytes and adipocytes.
3. 心筋細胞のみに分化誘導される心筋幹細胞である、 請求項 1記載の細胞。  3. The cell according to claim 1, which is a cardiac stem cell that is induced to differentiate only into a cardiac muscle cell.
4. 心筋細胞のみに分化誘導される心筋前駆細胞である、 請求項 1記載の細胞。 4. The cell according to claim 1, which is a cardiac progenitor cell that is induced to differentiate only into a cardiac muscle cell.
5. 心室筋細胞に分化する能力を有する、 請求項 1記載の細胞。 5. The cell of claim 1, which has the ability to differentiate into ventricular myocytes.
6. 洞結節細胞に分ィ匕する能力を有する、 請求項 1記載の細胞。  6. The cell according to claim 1, which has an ability to divide into sinus node cells.
7. 骨髄がほ乳動物由来のものである、 請求項 1記載の細胞。  7. The cell of claim 1, wherein the bone marrow is from a mammal.
8. ほ乳動物がヒト、 ラットおよびマウスから選ばれるものである、 請求項 7記載 の細胞  8. The cell according to claim 7, wherein the mammal is selected from human, rat and mouse.
9. 細胞が、 マウス骨髄由来幹細胞 BMSC(FERM BP- 7043)である、 請求項 1記載の 細胞。  9. The cell according to claim 1, wherein the cell is a mouse bone marrow-derived stem cell BMSC (FERM BP-7043).
10. 染色体 DNAの脱メチル化により心筋細胞に分化する能力を有する、 請求項 1記 載の細胞。  10. The cell according to claim 1, which has an ability to differentiate into cardiomyocytes by demethylation of chromosomal DNA.
11. 染色体 DNAの脱メチル化が、 デメチラ一ゼ、 5—ァザシチジンおよびジメチ ルスルフォキシド （DMSO) からなる群から選ばれる少なくとも 1種によるものであ ることを特徴とする、 請求項 10記載の細胞。  11. The cell according to claim 10, wherein the demethylation of chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and dimethyl sulfoxide (DMSO).
12. デメチラ一ゼが、 配列番号 1記載で表されるァミノ酸配列を有するデメチラ一 ゼである、 請求項 11記載の細胞。  12. The cell according to claim 11, wherein the demethylase is a demethylase having an amino acid sequence represented by SEQ ID NO: 1.
13. 胎児の心臓発生領域で発現している因子により心筋細胞への分化が促進される 請求項 1記載の細胞。  13. The cell according to claim 1, wherein differentiation into cardiomyocytes is promoted by a factor expressed in a fetal heart development region.
14. 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ夕ミン および転写因子からなる群から選ばれる少なくとも 1種であることを特徴とする、請 求項 13記載の細胞。  14. The cell according to claim 13, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of a cytokine, an adhesion molecule, bimin, and a transcription factor.
15. 胎児の心臓発生段階において心筋細胞への分化に働く因子により心筋細胞への 分化が促進される請求項 1記載の細胞。  15. The cell according to claim 1, wherein differentiation into cardiomyocytes is promoted by a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
16. 胎児の心臓発生段階において心筋細胞への分ィ匕に働く因子がサイトカイン、接 着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であること を特徴とする、 請求項 15記載の細胞。 16. In the fetal heart development stage, the factors that act to 16. The cell according to claim 15, wherein the cell is at least one selected from the group consisting of a deposition molecule, a vitamin, and a transcription factor.
17. サイトカインが血小板由来増殖因子（PDGF) である、 請求項 1 3または 16記 載の細胞。  17. The cell according to claim 13 or 16, wherein the cytokine is platelet-derived growth factor (PDGF).
18. PDGFが配列番号 3または 5で表されるァミノ酸配列を有する PDGFである、 請求項 17記載の細胞。  18. The cell according to claim 17, wherein the PDGF is PDGF having an amino acid sequence represented by SEQ ID NO: 3 or 5.
19. 接着分子がフイブロネクチンである、 請求項 14または 16記載の細胞。  19. The cell according to claim 14 or 16, wherein the adhesion molecule is fibronectin.
20. ビタミンがレチノイン酸である、 請求項 14または 16記載の細胞。  20. The cell according to claim 14 or 16, wherein the vitamin is retinoic acid.
21. 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF- 2A、 MEF- 2B、 MEF- 2Cヽ MEF- 2D、 dHAND、 eHAND、 TEF - 1、 TEF - 3および TEF- 5からなる群から選ばれるものである、 請求項 I4または I6記載の細胞。 21. The transcription factor is selected from the group consisting of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C ヽ MEF-2D, dHAND, eHAND, TEF-1, TEF-3, and TEF-5 are those, claim I 4 or I 6 wherein the cell.
22. Nkx2.5/Csxが配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csxである、 請求項 21記載の細胞。  22. The cell according to claim 21, wherein Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
23. GATA が配列番号 1 1で表されるアミノ酸配列を有する GATA4である、請求 項 21記載の細胞。  23. The cell according to claim 21, wherein GATA is GATA4 having the amino acid sequence represented by SEQ ID NO: 11.
24. MEF-2Aが配列番号 1 3で表されるアミノ酸配列を有する MEF 2Aである、 請 求項 21記載の細胞。 '  24. The cell according to claim 21, wherein MEF-2A is MEF 2A having an amino acid sequence represented by SEQ ID NO: 13. '
25. MEF-2Bが配列番号 1 5で表されるァミノ酸配列を有する MEF - 2Bである、 請 求項 21記載の細胞。  25. The cell according to claim 21, wherein MEF-2B is MEF-2B having an amino acid sequence represented by SEQ ID NO: 15.
26. MEF-2Cが配列番号 1 7で表されるアミノ酸配列を有する MEF- 2Cである、請 求項 21記載の細胞。  26. The cell according to claim 21, wherein MEF-2C is MEF-2C having the amino acid sequence represented by SEQ ID NO: 17.
27. MEF-2Dが配列番号 1 9で表されるアミノ酸配列を有する MEF-2Dである、請 求項 21記載の細胞。  27. The cell according to claim 21, wherein MEF-2D is MEF-2D having the amino acid sequence represented by SEQ ID NO: 19.
28. dHANDが配列番号 2 1で表されるアミノ酸配列を有する dHANDである、請求 項 21記載の細嗨。 28. DHAND is DHAND having the amino acid sequence represented by SEQ ID NO: 2 1, Hoso嗨of claim 2 1, wherein.
29. eHANDが配列番号 2 3で表されるァミノ酸配列を有する eHANDである、請求 項 21記載の細胞。  29. The cell according to claim 21, wherein the eHAND is an eHAND having an amino acid sequence represented by SEQ ID NO: 23.
30. TEF-1が配列番号 2 5で表されるアミノ酸配列を有する TEF-1である、 請求 項 21記載の細胞。 30. The TEF-1 having the amino acid sequence represented by SEQ ID NO: 25. Item 22. The cell according to Item 21.
31. TEF-3が配列番号 2 7で表されるァミノ酸配列を有する TEF - 3である、 請求 項 21記載の細胞。  31. The cell according to claim 21, wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
32. - TEF- 5が配列番号 2 9で表されるアミノ酸配列を有する TEF- 5である、 請求 項 21記載の細胞。  32. The cell according to claim 21, wherein-TEF-5 is TEF-5 having an amino acid sequence represented by SEQ ID NO: 29.
33. 線維芽細胞増殖因子— 2 (FGF-2) により心筋細胞への分ィ匕が抑制される請求 項 1記載の細胞。  33. The cell according to claim 1, wherein fibroblast growth factor-2 (FGF-2) suppresses division into cardiomyocytes.
34. FGF-2が配列番号 7または 8記載のアミノ酸配列を有する FGF- 2である、 請 求項 33記載の細胞。  34. The cell of claim 33, wherein FGF-2 is FGF-2 having the amino acid sequence of SEQ ID NO: 7 or 8.
35. 染色体 DNAの脱メチル化剤を用いて、 骨髄由来の細胞から心筋を形成する方 法。  35. A method of forming myocardium from bone marrow-derived cells using a chromosomal DNA demethylating agent.
36. 染色体 DNAの脱メチル化剤が、デメチラ一ゼ、 5—ァザシチジンおよび DMSO からなる群から選ばれる少なくとも 1種であることを特徴とする、 請求項 35記載の 方法。  36. The method according to claim 35, wherein the demethylating agent for chromosomal DNA is at least one selected from the group consisting of demethylase, 5-azacitidine and DMSO.
37. デメチラ一ゼが、 配列番号 1記載のアミノ酸配列で表されるデメチラーゼであ る、 請求項 36記載の方法。  37. The method according to claim 36, wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
38. 胎児の心臓発生領域で発現している因子を用いることを特徴とする、 骨髄由来 の細胞から心筋を形成する方法。  38. A method for forming myocardium from bone marrow-derived cells, comprising using a factor expressed in a fetal heart development region.
39. 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビタミン および転写因子からなる群から選ばれる少なくとも 1種であることを特徴とする、請 求項 38記載の方法。  39. The method according to claim 38, wherein the factor expressed in the fetal heart development region is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors.
40. 胎児の心臓発生段階において心筋細胞への分化に働く因子を用いることを特徴 とする、 骨髄由来の細胞から心筋を形成する方法。  40. A method for forming myocardium from bone marrow-derived cells, comprising using a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
41. 胎児の心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、接 着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であること を特徴とする、 請求項 40記載の方法。 41. Cardiac work during the development stage differentiation into cardiomyocytes factor cytokines fetal contact Chakubunshi, characterized in that at least one selected from the group consisting of vitamins and transcription factor of claim 4 0 wherein Method.
42. サイトカインが PDGFである、 請求項 39または 41記載の方法。  42. The method of claim 39 or 41, wherein the cytokine is PDGF.
43. PDGFが配列番号 3または 5記載のアミノ酸配列で表される PDGFである、 請 求項 42記載の方法。 ， 43. PDGF is PDGF represented by the amino acid sequence of SEQ ID NO: 3 or 5. 43. The method of claim 42. ,
44. 接着分子がフイブロネクチンである、 請求項 39または 41記載の方法。  44. The method of claim 39 or 41, wherein the adhesion molecule is fibronectin.
45. ビタミンがレチノイン酸である、 請求項 39または 41記載の方法。  45. The method according to claim 39 or 41, wherein the vitamin is retinoic acid.
46. 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF- 2A、 MEF- 2B、 MEF- 2C、 MEF- 2D、 dHAND、 eHAND、 TEF- 1、 TEF - 3および TEF - 5からなる群から選ばれる、 請求項 39 または 41記載の方法。  46. The transcription factor is selected from the group consisting of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5 42. The method of claim 39 or claim 41.
47. Nkx2.5/Csxが、 配列番号 9で表されるアミノ酸配列を有する Nkx2.5/Csxであ る、 請求項 46記載の方法。  47. The method according to claim 46, wherein Nkx2.5 / Csx is Nkx2.5 / Csx having the amino acid sequence represented by SEQ ID NO: 9.
48. GATA4が、 配列番号 1 1で表されるァミノ酸配列を有する GATA4である、 請 求項 46記載の方法。  48. The method according to claim 46, wherein GATA4 is GATA4 having an amino acid sequence represented by SEQ ID NO: 11.
49. MEF-2Aが、 配列番号 1 3で表されるァミノ酸配列を有する MEF - 2Aである、 請求項 46記載の方法。  49. The method according to claim 46, wherein MEF-2A is MEF-2A having an amino acid sequence represented by SEQ ID NO: 13.
50. MEF-2Bが、 配列番号 1 5で表されるァミノ酸配列を有する M E F— 2 Bであ る、 請求項 46記載の方法。  50. The method according to claim 46, wherein MEF-2B is MEFF-2B having an amino acid sequence represented by SEQ ID NO: 15.
51. MEF-2Cが、配列番号 1 7で表されるアミノ酸配列を有する MEF - 2Cである、 請求項 46記載の方法。  51. The method of claim 46, wherein MEF-2C is MEF-2C having the amino acid sequence set forth in SEQ ID NO: 17.
52. MEF-2Dが、配列番号 1 9で表されるァミノ酸配列を有する MEF-2Dである、 請求項 46記載の方法。  52. The method according to claim 46, wherein MEF-2D is a MEF-2D having an amino acid sequence represented by SEQ ID NO: 19.
53. dHANDが、 配列番号 2 1で表されるァミノ酸配列を有する dHANDである、請 求項 46記載の方法。  53. The method according to claim 46, wherein the dHAND is a dHAND having the amino acid sequence represented by SEQ ID NO: 21.
54. eHANDが、 配列番号 2 3で表されるアミノ酸配列を有する eHANDである、請 求項 46記載の方法。  54. The method according to claim 46, wherein the eHAND is an eHAND having the amino acid sequence represented by SEQ ID NO: 23.
55. TEF-1が、 配列番号 2 5で表されるアミノ酸配列を有する TEF-1である、 請 求項 46記載の方法。  55. The method according to claim 46, wherein TEF-1 is TEF-1 having an amino acid sequence represented by SEQ ID NO: 25.
56. TEF- 3が、 配列番号 2 7で表されるアミノ酸配列を有する TEF - 3である、 請 求項 46記載の方法。  56. The method according to claim 46, wherein TEF-3 is TEF-3 having an amino acid sequence represented by SEQ ID NO: 27.
57. - TEF - 5が、 配列番号 2 9で表されるアミノ酸配列を有する TEF - 5である、 請 求項 46記載の方法。 57. The method according to claim 46, wherein-TEF-5 is TEF-5 having the amino acid sequence represented by SEQ ID NO: 29.
58. 染色体 DNAの脱メチル化剤を有効成分として含有することを特徴とする心筋 形成剤。 58. A cardiomyogenic agent comprising a chromosomal DNA demethylating agent as an active ingredient.
59. 染色体 DNAの脱メチル化剤がデメチラーゼ、 5—ァザシチジンおよび DMSO からなる群から選ばれる少なくとも 1種である、 請求項 58記載の心筋形成剤。  59. The cardiomyogenic agent according to claim 58, wherein the demethylating agent for chromosomal DNA is at least one member selected from the group consisting of demethylase, 5-azacitidine and DMSO.
60. デメチラ一ゼが、配列番号 1記載のアミノ酸配列で表されるデメチラ一ゼであ る、 請求項 59記載の心筋形成剤。  60. The cardiomyogenic agent according to claim 59, wherein the demethylase is a demethylase represented by the amino acid sequence of SEQ ID NO: 1.
61. 胎児の心臓発生領域で発現している因子を有効成分として含有する心筋形成剤 c 61. Cardiomyogenic agent containing factor expressed in fetal heart development region as active ingredient c
62. 胎児の心臓発生領域で発現している因子がサイトカイン、 接着分子、 ビ夕ミン および転写因子からなる群から選ばれる少なくとも 1種であることを特徴とする、請 求項 61記載の心筋形成剤。 62. The cardiomyogenesis of claim 61, wherein the factor expressed in the fetal heart development region is at least one member selected from the group consisting of cytokines, adhesion molecules, bimin, and transcription factors. Agent.
63. 胎児の心臓発生段階において心筋細胞への分化に働く因子を有効成分として含 有することを特徴とする心筋形成剤。  63. A cardiomyogenic agent comprising, as an active ingredient, a factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development.
64. 胎児め心臓発生段階において心筋細胞への分化に働く因子がサイトカイン、接 着分子、 ビタミンおよび転写因子からなる群から選ばれる少なくとも 1種であること を特徴とする、 請求項 63記載の心筋形成剤。  64. The myocardium according to claim 63, wherein the factor that acts on differentiation into cardiomyocytes at the stage of fetal heart development is at least one selected from the group consisting of cytokines, adhesion molecules, vitamins and transcription factors. Forming agent.
65. サイトカインが PDGFである、 請求項 62または 64記載の心筋形成剤。  65. The cardiomyogenic agent according to claim 62 or 64, wherein the cytokine is PDGF.
66. PDGFが配列番号 3または 5記載のアミノ酸配列で表される、請求項 65記載の 心筋形成剤。  66. The cardiomyogenic agent according to claim 65, wherein PDGF is represented by the amino acid sequence of SEQ ID NO: 3 or 5.
67. 接着分子がフイブロネクチンである、 請求項 62または 記載の心筋形成剤。67. adhesion molecule is fibronectin, claim 6 2 or myocardial forming agent according.
68. ビタミンがレチノイン酸である、 請求項62または 記載の心筋形成剤。 68. The cardiomyogenic agent according to claim 62 , wherein the vitamin is retinoic acid.
69. 転写因子が、 Nkx2.5/Csx、 GATA4、 MEF- 2A、 MEF- 2B、 MEF-2C, MEF- 2D、 dHAND、 eHAND, TEF-1、 TEF - 3および TEF-5からなる群から選ばれる、 請求項 62 または64記載の心筋形成剤。 69. The transcription factor is selected from the group consisting of Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3 and TEF-5 65. The cardiomyogenic agent according to claim 62 or 64 .
70. Nkx2.5/Csxが、配列番号 9記載のァミノ酸配列で表される Nkx2.5/Csxである、 請求項 69記載の心筋形成剤。 70. Nkx2.5 / Csx is a Nkx 2. 5 / Csx represented by Amino acid sequence of SEQ ID NO: 9, cardiomyogenesis agent according to claim 69.
71. GATA4が、 配列番号 1 1記載のアミノ酸配列で表される GATA4である、 請求 項 69記載の心筋形成剤。  71. The cardiomyogenic agent according to claim 69, wherein GATA4 is GATA4 represented by the amino acid sequence set forth in SEQ ID NO: 11.
72. MEF-2Aが、 配列番号 1 3記載のァミノ酸配列で表される MEF- 2Aである、 請 求項 69記載の心筋形成剤。 72. MEF-2A is MEF-2A represented by the amino acid sequence set forth in SEQ ID NO: 13. 74. The myocardial agent according to claim 69.
73. MEF-2Bが、 配列番号 1 5記載のァミノ酸配列で表される MEF-2Bである、 請 求項 69記載の心筋形成剤。  73. The cardiomyogenic agent according to claim 69, wherein MEF-2B is MEF-2B represented by the amino acid sequence set forth in SEQ ID NO: 15.
74. MEF-2Cが、 配列番号 1 7記載のァミノ酸配列で表される MEF-2Cである、請 求項 69記載の心筋形成剤。  74. The cardiomyogenic agent according to claim 69, wherein MEF-2C is MEF-2C represented by the amino acid sequence of SEQ ID NO: 17.
75. MEF-2Dが、 配列番号 1 9記載のアミノ酸配列で表される MEF- 2Dである、請 求項 69記載の心筋形成剤。  75. The cardiomyogenic agent according to claim 69, wherein MEF-2D is MEF-2D represented by the amino acid sequence of SEQ ID NO: 19.
76. dHANDが、配列番号 2 1記載のァミノ酸配列で表される dHANDである、請求 項 69記載の心筋形成剤。  76. The cardiomyogenic agent of claim 69, wherein the dHAND is a dHAND represented by the amino acid sequence of SEQ ID NO: 21.
77. eHANDが、配列番号 2 3記載のァミノ酸配列で表される eHANDである、 請求 項 69記載の心筋形成剤。  77. The cardiomyogenic agent according to claim 69, wherein the eHAND is an eHAND represented by the amino acid sequence of SEQ ID NO: 23.
78. TEF-1が、 配列番号 2 5記載のァミノ酸配列で表される TEF-1である、 請求 項 69記載の心筋形成剤。  78. The cardiomyogenic agent according to claim 69, wherein TEF-1 is TEF-1 represented by the amino acid sequence of SEQ ID NO: 25.
79. TEF-3が、 配列番号 2 7記載のアミノ酸配列で表される TEF-3である、 請求 項 69記載の心筋形成剤。  79. The cardiomyogenic agent according to claim 69, wherein TEF-3 is TEF-3 represented by the amino acid sequence of SEQ ID NO: 27.
80. TEF-5が、 配列番号 2 9記載のアミノ酸配列で表される TEF - 5である、 請求 項 69記載の心筋形成剤。  80. The cardiomyogenic agent according to claim 69, wherein TEF-5 is TEF-5 represented by the amino acid sequence set forth in SEQ ID NO: 29.
81. 請求項 1〜34のいずれか 1項に記載の細胞を用いることを特徴とする、心臓疾 患により破壊された心臓を再生する方法。  81. A method for regenerating a heart destroyed by a heart disease, comprising using the cell according to any one of claims 1 to 34.
82. 請求項 1〜34のいずれか 1項に記載の細胞を有効成分とする心臓再生治療薬。82. A therapeutic agent for regenerating a heart, comprising the cell according to any one of claims 1 to 34 as an active ingredient.
83. 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入された請 求項 1〜34のいずれか 1項に記載の細胞を用いることを特徴とする、先天性遺伝子疾 患での変異遺伝子に対する野生型遺伝子を心筋へ特異的に輸送する方法。 83. Use of the cell according to any one of claims 1 to 34, wherein a wild-type gene for a mutant gene in a congenital heart disease is introduced. A method for specifically transporting a wild-type gene for a mutant gene to the heart muscle.
84. 心臓の先天性遺伝子疾患での変異遺伝子に対する野生型遺伝子が導入された請 求項 1〜Mのいずれか 1項に記載の細胞を有効成分として含有する心臓疾患治療薬。 84. A therapeutic agent for heart disease, comprising, as an active ingredient, the cell according to any one of claims 1 to M, into which a wild-type gene for a mutant gene in congenital heart disease has been introduced.
85. 請求項 1〜34のいずれか 1項に記載の細胞を免疫原として用いることを特徴と する、 該細胞を特異的に認識する抗体を取得する方法。 85. which comprises using the cell according to any one of claims 1 to 3 4 as an immunogen, a method of obtaining an antibody that specifically recognizes the cell.
86. 請求項 85記載の方法で取得された抗体を用いることを特徴とする、 ヒト骨髄 から心筋細胞への分化能を する成体狩髄 flj来細胞を単離 ·精製する方法。 86. Human bone marrow, characterized by using an antibody obtained by the method according to claim 85. A method for isolating and purifying adult myelinating flj-derived cells that have the potential to differentiate into cardiomyocytes from yeast.
87. 滅 1 -3 のいずれか 1项に記載の細胞を川いることを特徴とする、該細胞 に特 的な iiii抗^を取得する方法。  87. A method for obtaining a specific iiii antibody against a cell according to any one of 1 to 3 above, comprising:
88. 請求 ¾ 1〜： のいずれか 1项に記載の細胞を川いることを特徴とする、該細胞 を^ する【人卜了-をスクリーニングする方法。  88. A method for screening a human cell that regenerates the cell, characterized by flowing the cell according to any one of (1) to (1).
89. 项 1 -3 のいずれか 1 に記載の細胞を川いることを特徴とする、纖 I胞 の心筋細胞への分化を誘導する闪子をスクリーニングする方法。  89. A method for screening a cell that induces differentiation of a Fiber I vesicle into a cardiomyocyte, comprising flowing the cell according to any one of the items 1-3.
90. 詰求项 1〜： Mのいずれか 1 1¾に記載の細胞を川いることを特徴とする、該細胞 を不死化する wir-をスクリーニングする方法。  90. A method for screening wir- for immortalizing a cell, characterized by flowing the cell according to any one of 11-11 of M.
91 . 求项 1〜： Mのいずれか 1項に記載の細胞にテロメラ一ゼを究 ¾させることを 特徴とする、 該細胞の不死化方法。  91. Claim 1 to: A method for immortalizing a cell according to any one of M, characterized by examining the cell according to any one of M for telomerase.
92. テロメラ一ゼが、 配列桥号 3 1記載で ¾されるアミノ酸配列をおするテロメラ ーゼである詰求 ¾ 91記載の方法。  92. The method according to 91, wherein the telomerase is a telomerase having the amino acid sequence shown in SEQ ID NO: 31.
93. テロメラーゼを発現させることにより、不死化させた請求 ¾ 1〜： Mのいずれか 1 ¾'ίに記載の細胞を 効成分として含おする心臓疾 / 抬^薬。  93. A cardiomyopathy drug comprising the cell of any one of claims 1 to 1 as an active ingredient, which is immortalized by expressing telomerase.
9^1. テロメラ一ゼが、 配列桥 - 3 1記載で ¾されるアミノ酸配列をィ するテロメラ —ゼである請求項 93記載の^療薬。  94. The therapeutic drug according to claim 93, wherein the telomerase is a telomerase having an amino acid sequence represented by SEQ ID NO: 31.
95. 詰求项 1〜34のいずれか 1埙に記載の細胞を含んだ培養上洁。 95. A culture medium containing the cell according to any one of 1 to 34 .
96. 請求项 95記載の培養上消を用いることを特徴とする、 請求項 1記載の細胞を 心筋細胞に分化誘導する方法。  96. A method for inducing differentiation of the cell according to claim 1 into a cardiomyocyte, wherein the method according to claim 95 is used.
PCT/JP2000/001148 1999-12-28 2000-02-28 Adult bone marrow-origin cell capable of differentiating into heart muscle cell WO2001048149A1 (en)

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WO2002036772A1 (en) * 2000-10-30 2002-05-10 Takeda Chemical Industries, Ltd. Novel disease-associated gene and use thereof
WO2004000993A2 (en) * 2002-06-21 2003-12-31 Universite Du Quebec A Montreal Oxytocin as cardiomyogenesis inducer and uses thereof
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