JP5834297B2 - Collagen production enhancer - Google Patents

Description

The present invention relates to a collagen production enhancer .

  A tendon is a tissue that connects muscles and bones, and a ligament is a tissue that connects bones and bones. These organizations have an important function of transmitting power and movement. Tendons and ligaments are damaged and decreased by overuse and aging, but because there are few nutrient blood vessels, the healing power of tendons is very weak and it is extremely difficult to cure. Due to insufficient biological understanding of the mechanism of tendon cell differentiation and the like, a therapeutic method such as regenerative medicine has not been established.

  Tendons and ligaments are made of fibers formed by cross-linking collagen fibrils. There are a few tendon cells between the tendon fibers, producing extracellular matrix such as collagen and proteoglycan. Collagen that constitutes most of the tendon gives elasticity to the tendon, but most of it is type I collagen. Proteoglycans include decorin, fibromodulin, biglycan, and lumican in tendons, and are involved in the binding and assembly of collagen fibers. In proteoglycan gene-deficient mice, abnormalities of tendon collagen fibrils are observed. Tendon disorders have been reported in patients with diseases that have abnormalities in collagen production, such as the Ehlers-Danlos syndrome. The production of extracellular matrix in tendon cells is suggested to be important for tendon formation.

  In recent years, it has been reported that scleraxis (Scx), a transcriptional factor of basic helix-loop-helix (bHLH) expressed in tendon and ligament cells and tendon and ligament progenitor cells, is an important factor for tendon development. In mice, significant dysplasia of tendons was observed (Non-patent Document 1). It was also suggested to directly induce type I collagen, which is the main component of tendons (Non-patent Document 2). However, Scx knockout mice do not lose all type I collagen in the tendons, as shown in the examples below.

Cserjesi P, et al. (1995) Scleraxis: a basic helix-loop-helix protein that pre-shapes skeletal formation developing mouse development 121 (4) pp 1099110 Lejard V, et al. (2007) Scleraxis and NFATc regulate the expression of the pro-alpha1 (I) collagen gene in tendon fibroblasts J Biol Chem 282 (24) p17665-17675

  Tendon and ligament cells are cells that produce a substrate such as type I collagen and are involved in the formation and maintenance of tendon and ligament tissue, respectively. Tendons and ligaments are tissues with low regeneration ability as described above, and regenerative medicine for treatment of tendon and ligament diseases is desired. Scx is expressed in tendon and ligament cells and is suggested to induce the expression of type I collagen, which is the main component of tendon and ligament, but suggests the presence of other type I collagen expression regulators, etc. However, there are many unexplained mechanisms of tendon development. A method for inducing differentiation into tendon and ligament cells has not been established.

Accordingly, an object of the present invention is to provide a collagen production enhancer .

  The present inventors considered that it is important to identify a gene important for differentiation and introduce or induce the gene for establishing a differentiation method into tendon and ligament cells. As shown in Examples described later, the present inventors reduced the amount of type I collagen produced by tendon tissue and tendon cells when the Mkx gene was disrupted. Has been found to increase. Mkx has been known as a homeobox-type transcription factor that is expressed in tendons in the embryonic period. Mkx is an important factor that regulates the differentiation of type I collagen and regulates the expression of type I collagen in tendon cells. First revealed. Based on these findings, the present inventors have completed the following invention.

That is, the present invention
(A) Mkx protein,
(B) In the amino acid sequence of the Mkx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and tendons and / or ligaments A mutant having an activity to enhance the production of type I collagen in cells having the ability to differentiate into cells,
(C) a nucleic acid encoding the protein of (A) or (B), and
(D) A collagen production enhancer that enhances the production of type I collagen in cells capable of differentiating into tendon and / or ligament cells, comprising as an active ingredient at least one of the expression vectors containing the nucleic acid of (C). To do.

The collagen production enhancer is
(A1) Scx protein,
(B1) In the amino acid sequence of the Scx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and induce type I collagen A mutant having the activity of
(C1) a nucleic acid encoding the protein of (A1) or (B1), and
(D1) an expression vector comprising the nucleic acid of (C1),
May further include at least one of the following.

  In medicine such as joint regeneration, tendons and ligaments are important tissues that connect muscles and skeletons. When the Mkx gene is overexpressed, the expression of type I collagen, which is a major component of the tendon, increases, suggesting that Mkx is important in the tendon maturation process. It is expected that healing of tendons, regeneration of musculoskeletal organs, and reconstruction will be promoted by introducing a drug consisting of Mkx and its upstream / downstream genes into the damaged tendon or ligament site.

  Using cells collected from patients suffering from tendon and / or ligament-related diseases such as Erasdanros syndrome, autogenous tendons and / or ligament cells can also be produced with the differentiation inducer of the present invention. Regenerative medicine for the disease becomes possible.

  The genetically modified non-human animal of the present invention in which the Mkx gene is replaced with a reporter gene such as a fluorescent protein specifically for tendon cells and ligament cells is used for screening and evaluation of a test substance that regulates Mkx using the fluorescence of the fluorescent protein as an indicator It is useful as a model animal for monitoring the local expression of Mkx during the healing process in a tendon injury model experiment or the like.

  Analysis or analysis of tendon or ligament-related diseases using tendon and / or ligament cells obtained by differentiation-inducing agents and differentiation-inducing methods is the basis for understanding the molecular mechanism of tendon differentiation. This may lead to the discovery of therapeutic targets for tendon-related diseases such as

  Mkx is important for the development and maturation of tendons, so it causes abnormalities in the gene sequence of Mkx itself or genes such as upstream and downstream genes involved in the Mkx gene network due to diseases such as tendons and ligaments. The potential is great. The screening method of the present invention is applied to such searching, diagnosis and treatment.

  According to the substance selected by the screening method of the present invention and the medicament of the present invention, tendon- or ligament-related diseases, particularly diseases having abnormalities in Mkx can be prevented or treated.

An example of an Mkx targeting construct designed to generate Mkx mutant mice is shown. Here, arrows a to e indicate PCR primers for genotyping, □ indicates UTR, ■ indicates translation region, DT-A indicates diphtheria toxin A, WT indicates wild type allele, and TA indicates targeted allele. 1B shows genomic PCR (drawing substitute photograph) using primers a, b and c of FIG. 1A in wild type mice and Mkx mutant mice. The upper row shows Mkx WISH (whole mount in situ hybridization) in the E13.5 forelimb and tail of wild type mouse embryos (drawing substitute photo), and the lower row shows wild type mice and Mkx. The observation of the Venus fluorescence signal of a mutant mouse (drawing substitute photograph) is shown. The micrograph which observed the immunohistochemical staining using the myosin heavy chain antibody (MF20; red) and the Venus fluorescence signal in the tail of the embryo of an E16.5 Venus knock-in hetero mouse is shown. In the figure, the portion surrounded by a circle indicates a myosin heavy chain expressing cell that is a muscle marker gene, and the others indicate fluorescence signals of Venus expressing cells. Bar is 100 μm. The RT-PCR analysis (drawing substitute photograph) of Mkx in the Achilles tendon of the wild type mouse and the Mkx mutant mouse is shown. Gapdh indicates an endogenous control. In 3-month-old wild type mice, patella tendon (A arrow), Achilles tendon (C arrow, hind limb (E arrow), forelimb tendon (G arrow), tail tendon (I arrow), back tendon (K arrow) and cervical tendon tendon (M arrow) drawing substitute photograph, and 3-month-old Mkx knockout mouse, patella tendon (B arrow), Achilles tendon (D arrow, hind limb (F arrow) ), Forelimb tendon (H arrow), tail tendon (J arrow), back tendon (L arrow), and cervical tendon tendon (N arrow) are shown in Mkx knockout mice. Abnormal tendon is observed. It is the graph which measured the tensile strength of the Achilles tendon of a wild type mouse | mouth and a Mkx knockout mouse | mouth. It is the graph which measured the Young's modulus of the Achilles tendon of a wild type mouse and a Mkx knockout mouse. The H & E dyeing | staining image (drawing substitute photograph) of the patella tendon of a 3 month-old wild-type mouse | mouth and a Mkx knockout mouse | mouth is shown. The bars a and c mean 1 mm, and the bars b and d mean 50 μm. In Mkx-deficient mice, it can be seen that hypotenosis of the tendon occurs. It is the graph which measured the thickness of the patella tendon of a 7-day-old wild type mouse | mouth and a Mkx knockout mouse | mouth. Mkx knockout mice have significantly reduced patella tendon thickness. The H & E dyeing | staining image (drawing substitute photograph) of the tail tendon of a 3 month-old wild type mouse | mouth and a Mkx knockout mouse | mouth is shown. The bars a and c mean 200 μm, and the bars b and d mean 50 μm. The arrowhead indicates the fiber bundle of the tail tendon. The fiber bundles of the tail tendon of Mkx knockout mice are significantly thinner. It is the graph which measured the cell density of the tail tendon of a 3 month-old wild-type mouse | mouth and a Mkx knockout mouse | mouth. Azan staining images (drawing substitute photos) of tail tendons of embryos of E18.5 wild type mice and Mkx knockout mice are shown. The arrowhead indicates the fiber bundle of the tail tendon. The bar in the left figure means 500 μm, and the bar in the right figure means 200 μm. The electron microscope observation image (drawing substitute photograph) of the collagen fibril of the Achilles tendon of a wild type mouse | mouth and a Mkx knockout mouse | mouth is shown. Here, the bar means 200 nm. The measurement result of the collagen amount of the Achilles tendon of an 8 week-old wild type mouse | mouth and a Mkx knockout mouse | mouth is shown. It can be seen that the ability to produce type I collagen decreases in Mkx-deficient tendon cells. The results of Western blot analysis of type I collagen (Col1) and β-actin of the Achilles tendon of 8-week-old wild type mice and Mkx knockout mice (drawing substitute photo) are shown. The results of real time PCR analysis of Col1a1, Col1a2 and Scx of Achilles tendon of 8-week-old wild type mice and Mkx knockout mice are shown. The result of Western blotting of type I collagen (Col1), FLAG and β-actin of C3H10T1 / 2 cells transfected with FLAG-Mkx expression vector (Mkx) or empty vector (Empty) is shown (photograph substituted for drawing). A schematic diagram of a tendon differentiation network is shown. For the Mkx mutant ES or mouse Southern blot analysis, the wild-type (WT) and targeted allele (TA) and the location of each probe are indicated. Here, N means NcoI, and S means SacI. The Southern blot analysis result (drawing substitute photograph) of a recombinant embryonic stem cell is shown. Here, TA means targeted allyl and WT means wild type allele. The Southern blot analysis result (drawing substitute photograph) of a wild type mouse | mouth and a Mkx mutant mouse | mouth is shown. The RT-PCR analysis (drawing substitute photograph) of Mkx in the muscle, an Achilles tendon, and a tail tendon of a wild type mouse (adult) is shown. It can be seen that Venus expression in Mkx heterozygous mice (adult) is linked to Mkx expression. Gapdh represents an endogenous control. The expression and appearance photograph of Venus in Achilles tendon (BE), tail tendon (FI) and trunk tendon (JM) of Mkx hetero mice and wild type mice are shown. The graph which compared the body weight of the Mkx ^{<+ / +>} , Mkx < ^{+/- >} , and Mkx <^-/-> mouse | mouth in each stage after birth is shown. There is no difference in body weight between wild type mice and Mkx knockout mice. Appearance photograph of 3-month-old wild type mouse (WT), Achilles tendon (A), tail tendon (C), patella tendon (E) and trunk tendon (G), and 3-month-old neomycin resistance gene were removed. The appearance photograph of the Achilles tendon (B), tail tendon (D), patella tendon (F), and trunk tendon (H) of the Mkx knockout mouse (KO) is shown. Tendon abnormalities also occur in knockout mice from which the neomycin resistance gene has been removed. The H & E dyeing | staining image (drawing substitute photograph) of the knee cruciate ligament of a wild type mouse | mouth and a Mkx knockout mouse | mouth is shown. The left bar means 500 μm, and the right bar means 200 μm. The knee cruciate ligament has no effect in Mkx knockout mice.

The differentiation-inducing agent from cells having the ability to differentiate into tendon and / or ligament cells of the present invention to tendon and / or ligament cells contains at least one of the following (A) to (D) as an active ingredient.
(A) Mkx protein,
(B) In the amino acid sequence of the Mkx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and tendons and / or ligaments A mutant having an activity of inducing differentiation into a tendon and / or ligament cell from a cell capable of differentiating into a cell;
(C) a nucleic acid encoding the protein of (A) or (B), and
(D) an expression vector comprising the nucleic acid of (C)

  Mkx (Mohawk, also known as Irxl1) is a homeobox-type transcription factor that is expressed in tendons during the embryonic period. Mkx is the only gene belonging to a newly classified class within the TALE superclass of atypical homeobox genes (Anderson DM, et al. (2006). Dev Dyn 235 (3) p792-801). Mkx has been reported to be expressed in tendon, muscle, cartilage progenitor cells, male gonads, and renal ureteric bud tips (Dev Dyn 235 (3) p792-801, etc.). The present inventors also used a tendon in the process of constructing the whole mount in situ hybridization database (EMBRYS) of 1520 transcription factors and transcription cofactors using E9.5, E10.5 and E11.5 mouse embryos. It was identified as a transcription factor to be expressed (Yokoyama S, et al. 2009 Dev Cell 17 (6) p836-848).

  In the present invention, Mkx protein is used as a differentiation inducer for producing, regenerating, enhancing, etc. tendon cells and / or ligament cells from cells.

  When the differentiation-inducing agent of the present invention is used for experiments and research, the origin of the Mkx protein does not matter. Examples thereof include mammals such as humans, mice, monkeys, chimpanzees, pigs, and dogs. When the differentiation-inducing agent of the present invention is used for medical treatment such as regeneration of human tendon cells and / or ligament cells, it is preferable to use human Mkx protein.

  Information regarding the human Mkx protein is available from NCBI as accession number NP — 775847, which is a protein composed of 352 amino acids. Information on the human Mkx gene is available from NCBI as accession number NM_173576. The human Mkx gene exists on human chromosome 10 and consists of about 3658 base pairs in total length and is composed of 7 exons. The mouse Mkx gene can be obtained from NCBI as accession number NM_177595.

  The (A) Mkx protein used in the differentiation inducer of the present invention may be a recombinant protein in addition to a naturally derived protein. The recombinant protein can be obtained by culturing a transformant incorporating a nucleic acid encoding the Mkx protein by a conventional method.

  In the amino acid sequence of (B) Mkx protein used for the differentiation-inducing agent of the present invention, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, And the variant which has the activity which induces the differentiation from a cell which has the differentiation ability to a tendon and / or a ligament cell to a tendon and / or a ligament cell can be easily acquired by a method well-known to those skilled in the art. Whether the mutant has differentiation-inducing activity can be measured by Western blot, Northern blot, real-time PCR, or the like.

  The variant of Mkx protein is also composed of an amino acid sequence having usually 70% or more, preferably 80% or more, more preferably 90% homology with the amino acid sequence of Mkx protein, and tendon and / or ligament cell It may have an activity of inducing differentiation from a cell having the ability to differentiate into a tendon and / or a ligament cell.

  The differentiation inducer of the present invention may be a nucleic acid (C) encoding the protein (A) or (B). Nucleic acids include chromosomal DNA and cDNA. Chromosomal DNA is obtained, for example, by preparing a DNA library from PCR amplification or cells and screening with a probe that hybridizes to DNA encoding the Mkx protein. cDNA is obtained by, for example, a PCR method using RNA extracted from tendon cells or the like and using a primer that hybridizes to DNA encoding Mkx protein.

  The differentiation-inducing agent of the present invention may be (D) an expression vector containing the nucleic acid of (C). An expression vector is obtained by linking an Mkx gene to a known vector so as to allow expression. The vector is not particularly limited as long as it can be replicated in a host, and examples thereof include a plasmid vector, a virus vector, and a phage vector.

  Examples of the plasmid vector include plasmids derived from E. coli (eg, pBR322, pUC18, pUC119, pTrcHis, pBlueBacHis, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.), and yeast-derived plasmids (eg, YEp13, YEp24, YCp50, pYE52). Etc.). Examples of the phage vector include λ phage and the like. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Sendai viruses, baculoviruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, and potato exviruses.

  In order to insert the Mkx gene into a vector, a method of cutting the DNA with a restriction enzyme and inserting it into an appropriate restriction enzyme site of the vector DNA or a cloning method using a recombinase, a Gateway system, or the like is employed. At that time, it is necessary to incorporate the vector into the vector so that the function of the Mkx gene can be exhibited. Therefore, in addition to the Mkx gene, the vector may contain a cis element such as a promoter and an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, neomycin (neo) resistance gene, dihydrofolate reductase gene, and the like.

  The expression vector may be one that directly expresses the Mkx gene, or one that is expressed as a fusion protein with another protein or peptide such as a FLAG tag or Myc tag.

The differentiation inducer of the present invention may further contain at least one of the following (A1) to (D1).
(A1) Scx protein,
(B1) In the amino acid sequence of the Scx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and induce type I collagen A mutant having the activity of
(C1) a nucleic acid encoding the protein of (A1) or (B1), and (D1) an expression vector comprising the nucleic acid of (C1).

  As described above, it is suggested that the Scx protein directly induces type I collagen, which is the main component of tendons (Non-patent Document 1). Scx knockout mice partially lose their tendons. On the other hand, in the Mkx knockout mouse, as shown in Examples, hypotenosis of the tendon is observed, but the number of tendon cells is not changed. This is an essential factor from the early stage of Scx tendon cell differentiation, and Mkx is expected to be important for late differentiation / maturation. In addition, for the differentiation of tendon cells and the production of type I collagen therein, it can be said that the cooperative work of both initial differentiation by the Scx gene and maturation by Mkx is important. Therefore, the inclusion of the Scx-related substance (A1) to (D1) in the differentiation-inducing agent of the present invention makes it possible to more differentiate cells from tendons and / or ligament cells into tendons and / or ligament cells. It is preferable in terms of efficient execution.

  Information on the Scx protein is available from NCBI as accession number NP_001008272. Information on the Scx gene is available from NCBI as accession number NM_001008271.

  The production method of (A1) to (D1) is the same as that described in (A) to (D) except that the protein and gene are different from Mkx.

  The present invention also provides a step of culturing the cells using the differentiation inducer under conditions suitable for growth of cells capable of differentiating into tendon and / or ligament cells, and among the cultured cells. A method for inducing differentiation from tendon and / or ligament cell to tendon and / or ligament cell, comprising the step of selecting tendon and / or ligament cell.

  Examples of the cells having the ability to differentiate into tendon and / or ligament cells include fibroblasts (C3H10T1 / 2 and the like), tendon progenitor cells, ligament progenitor cells, embryonic stem cells, embryonic germ cells, somatic stem cells, iPS, and the like. Examples thereof include cells.

  Hereinafter, one aspect of the differentiation induction method will be described. First, a differentiation-inducing agent is introduced into cells capable of differentiating into tendon and / or ligament cells and cultured. Specifically, the Mkx expression vector is transfected into 50% confluent C3H10T1 / 2 cells obtained by culturing C3H10T1 / 2 cells in DMEM medium or the like. Transfection can be performed by known methods, and examples of introduction methods other than transfection include introduction methods using viral vectors, electroporation methods, calcium phosphate precipitation methods, microinjection methods, and liposome methods.

  The culture method is not particularly limited as long as it is suitable for the differentiation induction method. For example, a flat culture method, a floating aggregation culture method, a suspension culture method, a co-culture method with feeder cells, a swirl culture method, a soft agar culture method, a microcarrier culture method and the like can be mentioned.

  After culturing, the collected cells are lysed with a cell lysis buffer to obtain a total soluble protein. It is preferable to check the presence of type I collagen by subjecting all soluble proteins to Western blotting or the like.

  When the Mkx gene is overexpressed using the differentiation inducer, the expression of type I collagen, which is the main component of tendon, is increased. If a tendon cell and / or a ligament cell produced using a differentiation-inducing agent or a differentiation-inducing agent is introduced into a damaged tendon site, it can be expected to promote healing of the tendon and regeneration and reconstruction of the musculoskeletal organ. Therefore, the present invention provides a medicament for preventing or treating a tendon or ligament-related disease, comprising the differentiation inducer or a tendon and / or ligament cell produced using the differentiation inducer.

  The differentiation inducer of the present invention is also suitable for gene therapy. A candidate for gene therapy tendon or ligament-related disease is Erasdanros syndrome. Erasdanros syndrome is a disease in which connective tissue components such as collagen forming human skin and tissues exhibit inborn errors of metabolism, resulting in abnormal ligament and joint mobility enhancement. At present, no fundamental cure has been found. Regenerative medicine for the disease is expected by producing autologous tendon and / or ligament cells with a differentiation-inducing agent using cells collected from patients suffering from tendon and / or ligament in Eras Dunros syndrome. .

  The administration form of the medicine is not particularly limited, but is preferably direct administration to a damaged tendon and / or ligament site. Specific examples include injections such as subcutaneous injections and intramuscular injections, drops, transdermal administration agents, external preparations such as ointments, and single-layer or multi-layer sheets of cells.

  As the auxiliary agent and the preparation technique applied to these dosage forms, those known in the art can be used. For example, in the case of injections, suitable buffers, stabilizers, preservatives, soothing agents, isotonic agents, solubilizing agents, and the like can be mentioned.

  The pharmaceutical of the present invention is administered in a pharmacologically necessary amount to animals including humans. The pharmacologically necessary amount is appropriately determined according to the judgment of a doctor or the like in consideration of the dosage form, administration method, symptoms of the patient or laboratory animal, the treatment site, the age, sex, weight, etc. of the patient or laboratory animal.

  The present invention also provides a genetically modified non-human animal for the study of tendon or ligament related diseases comprising a functional defect of the Mkx gene. The “functional deficiency of Mkx gene” in this specification means a state in which the normal function originally possessed by the Mkx gene is not sufficiently exhibited or not exhibited at all.

There are two types of genetically modified non-human animals: heterozygotes for Mkx-deficient genes (sometimes denoted as Mkx ^+/− ) and homozygotes (sometimes denoted as Mkx ^{− / −} ). In the present specification, the description of “Mkx mutation” (for example, Mkx mutant mouse) is used in the meaning including both.

  Homozygotes such as knockout mice show significantly lower tendons and / or ligaments in genetically modified non-human animals compared to wild type animals. Specifically, in the Mkx knockout mouse, the number of tendon cells is similar to that of the wild type, although the tendon of the whole body becomes hypoplastic and the fiber bundle of the tail tendon becomes thin. Furthermore, the tendons of Mkx knockout mice have a small collagen fibril diameter and a reduced amount of type I collagen. The above results suggest that Mkx contributes to the ability to produce type I collagen in tendon cells and plays an important role in the differentiation of tendon cells. Based on this finding, the Mkx knockout mouse of the present invention is useful for studies on the induction of differentiation of Mkx into tendon and / or ligament cells and for searching for a drug for preventing or treating a tendon or ligament-related disease.

  On the other hand, heterozygotes such as hetero mice do not show abnormal tendon formation. Heterogeneous mice have no abnormalities in tendons, and the fluorescent protein Venus is expressed in cells that express Mkx, such as tendons and ligaments. Therefore, where Mkx is expressed, and what substances and genes are used to induce expression of Mkx It is useful for analysis of such as, observation of how tendon tissue can be formed, separation of tendon cells and investigation of cell properties.

  Examples of non-human animals are experimental animals such as mice, rats, hamsters, guinea pigs, chimpanzees, monkeys and rabbits, domestic animals such as pigs, cows and sheep, pets such as dogs and cats, preferably mice. .

  Any method known in the art can be employed as a method for functionally deleting the Mkx gene. Specifically, a method of homologous recombination of a region containing exon of genomic DNA containing Mkx gene with a targeting vector is exemplified.

  When the Mkx gene is functionally deleted, the reporter gene is preferably expressed in a tendon cell-specific manner by inserting a reporter gene downstream of the promoter of the Mkx gene. When the Mkx-deficient site of the heterozygous Mkx hetero mouse is replaced with a reporter gene such as a fluorescent protein, the reporter gene is expressed specifically in tendon cells and normal tendon cells can also be visually recognized (FIG. 1D). Such Mkx hetero mice are extremely useful for studies on the induction of differentiation of Mkx into tendon and / or ligament cells, and for searching for drugs for preventing or treating tendon- or ligament-related diseases.

  The reporter gene includes Venus, BFP, CFP, dsRed, EBFP, ECFP, EGFP, EYFP, FlAsH, GFP, HcRed1, mRFP1, mPlum, PhiYFP, RFP, YFP and other fluorescent proteins, luciferase gene, catalase gene, LacZ gene Etc. can be used.

  Furthermore, it is preferable to insert a selection marker such as a neomycin resistance gene downstream of the Mkx gene.

  More preferably, both ends of the selection marker are sandwiched between loxP sequences. In this way, the selection marker can be removed using the Cre-loxP system.

The genetically modified non-human animal can be produced by a method known per se. In particular,
1. Producing an embryonic stem cell clone containing a functional defect of the Mkx gene;
2. Injecting the embryonic stem cell clone into an embryo,
3. Transferring the chimeric embryo to the uterus of the animal to create a chimeric animal,
4). 4. mating the chimeric animals to obtain heterozygotes; and Mating heterozygotes to obtain a homozygote,
including.

  The genetically modified non-human animal will be described more specifically by taking Mkx hetero mice and Mkx knockout mice as examples. In order to delete the Mkx gene of genomic DNA, a partial region of Mkx gene (including a part of exon) in homologous recombination in embryonic stem cells, eg, a fluorescent protein gene and a positive selection marker (eg, neomycin resistance gene, puromycin) A targeting vector is designed to replace the cassette with a resistance gene or the like (FIG. 1A). In addition, when a recombinase target sequence such as a loxP sequence used in the Cre / loxP system is added to both sides of the positive selection marker gene, the marker gene can be excised via the Cre enzyme. Diphtheria toxin A (DT-A) gene, thymidine kinase (TK) gene and the like are incorporated for negative selection.

  The above targeting vector is introduced into somatic cells such as embryonic stem cell clones and primary cell lines. Embryonic stem cell clones can be established by culturing inner cell mass separated from blastocysts of animals such as mice, humans, hamsters and pigs on feeder cells. Commercially available embryonic stem cell clones can be used, and examples thereof include TT2, ES-D3, SCC-PSA1, and the like.

  The embryonic stem cells into which the targeting vector is incorporated are cultured in an appropriate selection medium such as a medium containing G418 antibiotics for neomycin resistance selection, and then a recombinant embryonic stem cell clone is isolated.

  Screening for appropriately integrated recombinant embryonic stem cell clones is performed by Southern blot or PCT. FIG. 5A shows the position of each probe with wild type (WT) and targeted allele (TA) for Southern blot analysis.

  Embryonic stem cell clones confirmed to have homologous recombination are introduced into mouse embryos at the 8-cell stage, etc. by microinjection, aggregation, etc. to produce chimeric embryos.

  The resulting chimeric embryo is transferred to the animal's uterus to create a chimeric animal. The resulting chimeric animal is crossed with a wild type animal (eg, C57BL / 6 mouse). Transmission to germ cells is confirmed by genomic PCR or Southern blot analysis. The neomycin resistance gene cassette flanked by LoxP sequences is removed by crossing with Meox-Cre transgenic animals. The removal of the neomycin resistance gene via Cre is confirmed by genomic PCR. Chimeric animals are mated with wild type animals to obtain Mkx heterozygotes.

  Mkx heterozygotes are mated to obtain a homozygote (Mkx knockout mouse).

  The present invention is also characterized by using the differentiation-inducing agent, tendon and / or ligament cells obtained by the differentiation-inducing method, or tendons and / or ligaments of genetically modified non-human animals, tissues or cells thereof, Methods of analyzing or analyzing tendon or ligament related diseases are provided. Analysis and analysis targets include embryos and tissues of Mkx hetero mice obtained by differentiating cells by causing differentiation inducers to differentiate, tendons and / or ligaments obtained from transgenic non-human animals, tissues or cells thereof, etc. It is. Examples of analysis and analysis methods are given below.

  Whole mount in situ hybridization (WISH) is effective for analyzing gene expression patterns in mouse embryos. For WISH, a conventional method can be used. For example, Yokoyama S, et al. (2008) Dynamic gene expression of Lin-28 during embedding development in mouse and chicken. The method described in Gene Expr Patterns 8 (3): 155-160 is followed. Details of the method of synthesizing the probe used for this can be viewed on the EMBRYS website (http://embrys.jp/html/MainMenu.html?).

  A fluorescent protein such as Venus is effective for observing the expression pattern of each tissue of the Mkx gene.

  Immunohistochemical staining is effective for observing mouse embryos and adult mouse tissues. Markers specific to each tissue include myosin heavy chain in muscle and PECAM1 in blood vessel. The expression of these marker genes is immunohistologically stained using marker-specific polyclonal or monoclonal antibodies. First, a tissue in which a mouse embryo is fixed to paraformaldehyde is embedded in a frozen tissue section preparation embedding agent and frozen. The specimen is sliced and air-dried, and then blocked with a blocking agent for 1 hour. The sections are incubated with anti-myosin heavy chain antibody (MF20, DSHB) and further incubated with a secondary antibody. In Venus knock-in Mkx hetero mice, both red staining of myosin heavy chain antibody (MF20) and Venus green signal are observed.

  In order to investigate abnormalities of tendons in Mkx knockout mice, it is effective to perform histological observation of tendons and ligaments. Examples of such methods include electron microscope observation, H & E staining, and Azan staining.

  Using an electron microscope, collagen fibrils in the tendons of Mkx knockout mice can be observed. First, an Achilles tendon fragment is collected and fixed with a buffer solution. Wash with buffer and physiological saline, dehydrate with ethanol, embed in resin and cut into very thin slices. The obtained slice is placed on a copper lattice and observed with a transmission electron microscope.

  From the H & E staining, the cell density of the tail tendon of wild type mice and Mkx knockout mice can be determined. First, tissues such as adult knees and tails are collected and fixed in paraformaldehyde or the like. The tissue is dehydrated, embedded in paraffin, sliced and stained with hematoxylin and eosin (H & E).

  Azan staining detects collagen. First, mouse tails are collected and fixed in 4% paraformaldehyde. The tissue is dehydrated, embedded in paraffin, sliced and stained with azocarmine-aniline blue (Azan).

  In order to examine the mechanical characteristics of a tendon lacking Mkx, for example, a tensile test is performed using all tendons from a muscle tendon junction to a knot. Tension determination is described by Gentleman E, et al. (2003) Mechanical characterization of collagen fibers and scaffolds for tissue engineering. A uniaxial material test system (Autograph AGS-G, Shimadzu Corp. Ltd.) may be used according to the method reported in Biomaterials 24 (21): 3805-3813. The initial length and cross-sectional area of each specimen is measured by microscopic examination of H & E stained sections using a digital caliper. For example, Trapezium software (registered trademark, Shimadzu) is used for data collection. The stress-strain curve of each specimen is created from a load-displacement curve, and the Young's modulus is calculated from each stress-strain curve and tendon cross-sectional area.

  The present invention also provides the above-described differentiation induction method in the presence or absence of a test substance, and a cell having the ability to differentiate into a tendon and / or ligament cell in the presence or absence of the test substance. Screening for or evaluating a substance related to the regulation of differentiation from tendon and / or ligament cells into tendon and / or ligament cells, characterized by comparing differentiation into ligament cells Provide a method.

  Preferably, the expression level of Mkx and / or the transcription amount of mRNA for the base sequence encoding Mkx in a cell cultured in the presence of the test substance is measured, and the cell cultured in the absence of the test substance Including comparing to the amount of. When the amount to be measured is large or small, the test substance is selected as a substance related to regulation of differentiation from tendon and / or ligament cell to tendon and / or ligament cell.

  The culture medium is appropriately determined according to the cells used. For example, MEM medium, DMEM medium, RPM1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the cells.

  The present invention also provides a tendon and / or ligament cell from a cell capable of differentiating into a tendon and / or ligament cell, characterized by using a tendon obtained from the genetically modified non-human animal, a tissue thereof or a cell thereof. Provided is a method for screening or evaluating a substance related to regulation of differentiation into a cell. Preferably, the test substance is brought into contact with a tendon collected from a genetically modified non-human animal, its tissue or its cell, and the test substance that increases or decreases the expression of a reporter gene such as a knocked-in fluorescent protein is selected. Including.

  The test substance to be used for the screening or evaluation method is not particularly limited. For example, natural or artificial organic compounds and inorganic compounds, nucleic acids, peptides, proteins, carbohydrates, lipids, cells, plants, animals and other extracts.

  The contact of the test substance with the cell to induce differentiation or the tissue of the genetically modified non-human animal is not particularly limited, but includes addition, transfection, introduction with a viral vector, and the like.

  The expression level of Mkx and / or the amount of mRNA transcription with respect to the base sequence encoding Mkx and the measurement of the increase or decrease in the expression of the reporter gene are based on conventional methods, for example, Northern blotting, real-time PCR, RT-PCR, Western blot Law, immunological techniques, etc.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
[Example 1] (Production and evaluation of differentiation inducer)
A differentiation inducer comprising an expression vector containing a nucleic acid encoding Mkx protein was introduced into C3H10T1 / 2 mouse fibroblasts to overexpress Mkx, thereby examining the effect of the differentiation inducer.

First, a FLAG-Mkx expression vector was prepared by incorporating the entire protein coding region of the mouse Mkx gene (NCBI accession number NM — 177595) into the p3XFLAG-CMV ^™ -7.1 vector (SIGMA). An empty vector that does not incorporate the FLAG-Mkx gene was used as a negative control.

  C3H10T1 / 2 cells (obtained from ATDC) were cultured in DMEM medium containing 10% FBS. 50% confluent C3H10T1 / 2 cells cultured in a 6-well plate were transfected with 10 μl of a transfection reagent (product name, FugeneHD, Roche) and 1 μg of the FLAG-Mkx expression vector or empty vector.

  Cells were harvested after 48 hours of culture. Total soluble protein in C3H10T1 / 2 cells by lysing cells with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% DOC, 0.1% SDS, 1% NP40; pH 8.0) Were obtained and subjected to the following Western blot analysis.

  The obtained total soluble protein was separated by SDS-PAGE and blotted on a PVDF membrane by a semi-dry method. The membrane was blocked with a blocking agent (product name: Blocking One, Nacalai Tesque) for 30 minutes and incubated with anti-type I collagen antibody or anti-β-actin antibody at 4 ° C. overnight.

  After washing, it was incubated with horseradish peroxidase (HRP) -labeled anti-rabbit immunoglobulin G (IgG) antibody (A6154, SIGMA) or HRP-labeled anti-mouse IgG antibody (A2304, SIGMA) for 1 hour. For the color development of the antigen-antibody, a chemiluminescent reagent (product name Chemi-Lumi One, Nacalai Tesque) was used.

  FIG. 4D shows the results of Western blotting of type I collagen (Col1), FLAG and β-actin of C3H10T1 / 2 cells transfected with the FLAG-Mkx expression vector or empty vector. As shown in FIG. 4D, when Mkx was overexpressed in C3H10T1 / 2 cells, the amount of type I collagen increased.

[Example 2] (Production and evaluation of Mkx mutant mice)
In order to functionally delete the Mkx gene on the mouse chromosome, FIG. 1A shows the relationship between the Mkx gene from the start of translation of Mkx to the end of exon in the homologous recombination in embryonic cells. A targeting vector was designed to replace the cassette.

  Based on the targeting construct of FIG. 1A, PCR was performed using a C57BL / 6 genomic BAC clone (BACPAC Resource Center) as a template to amplify the 5 ′ and 3 ′ adjacent regions of the Mkx gene. A targeting vector was prepared by incorporating these homologous recombination regions into a vector derived from pBluescript containing a Venus gene, a neomycin resistance gene for positive selection, and a diphtheria toxin A (DT-A) gene for negative selection.

  The above targeting vector was linearized with the restriction enzyme NotI and introduced into TT2F embryonic stem cells by electroporation. TT2F embryonic stem cells incorporating the targeting vector were cultured in a medium containing G418 to isolate recombinant embryonic stem cell clones.

  Screening for properly integrated recombinant embryonic stem cell clones was performed by Southern blotting using 5 'probe, 3' probe and neo probe.

  FIG. 5A shows the position of each probe for wild-type (WT) and targeted allele (TA) for Southern blot analysis. FIG. 5B shows the results of Southern blotting of recombinant embryonic stem cells. FIG. 5C shows the results of Southern blotting of wild type mice and Mkx mutant mice.

  Two embryonic stem cell clones confirmed to have homologous recombination were injected into 8-cell mouse embryos. The resulting chimeric mice were crossed with C57BL / 6 mice, and transmission to germ cells was confirmed by genomic PCR. The neomycin resistance gene cassette sandwiched between the LoxP sequences was removed by crossing with Meox-Cre transgenic mice, and the removal of the neomycin resistance gene via Cre was confirmed by genomic PCR. Table 1 shows the sequences of PCR primers used for genotyping.

  FIG. 1B shows the results of genomic PCR using primers a, b and c of wild type mice and Mkx mutant mice.

  Venus knock-in Mkx hetero mice produced using the targeting vector are positive for Mkx expression, and expression of the knocked-in Venus gene is also expected. Therefore, the fluorescence signal of the mouse embryo was observed. Whole mount in situ hybridization was performed using the Mkx probe.

  FIG. 1C shows Mkx whole mount in situ hybridization of wild type and Mkx heterozygous embryos in the E13.5 forelimb and tail. In the forelimb and tail of Venus knock-in Mkx hetero mice, Venus expression was observed at a location similar to that of Mkx.

  For immunohistochemical staining of Mkx heteromouse embryos, the tails of E16.5 mouse embryos were collected and fixed in 4% paraformaldehyde at 4 ° C. for 2 hours. The tissue was embedded in an embedding agent for frozen tissue section preparation (product name OCT compound, Sakura Finetec) and immediately frozen in liquid nitrogen. The specimen was sliced into 10 μm, and the frozen section was air-dried and then blocked with Blocking One (Nacalai Tesque) for 1 hour. The sections were incubated overnight at 4 ° C. with anti-myosin heavy chain antibody (MF20, DSHB). After washing, it was incubated with a secondary antibody fluorescent reagent (product name: Molecular Probes Alexa 594) for 1 hour.

  FIG. 1D shows a micrograph observing immunohistochemical staining and anti-Venus fluorescence signal using anti-myosin heavy chain antibody (MF20; red) in the tail of an embryo of E16.5 Venus knock-in Mkx heteromouse. In the figure, the portion surrounded by a circle indicates MF20 immunohistochemical staining (actually colored in red), and the others indicate a Venus fluorescent signal (actually colored in green). Venus knock-in Mkx heteromouse embryos showed tendon-specific Venus expression in the E16.5 tail.

  In addition to analysis of Mkx heteromouse embryos, the expression of Venus in the adult mice was also investigated. Since the expression of Mkx in the adult tendon was not clear, the expression of Mkx in the adult tendon was investigated by RT-PCR. FIG. 6A shows RT-PCR analysis of Mkx and Gapdh in muscle, Achilles tendon and tail tendon of wild type mice (adult). From FIG. 6A, it was confirmed that Mkx is strongly expressed in the Achilles tendon and the tail tendon.

  FIG. 6B shows Venus expression and appearance photographs in the Achilles tendon (BE), tail tendon (FI), and trunk tendon (JM) of Mkx hetero mice and wild type mice. From FIG. 6B, Venus in Mkx hetero mice showed specific expression in Achilles tendon, tail tendon and trunk tendon.

  In order to confirm the deficiency of the Mkx gene, expression analysis by RT-PCR using Mkx mutant mice was performed. Total RNA was isolated using a nucleic acid extractant (product name ISOGEN, Nippongene). From the isolated total RNA, reverse transcription was performed using a cDNA synthesis kit (product name Ready-To-Go You-Prime First-Strand Beads, GE Healthcare), and the cDNA was used as a template for DNA polymerase (product name Go-Taq (registered) (Trademark) and Promega). The primer sequences are shown in Table 2. Gapdh was used as mRNA control.

  FIG. 1E shows the results of RT-PCR analysis of Mkx and Gapdh in the Achilles tendon of wild type mice and Mkx mutant mice. From FIG. 1E, it was found that the expression of Mkx was completely lost in the Achilles tendon of the Mkx knockout mouse (adult).

  From the above results, it was shown that Mkx mutant mice (Venus knock-in Mkx hetero mice and Mkx knock-out mice) are useful not only for functional analysis of Mkx but also for biological analysis of tendons and expression analysis of Mkx.

[Example 3] (Production and evaluation of Mkx knockout mouse)
Mkx knockout mice were produced by crossing the Mkx hetero mice produced in Example 2. Table 3 shows the number and ratio of newborn mice of each genotype.

  The results of genotyping 206 newborn mice born by crossing heterozygous mice followed Mendel's law.

  Mkx heterozygous mice and Mkx homozygous mice (knockout mice) survived and reproduced without problems, and the results of weighing were the same as those of wild-type mice (FIG. 7).

  No tendon abnormality was observed in the Mkx hetero mice (FIG. 6B). In order to investigate the role of Mkx in tendon formation, it was examined whether or not tendon abnormalities were observed in Mkx knockout mice.

  FIG. 2A shows patella tendon (A arrow), Achilles tendon (C arrow, hind limb (E arrow), forelimb tendon (G arrow), tail tendon (I arrow), back tendon in wild type mouse. (K arrow) and cervical tendon tendon (M arrow), and Mkx knockout mice, patella tendon (B arrow), Achilles tendon (D arrow, hind limb (F arrow), forelimb tendon ( (H arrow), tail tendon (J arrow), back tendon (L arrow) and cervical tendon tendon (N arrow) are shown in Mkx knockout mice, patella tendon, Achilles tendon, tail tendon, Leg tendons, trunk tendons and cervical tendon tendons were hypoplastic and pale in white compared to wild-type mice, and tendon abnormalities were observed in Mkx knockout mice from 7 days to 8 months of age. And mice from which the neomycin resistance gene has been removed (FIG. 8) It has been police.

  In order to investigate the mechanical properties of tendons lacking Mkx, a tensile test was performed using all tendons from the muscle tendon junction to the fistula. Tension determination is described by Gentleman E, et al. (2003) Mechanical characterization of collagen fibers and scaffolds for tissue engineering. According to the method reported in Biomaterials 24 (21): 3805-3813, it was performed using a uniaxial material test system (Autograph AGS-G, Shimadzu). The specimen was pulled at a heavy pressure rate of 0.5 mm / sec.

  The initial length and cross-sectional area of each specimen was measured by microscopic examination of H & E stained sections using a digital caliper. Data collection was performed using Trapeium (registered trademark, Shimadzu) software. The stress-strain curve of each specimen was created from a load-displacement curve, and the Young's modulus was calculated from each stress-strain curve and tendon cross-sectional area.

  FIG. 2B shows the tensile strength of the Achilles tendon of wild type mice and Mkx knockout mice. The Achilles tendon of the Mkx knockout mouse had a low tensile strength and a reduced function. FIG. 2C shows the Young's modulus of the Achilles tendon of wild type mice and Mkx knockout mice. The values of Young's modulus indicating the tensile strength per unit area and the elasticity per unit area were almost the same in the wild type mouse and the Mkx knockout mouse. From the above results, it was suggested that the decrease in the tensile strength in the Achilles tendon of the Mkx knockout mouse was due to the tendon becoming thinner.

  In order to investigate the tendon abnormalities of Mkx knockout mice in more detail, histological analysis was performed. Adult mouse knees or tails were collected and fixed overnight in 4% paraformaldehyde at 4 ° C. The tissue was dehydrated, embedded in paraffin, sliced and stained with hematoxylin and eosin (H & E).

  FIG. 3A shows H & E-stained images of the patella tendon of 3 month old wild type mice and Mkx knockout mice. As a result of H & E staining, it was found that the patellar tendon of the 3-month-old Mkx knockout mouse was thinner than that of the wild-type mouse.

  FIG. 3B shows the patella tendon thickness of 7-day-old wild type mice and Mkx knockout mice. Even in the 7-day-old knockout mice, the patella tendon was thin and about half the thickness of the wild-type mouse tendon (FIG. 3B).

  FIG. 9 shows H & E-stained images of knee cruciate ligaments of wild type mice and Mkx knockout mice. In the knee cruciate ligament, which is one of the ligaments having a composition similar to that of the tendon, no difference was observed between the wild type mouse and the Mkx knockout mouse.

  FIG. 3C shows H & E-stained images of tail tendons of 3 month-old wild-type mice and Mkx knockout mice. In the tail tendon of the Mkx knockout mouse, the diameter of the tendon fiber bundle was reduced (Δ arrow in FIG. 3C).

  FIG. 3D shows the cell density of the tail tendon of 3 month old wild type mice and Mkx knockout mice. In the tail tendon of the Mkx knockout mouse, the cell density was high, and the number of cells in one tail tendon fiber bundle was similar to that of the wild type mouse. This suggests that Mkx knockout tendon cells are functionally incomplete.

  In order to investigate whether tendon dysplasia in Mkx knockout mice was observed even in embryonic period, Azan staining of tail tendon of E18.5 Mkx-deficient embryos was performed. Specifically, tails were collected and fixed overnight in 4% paraformaldehyde at 4 ° C. The tissue was dehydrated, embedded in paraffin, sliced, and stained with azocarmine-aniline blue (Azan).

  FIG. 3E shows an Azan staining image of the tail tendon of embryos of E18.5 wild-type mice and Mkx knockout mice. From FIG. 3E, the size of the tail tendon is not different from that of the wild type mouse. In the Mkx knockout embryo, it was found that the density of aniline blue staining for detecting collagen was low (Δ arrow head).

To analyze the collagen fibrils in the tendons of Mkx knockout mice, the Achilles tendons were observed with an electron microscope. An Achilles tendon fragment of about 1 mm ³ was collected and fixed overnight in 0.1 M cacodylate buffer containing 4% paraformaldehyde and 2.5% glutaraldehyde. It was washed with 0.1 M cacodylate buffer and physiological saline, dehydrated with ethanol, embedded in an epoxy resin and cut into ultrathin slices. The obtained slice was placed on a copper lattice, compared with aqueous uranyl acetate and lead citrate, and observed with a transmission electron microscope (H-7100, Hitachi).

  FIG. 3F shows electron microscope observation images of collagen fibrils of the Achilles tendon of wild type mice and Mkx knockout mice. It was found that the diameter of collagen fibrils in the tendon of the knockout mouse was uniformly smaller than that of the wild type mouse. These results suggested that collagen was decreased in the tendons of Mkx knockout mice and that Mkx was important for tendon differentiation.

  As shown in the tail tendon cell density of the 3-month-old Mkx knockout mouse (FIG. 3D), the number of tendon cells in the Mkx knockout mouse is the same as that in the wild-type mouse, although the amount of tendon is decreased. It didn't change. This suggests that the ability to produce extracellular matrix is reduced in Mkx knockout tendon cells. Further, from the Azan staining image of the tail tendon of the embryo of E18.5 Mkx knockout mouse shown in FIG. 3E and the fine structure analysis of collagen fibrils of the Achilles tendon of Mkx knockout mouse shown in FIG. It is thought that productivity is decreasing.

  In order to investigate the collagen-producing ability of tendon cells of Mkx knockout mice, the amount of collagen in tendons of 8-week-old wild type mice and Mkx knockout mice was measured. The collagen was quantified according to the instructions of a commercially available collagen quantification kit (product name: Sircol Soluble Collagen Assay, Biocolor). The results are shown in FIG. 4A. It was found that the amount of collagen in the tendon of the knockout mouse was smaller than that in the wild type mouse.

  FIG. 4B shows Western blot analysis of type A collagen (Col1) and β-actin in the Achilles tendon of 8-week-old wild type mice and Mkx knockout mice. Both protein and mRNA in Mkx knockout mice were decreased, as shown by the expression level of type I collagen, the major component of the tendon extracellular matrix.

  Col1a1, Col1a2 and Scx real time PCR of Achilles tendon of 8-week-old wild type mice and Mkx knockout mice was performed using SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences are shown in Table 4. Gapdh was used as mRNA control.

  FIG. 4C shows a graph of the relative expression level measured by the delta-delta CT method. In FIG. 4C, the expression of Scx, a transcription factor suggested to induce expression of type I collagen, was not reduced. This suggested that the decrease in type I collagen in Mkx knockout mice was not a decrease in Scx.

  From the above results, it was demonstrated that Mkx is an important factor that regulates the expression of type I collagen in tendon cells.

Claims (2)

(A) Mkx protein,
(B) In the amino acid sequence of the Mkx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and tendons and / or ligaments A mutant having an activity of enhancing the production of type I collagen in cells having the ability to differentiate into cells,
(C) a nucleic acid encoding the protein of (A) or (B), and
(D) A collagen production enhancer that enhances the production of type I collagen in cells capable of differentiating into tendon and / or ligament cells, comprising as an active ingredient at least one expression vector comprising the nucleic acid of (C). (A1) Scx protein,
(B1) In the amino acid sequence of the Scx protein, one to several amino acids have at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion, and induce type I collagen A mutant having the activity of
(C1) a nucleic acid encoding the protein of (A1) or (B1), and
(D1) an expression vector comprising the nucleic acid of (C1),
The collagen production enhancer which enhances the production of type I collagen in cells having the ability to differentiate into tendon and / or ligament cells according to claim 1, comprising at least one of the above.