HK1240598B - Peptide for inducing regeneration of tissue and use thereof - Google Patents

Description

Peptides for inducing tissue regeneration and their applications

This application is a divisional application of the Chinese patent application with the application date of April 3, 2012, the priority date of April 26, 2011, the Chinese patent application number 201280031164.X, and the invention name “Peptides for inducing tissue regeneration and their applications”.

Technical Field

The present invention relates to peptides for inducing tissue regeneration and uses thereof.

Background Art

It has become increasingly clear that tissue stem cells exist in various organs and tissues within a living organism, maintaining the homeostasis of their structure and function. For example, myocardial stem cells exist in the heart, neural stem cells exist in the brain, and epidermal stem cells and hair follicle stem cells exist in the skin. These cells supply myocardial cells, neural cells, epidermal cells, and hair follicle epithelial cells throughout life, maintaining the structure and function of the heart, brain, and skin. Meanwhile, hematopoietic stem cells exist in the bone marrow, which differentiate into blood cells such as red blood cells, white blood cells, and platelets. Blood cells derived from these hematopoietic stem cells circulate through the bloodstream throughout all organs and tissues in the body, performing functions essential for maintaining life, such as oxygen supply, immune response, hemostasis, and repair of damaged tissue. In other words, rather than saying that bone marrow hematopoietic stem cells contribute to the homeostasis of the tissues in which they are locally located, namely the bone marrow and bone tissue, it is more accurate to say that they contribute to the homeostasis of all tissues in the body through the peripheral circulation.

In recent years, it has been established that, in addition to hematopoietic stem cells, the bone marrow also contains mesenchymal stem cells, which can differentiate into mesodermal tissues such as bone, cartilage, and fat, and further into ectodermal tissues such as nerves and epidermis. However, the significance of their existence in the body remains largely unknown. However, since hematopoietic stem cells exist in the bone marrow and maintain the homeostasis of all tissues and organs by supplying blood cells through the peripheral circulation, it is expected that mesenchymal stem cells, also present in the bone marrow, may also contribute to the maintenance of tissue homeostasis by supplying cells capable of differentiating into bone, cartilage, fat, nerves, epithelium, and other cells to the required tissues and organs through the peripheral circulation.

Currently, efforts are underway to develop regenerative medicine that involves collecting bone marrow mesenchymal stem cells (BMSCs) through bone marrow blood collection, growing them through cell culture, and then transplanting them into sites of refractory tissue damage or into the peripheral circulation to induce regeneration of damaged tissue. Clinical applications of BMSC transplantation have already made progress in regenerative medicine for conditions such as cerebral infarction, myocardial infarction, and intractable skin ulcers. Furthermore, transplanted BMSCs have been shown to suppress inflammation and immune responses and fibrous scar formation locally within the body. Clinical trials of BMSC transplantation therapy have begun as a new treatment for graft-versus-host disease (GVHD), a serious side effect of bone marrow transplantation or blood transfusion, and the autoimmune disease scleroderma. However, BMSCs can only be collected through the invasive method of repeatedly inserting a thick needle into the iliac bone. Furthermore, BMSCs gradually lose their ability to proliferate and differentiate into multiple cellular phenotypes with continued in vitro subculture. Furthermore, the cultivation of mesenchymal stem cells (MSCs) based on high-quality management to ensure safety for in vivo transplantation requires specialized culture facilities such as CPCs (cell processing centers), and therefore is currently only available at a very limited number of universities and companies. In other words, in order for the vast number of patients worldwide suffering from intractable tissue damage to benefit from regenerative medicine using MSCs, the urgent challenge is to develop technologies for MSC-based regenerative medicine that can be implemented in all medical institutions.

HMGB1 (High Mobility Group Box 1) was identified approximately 30 years ago as a non-histone chromatin protein that controls gene expression and DNA repair by regulating nuclear chromatin structure. The HMGB1 protein structure primarily consists of two DNA-binding domains: the N-terminal domain is called the A-box, and the C-terminal domain is called the B-box. Previous studies have established that the domain within the HMGB1 molecule that binds to TLRs and triggers inflammatory responses is located within the B-box.

Prior art literature

Patent Literature

Patent Document 1: WO2008/053892

Patent Document 2: WO2007/015546

Patent Document 3: WO2009/133939

Patent Document 4: WO2009/133943

Patent Document 5: WO2009/133940

Patent Document 6: Japanese Patent Application No. 2005-537253

Non-patent literature

Non-patent document 1: Bustin et al., Mol Cell Biol, 19: 5237-5246, 1999

Non-patent document 2: Hori et al., J. Biol. Chem., 270, 25752-25761, 1995

Non-patent literature 3: Wang et al., Science, 285: 248-251, 1999

Non-patent document 4: Muller et al., EMBO J, 20: 4337-4340, 2001

Non-patent literature 5: Wang et al., Science, 285: 248-251, 1999

Non-patent literature 6: Germani et al., J Leukoc Biol. Jan; 81(1):41-5, 2007

Non-patent document 7: Palumbo et al., J. Cell Biol., 164: 441-449, 2004

Non-patent document 8: Merenmies et al., J. Biol. Chem., 266: 16722-16729, 1991

Non-patent document 9: Wu Y et al., Stem cells, 25: 2648-2659, 2007

Non-patent document 10: Tamai et al., Proc Natl Acad Sci U S A. 2011 Apr 4. [Epub ahead of print], 108: 6609-6614, 2011

Non-patent literature 11: Yang et al., J Leukoc Biol. Jan; 81(1):59-66, 2007

Summary of the Invention

Problems to be solved by the invention

Recently, the present inventors investigated the regenerative mechanism of desquamated epidermis in epidermolysis bullosa, an intractable hereditary skin disease characterized by whole-body skin desquamation from birth and symptoms resembling thermal burns, due to genetic abnormalities in adhesion molecules in the basement membrane region of the skin. Using epidermolysis bullosa model mice transplanted with GFP (green fluorescent protein) transgenic bone marrow cells, they demonstrated that HMGB1 (High Mobility Group Box 1) released from desquamated epidermis into the blood stimulates the mobilization of PDGFRα (platelet-derived growth factor receptor alpha)-positive cells in the bone marrow and induces their accumulation in the desquamated epidermis. Furthermore, the PDGFRα-positive cells accumulated in the desquamated epidermis differentiate into fibroblasts or epidermal cells, strongly contributing to the regeneration of damaged skin. Furthermore, by administering recombinant HMGB1 protein via the tail vein after inducing skin ulcers or cerebral infarction in mice, researchers demonstrated that PDGFRα-positive cells were mobilized from the bone marrow into the bloodstream and subsequently accumulated at the site of the skin ulcer or cerebral infarction, strongly inducing regeneration of the skin ulcer or cerebral infarction. PDGFRα-positive cells in the bone marrow have been reported to be mesenchymal stem cells capable of differentiating into bone, cartilage, fat, nerves, and epithelium. In other words, by mobilizing PDGFRα-positive mesenchymal stem cells in the bone marrow into the peripheral circulation through the administration of HMGB1, it is possible to accumulate large numbers of mesenchymal stem cells in damaged tissues in vivo without requiring specialized culture or removal outside the body.

By developing HMGB1 as a drug for inducing damaged tissue regeneration by mobilizing bone marrow mesenchymal stem cells from the blood, regenerative medicine using bone marrow mesenchymal stem cells will become available at all medical institutions. Consequently, many of the challenges currently facing regenerative medicine using bone marrow mesenchymal stem cells will be resolved.

As described above, HMGB1 drugs are groundbreaking therapeutics that promote the mobilization of bone marrow mesenchymal stem cells into the blood and their accumulation in damaged tissues, thereby inducing tissue regeneration in the body. In the inventors' studies to date, no side effects have been observed even when high concentrations of recombinant HMGB1 protein were administered to mice or rats. Furthermore, considering the inventors' observation that extremely high concentrations of HMGB1 are present in the peripheral blood of patients with epidermolysis bullosa who have no severe symptoms other than epidermal exfoliation, it is speculated that HMGB1 administration is highly safe. However, there are also reports that HMGB1 has inflammatory effects. Therefore, while numerous discoveries have been made regarding HMGB1, no findings have been made regarding the effects of HMGB1 protein fragments on mesenchymal stem cells or their role in tissue regeneration.

Means used to solve problems

The inventors secreted recombinant proteins consisting of a peptide composed of amino acids 1 to 84 and a peptide composed of amino acids 85 to 169 from the HMGB1 protein into the culture medium of HEK293 cells. The target proteins were purified from the culture medium by column chromatography and their migratory activity in a PDGFRα-positive bone marrow mesenchymal stem cell line (MSC-1) was confirmed. The peptide composed of amino acids 1 to 84 exhibited migratory activity.

Next, the inventors prepared peptides consisting of amino acids 1 to 44 and 45 to 84 of the peptide consisting of amino acids 1 to 84 that had been shown to have migratory activity against MSC-1 and examined their migratory activity. The results confirmed that all of the prepared peptide fragments exhibited migratory activity against the PDGFRα-positive bone marrow mesenchymal stem cell line (MSC-1).

Therefore, the present inventors chemically synthesized various peptide fragments, mainly containing fragments with slight overlap, and attempted to evaluate their migration activity on the PDGFRα-positive bone marrow mesenchymal stem cell line (MSC-1). As a result, they successfully identified several peptides that exhibited migration activity.

Furthermore, the present inventors have confirmed that the identified peptide also has migration activity on skin fibroblasts, which are PDGFRα-positive cells, and has an effect of reducing the size of cerebral infarction lesions in cerebral infarction model mice.

The inventors expressed recombinant proteins consisting of a peptide composed of amino acids 2 to 84 and a peptide composed of amino acids 89 to 215 of the HMGB1 protein in Escherichia coli. The expressed proteins were purified by column chromatography and their migration activity was confirmed in PDGFRα-positive bone marrow mesenchymal stem cell line (MSC-1) and human bone marrow mesenchymal stem cells. The inventors confirmed migration activity for the peptide composed of amino acids 2 to 84 and the peptide composed of amino acids 89 to 215.

Next, the inventors prepared peptides consisting of amino acids 2 to 44 and 45 to 84 of the peptide consisting of amino acids 2 to 84, which had been shown to exhibit migratory activity in MSC-1 and human bone marrow mesenchymal stem cells, and examined their migratory activity. The results confirmed that each of the prepared peptide fragments exhibited migratory activity in both the PDGFRα-positive bone marrow mesenchymal stem cell line (MSC-1) and human bone marrow mesenchymal stem cells.

Next, the inventors prepared peptides consisting of amino acids 89 to 215, which had been shown to exhibit migratory activity in MSC-1 and human bone marrow mesenchymal stem cells, by gradually shortening the C-terminus. These peptides consisted of amino acids 89 to 205, 89 to 195, and 89 to 185, and examined their migratory activity. The results showed that the shorter the C-terminus of the prepared peptide fragments, the greater their migratory activity in PDGFRα-positive bone marrow mesenchymal stem cells (MSC-1) and human bone marrow mesenchymal stem cells.

In addition, three fusion peptides were prepared in which all or part (10, 20, or 30 amino acid sequences) of the acidic tail composed of aspartic acid and glutamic acid at the C-terminus were added to a peptide consisting of amino acids 2 to 84. Surprisingly, in all cases, the migration activity of the peptides at positions 2 to 84 was greatly reduced. This suggests that all or part of the acidic tail inhibits migration activity in full-length HMGB1. This time, the fragmentation has clarified at least three or more migration-active domains, suggesting the possibility that any one domain is inhibited by the acidic tail in the full-length state.

Furthermore, the present inventors confirmed that the identified peptides have therapeutic effects on a skin injury model.

Based on this finding, the present application provides the following inventions.

[1] A composition for stimulating cell migration, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

[2] A composition for mobilizing cells from bone marrow into peripheral blood, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

[3] A composition for tissue regeneration, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

[4] The composition according to any one of [1] to [3], wherein the cells stimulated to migrate or mobilized from the bone marrow into the peripheral blood are PDGFRα-positive cells.

[5] The composition according to any one of [1] to [4], wherein the cells stimulated to migrate or mobilized from the bone marrow into the peripheral blood are stem cells.

[6] The composition according to any one of [1] to [5], wherein the cells stimulated to migrate or mobilized from the bone marrow into the peripheral blood are bone marrow cells.

[7] The composition according to any one of [1] to [6], wherein the cells stimulated to migrate or mobilize from the bone marrow into the peripheral blood are bone marrow mesenchymal stem cells.

[8] A composition as described in any one of [1] to [7], wherein the peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein is a peptide having cell migration stimulating activity consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 in the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5.

[9] The composition according to any one of [1] to [7], wherein the peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein is a peptide having cell migration stimulating activity comprising any of the following amino acid sequences:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[10] The composition according to any one of [1] to [7], wherein the peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein is a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, and is a peptide having cell migration stimulating activity comprising any one of the following amino acid sequences:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[11] A composition for stimulating cell migration, comprising a peptide comprising any one of the following amino acid sequences and having cell migration stimulating activity:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[12] A composition for mobilizing cells from bone marrow into peripheral blood, comprising a peptide having cell migration stimulating activity comprising any one of the following amino acid sequences:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[13] A composition for tissue regeneration, comprising a peptide comprising any one of the following amino acid sequences and having cell migration stimulating activity:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[14] The composition according to any one of [1] to [13], wherein the peptide is a synthetic peptide.

[15] The composition according to any one of [1] to [14], wherein the peptide is produced using cells.

[16] The composition according to any one of [1] to [15], wherein the peptide is a peptide to which a tag is attached

[17] The composition according to any one of [1] to [15], wherein the peptide is a peptide to which a peptide fragment derived from a tag is attached.

[18] A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein.

[19] A peptide as described in [18], which is a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 in the amino acid sequence shown in any one of sequence numbers 1, 3, and 5 and has cell migration stimulating activity.

[20] The peptide according to [18], which is a peptide comprising any one of the following amino acid sequences and having cell migration stimulating activity:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[21] The peptide according to [18], which is a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, and is a peptide having cell migration stimulating activity comprising any one of the following amino acid sequences:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[22] A peptide comprising any one of the following amino acid sequences and having cell migration stimulating activity:

(1) the amino acid sequence from positions 17 to 25 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(2) the amino acid sequence from position 45 to position 74 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(3) the amino acid sequence from position 55 to position 84 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5;

(4) the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; and

(5) The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5.

[23] The peptide according to any one of [18] to [22], wherein the peptide is a synthetic peptide.

[24] The peptide according to any one of [18] to [22], wherein the peptide is produced using cells.

[25] The peptide according to any one of [18] to [22], wherein the peptide is a peptide to which a tag is attached.

[26] The peptide according to any one of [18] to [22], wherein the peptide is a peptide to which a peptide fragment derived from a tag is attached.

[27] A DNA encoding the peptide according to any one of [18] to [26].

[28] A vector comprising the DNA described in [27].

[29] A transformed cell containing the DNA of [27] or the vector of [28].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing expression vectors for producing peptides and proteins using HEK293 cells.

Figure 2A is a photograph showing the migratory activity of peptides on a PDGFRα-positive bone marrow mesenchymal stem cell line. Positive controls included full-length HMGB1 (1-215), a peptide consisting of amino acids 1 to 84 (1-84), a peptide consisting of amino acids 85 to 169 (85-169), a peptide consisting of amino acids 1 to 44 (1-44), and a peptide consisting of amino acids 45 to 84 (45-84). These peptides were all produced using HEK293. Figure 2B is a western blot analysis confirming the presence of PDGFRα expression in human bone marrow mesenchymal stem cells. PDGFRα expression was confirmed in human bone marrow mesenchymal stem cells.

Figure 3 shows photographs showing the migratory activity of peptides on a PDGFRα-positive bone marrow mesenchymal stem cell line. A peptide consisting of amino acids 45 to 215 of HMGB1 (45-215) and a peptide consisting of amino acids 63 to 215 (63-215) were compared. These peptides were produced using HEK293.

FIG4 shows photographs of bone marrow cells collected from PDGFRα-GFP mice, cultured for a certain period of time in adherent cell culture dishes, and then separated from CD11b-positive cells using MACS to observe GFP fluorescence in each cell.

FIG5 is a photograph showing the migration activity of HMGB1_1-44 peptide on primary cultured bone marrow mesenchymal stem cells.

FIG6 shows photographs showing the osteogenic differentiation ability (A) and adipogenic differentiation ability (B) of primary cultured bone marrow mesenchymal stem cells (PDGFRα-positive, Lin-negative, c-kit-negative).

FIG. 7 presents photographs showing the migration activity of various synthetic peptides on PDGFRα-positive bone marrow mesenchymal stem cell lines.

Figure 8A is a photograph showing the migration activity of various synthetic peptides on PDGFRα-positive bone marrow mesenchymal stem cell lines. Figure 8B is a graph obtained by quantifying the migration activity of the lower left photograph in Figure 8A. The number of cells migrating toward each synthetic peptide and the negative control was counted under a microscope. The values were plotted with the average value of the negative control as 100. Figure 8C is a photograph showing the results of the migration activity of various peptides on bone marrow mesenchymal stem cells (MSC-1). The number of cells in each spot in the photograph was counted and the average was taken, and expressed as a percentage relative to the negative control.

FIG. 9 presents photographs showing the migration activity of various synthetic peptides on PDGFRα-positive bone marrow mesenchymal stem cell lines.

Figure 10 shows photographs and graphs showing the migratory activity of various peptides on PDGFRα-positive bone marrow mesenchymal stem cell lines. HMGB1_1-44 peptides (1-44) produced using various synthetic peptide production systems, including HEK293 cells that stably secrete the peptide, HEK293 cells that transiently transfect with a plasmid and secrete the peptide, and E. coli, were compared with full-length HMGB1 as a positive control.

FIG. 11 is a diagram showing FACS analysis of PDGFRα, cell lineage markers, and CD44 in a PDGFRα-positive bone marrow mesenchymal stem cell line.

Figure 12A is a photograph showing the migration activity of HMGB1_1-34 peptide (1-34) on mouse keratinocytes. Figure 12B is a photograph showing immunohistochemical images of keratin 5 in the skin of PDGFRα-GFP mice. Keratinocytes, which are keratin 5-positive cells, do not express PDGFRα.

Figure 13A is a photograph showing the migration activity of HMGB1_1-34 peptide (1-34) on mouse skin fibroblasts. Figure 13B is a photograph showing immunohistochemical analysis of vimentin in the skin of PDGFRα-GFP mice. Some vimentin-positive skin fibroblasts express PDGFRα.

Figure 14 shows FACS analysis of skin fibroblasts from PDGFRα-GFP mice and fibroblasts from wild-type mice (C57/Bl6 mice). PDGFRα is expressed in almost 98% of mouse skin fibroblasts.

FIG. 15 is a graph showing the mobilization of PDGFRα-positive CD44-positive cells into the blood by the synthetic peptide (1-44) using FACS.

Figure 16 shows cross-sectional photographs of the brain of a rat cerebral infarction model to which synthetic peptide (1-44) and negative control PBS were administered. A reduction in the size of cerebral infarction was observed in the synthetic peptide (1-44).

Figure 17 is a graph and photograph showing the ratio of the infarct area to the right brain area in rat cerebral infarction models administered with synthetic peptide (1-44) and negative control PBS. Four cross sections were prepared from the same brain and the area of each section was measured.

Figure 18A shows a human HMGB1 with a 6xHis tag and a TEV protease cleavage sequence added to the N-terminus. A cDNA expressing this protein was newly prepared and inserted into an E. coli expression vector.

Figure 18B is a photograph showing the migratory activity of HMGB1 fragments on PDGFRα-positive bone marrow mesenchymal stem cell lines. These fragments were all produced using E. coli. Figure 18C is a graph showing the quantification of the migratory activity of HMGB1 fragments on PDGFRα-positive bone marrow mesenchymal stem cell lines and the average values of each activity. Figure 18D is a table showing the quantification of the migratory activity of HMGB1 fragments on PDGFRα-positive bone marrow mesenchymal stem cell lines and the average values.

Figure 19, Panel A, shows a photograph of an HMGB1 fragment consisting of amino acids 89 to 215 produced in E. coli, further purified using an anion exchange column after nickel affinity purification (I: input), and subjected to SDS-PAGE of the fractions. M represents a molecular weight marker. At low salt concentrations, a 15.5 kDa fragment (*3) elutes, while as the salt concentration increases, fragments of 16 kDa (*2) and 17 kDa (*1) elute in that order. (*3) and (*2) are presumed to be degradation products of (*1). Panel B shows the migratory activity of the fractions obtained in Panel A on a PDGFRα bone marrow mesenchymal stem cell line. NC is a negative control, and 2-215 is a positive control. The lowest molecular weight fragment (*3), believed to be a cleaved fragment, exhibits greater activity than the longer fragments (*1) and (*2). This activity is stronger than that of 2-215. Figure C shows a photograph of an HMGB1 fragment consisting of amino acids 89 to 205 produced in E. coli, further purified using an anion exchange column after nickel affinity purification, and subjected to SDS-PAGE of the fractions. The shortest fragment (*4) eluted at low salt concentrations, while longer fragments (*5) and (*6) eluted as the salt concentration increased. It is speculated that (*5) and (*6) are degradation products of (*4). Furthermore, purified HMGB1 fragments 89-195 and 89-185 were simultaneously subjected to SDS-PAGE. M represents a molecular weight marker. Figure D shows the migratory activity of the fractions obtained in Figure C on a PDGFRα bone marrow mesenchymal stem cell line. NC is a negative control; the lowest molecular weight fragment (*4), considered to be the cleaved fragment, demonstrated greater activity compared to the longer fragments (*5) or (*6). Furthermore, stronger activity was confirmed in HMGB1 fragments 89-195 and 89-185, which were obtained by further shortening the C-terminus.

In Figure 20, Panel A is a photograph showing the migration activity of HMGB1 fragment 85-169. Activity exceeding that of the positive control, HMGB1 fragment 2-215, was confirmed. Panel B is a graph showing the quantified migration activity of Panel A and the average values plotted. Panel C is a table showing the average values of Panel B.

In Figure 21-1, Panel A is a graph showing the migration activity of HMGB1 fragments 2-215, 2-205, 2-195, and 2-185 produced by E. coli on a bone marrow mesenchymal stem cell line (MSC-1). Panel B is a photograph showing the migration activity of HMGB1 fragments produced by E. coli on human bone marrow mesenchymal stem cells.

In Figure 21-2, Panel C shows the fusion fragments "(2-84) + (186-215), (2-84) + (186-205), (2-84) + (186-195)" obtained by subjecting the purified human HMGB1 fragment (2-84) to the acidic tail fragments (186-215), (186-205), and (186-195) of human HMGB1 to SDS-PAGE, and then staining the electrophoresis gel with CBB for protein. Each purified fragment can be confirmed. Panel D shows the migration activity of the purified fragments on MSC-1. None of the fusion fragments with the acidic tail sequence attached to the 2-84 fragment showed migration activity. On the other hand, the 2-84 fragment itself showed migration activity.

Figure 22 shows a photograph of a skin flap created on the back of a rat one week after surgery. PBS served as the negative control group. Furthermore, a comparison was made between a group administered with a full-length HMGB1 produced using HEK293 cells (1-215(HEK)) and a group administered with a peptide synthesized from amino acids 1 to 44 (1-44(synthetic peptide)). Arrows indicate necrotic skin tissue.

Figure 23 shows a photograph of a skin flap created on the back of a rat five weeks after treatment. PBS served as the negative control group. Furthermore, a comparison was made between a group administered with a peptide containing full-length HMGB1 produced using HEK293 cells (1-215(HEK)) and a group administered with a peptide synthesized from amino acids 1 to 44 (1-44(synthetic peptide)). The reddish areas indicate areas of skin ulceration.

Figure 24 shows a photograph of a skin flap created on the back of a rat 7 weeks after surgery. PBS served as the negative control group. Furthermore, a comparison was made between a group administered with a full-length HMGB1 produced using HEK293 cells (1-215(HEK)) and a group administered with a peptide synthesized from amino acids 1 to 44 (1-44(synthetic peptide)).

Figure 25 quantifies the area of wounds (necrotic areas) in skin flaps created on the backs of rats. One to three weeks after flap creation, groups receiving full-length HMGB1 produced in HEK293 cells (1-215(HEK)) and a peptide synthesized from amino acids 1 to 44 (1-44(synthetic peptide)) demonstrated a reduction in wound size compared to the negative control group. Four weeks later, 1-44(synthetic peptide) demonstrated a further reduction in size compared to the other two groups.

FIG26 shows the ratio (%) of the area of each skin injury site to the area of the entire skin flap 2 weeks and 6 weeks after skin injury in which chemically synthesized HMGB1 peptides (1-44) and (17-25) were administered from the tail vein of rats.

DETAILED DESCRIPTION

The present invention provides a composition for stimulating cell migration, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

The compositions for stimulating cell migration of the present invention include reagent compositions and pharmaceutical compositions. In this specification, reagent compositions are also referred to as reagents, and pharmaceutical compositions are also referred to as drugs, pharmaceutical agents, or pharmaceutical compositions.

The reagent composition for stimulating cell migration of the present invention can be used as a reagent for basic research and clinical studies, such as those related to regenerative medicine and regenerative induction medicine. For example, by using this reagent composition, cells can be mobilized into the biological tissues of experimental animals, thereby studying the extent of tissue repair and tissue function restoration. Furthermore, by using this reagent composition, tissue regeneration induction studies using cell mobilization can be conducted in vitro.

Furthermore, the pharmaceutical composition for stimulating cell migration of the present invention can be used as a drug in, for example, regenerative medicine and regenerative induction medicine. For example, the use of this pharmaceutical composition can lead to tissue regeneration. Furthermore, for example, this pharmaceutical composition can be used as a so-called preventive drug to prevent the decline in tissue and organ function caused by a decrease in tissue stem cells, or as an anti-aging drug to slow the progression of aging-related changes.

In this specification, the composition for stimulating cell migration is also referred to as an agent for stimulating cell migration, a stimulator of cell migration, a composition for inducing cell migration, an agent for inducing cell migration, an inducer of cell migration, or a cell inducer.

In the present invention, cell migration stimulating activity refers to activity that stimulates cell migration. In this specification, cell migration stimulating activity is also expressed as cell migration inducing activity or cell inducing activity.

The present invention provides a composition for mobilizing myeloid cells from the bone marrow into peripheral blood, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

The composition for mobilizing myeloid cells from the bone marrow into peripheral blood of the present invention includes a reagent composition and a pharmaceutical composition.

The reagent composition for tissue regeneration of the present invention can be used as a reagent required for basic research and clinical research for the development of, for example, regenerative medicine and regenerative induction medicine. In addition, the pharmaceutical composition for tissue regeneration of the present invention can be used as a drug in, for example, regenerative medicine and regenerative induction medicine. For example, by using this pharmaceutical composition, medullary tissue stem cells can be mobilized into the peripheral circulation, thereby regenerating tissue. For example, cells mobilized into peripheral blood using this pharmaceutical composition can be recovered in vitro and then concentrated and applied to tissue for treatment. In previous methods, in order to recover cells from the bone marrow deep in the body, it is invasive to the organism. However, if the pharmaceutical composition of the present invention is used, medullary cells can be recovered from peripheral blood with low invasiveness and used for medullary cell transplantation, etc. In addition, in this specification, the composition for mobilizing medullary cells from the bone marrow into peripheral blood is also described as a composition for inducing medullary cells from the bone marrow into peripheral blood.

The present invention provides a composition for tissue regeneration, comprising any one of the following substances (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

The composition for tissue regeneration of the present invention includes a reagent composition and a pharmaceutical composition.

The reagent composition for tissue regeneration of the present invention can be used as a reagent required for basic research and clinical research in the development of, for example, regenerative medicine and regenerative induction medicine. Furthermore, the pharmaceutical composition for tissue regeneration of the present invention can be used as a pharmaceutical in, for example, regenerative medicine and regenerative induction medicine.

In addition, in this specification, a composition for tissue regeneration is also expressed as a composition for inducing or promoting tissue regeneration, an agent for inducing or promoting tissue regeneration, a tissue regeneration inducer, or a tissue regeneration promoter. In addition, tissue regeneration also includes tissue repair.

The composition for tissue regeneration of the present invention does not select the site of application/addition. That is, the composition can be applied to any tissue such as the tissue in need of regeneration, a tissue different from the tissue in need of regeneration, or blood, and its effect can be exerted. For example, by applying/adding the composition, cells are mobilized to the tissue at or near the site of application/addition, thereby inducing or promoting tissue regeneration. For another example, by applying/adding the composition to or near the site of damaged tissue, cells are mobilized to the damaged tissue, thereby inducing or promoting tissue regeneration. For another example, by applying/adding the composition to a tissue different from the tissue in need of regeneration, bone marrow cells are mobilized from the bone marrow to the tissue in need of regeneration through the peripheral circulation, thereby inducing or promoting tissue regeneration. Here, "peripheral circulation" is also referred to as "blood circulation" or "peripheral circulation blood flow".

Examples of tissues that require regeneration include damaged tissue, necrotic tissue, postoperative tissue, tissue with reduced function, fibrotic tissue, aged tissue, diseased tissue, and the like. Examples include skin tissue of a living organism, in vivo biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testicles, blood, etc.).

Being applied to the tissue different from the tissue that needs to be regenerated refers to being applied to the position (the position different from the position that needs to be regenerated) other than the position that needs to be regenerated. Therefore, " the tissue different from the tissue that needs to be regenerated " can also be expressed as the position different from the tissue that needs to be regenerated, the position different from the position that needs to be regenerated, the position separated from the tissue that needs to be regenerated, the position separated from the position that needs to be regenerated, the position located at the distant position of the position that needs to be regenerated, the tissue located at the distant position of the tissue that needs to be regenerated, the distant portion, the distant tissue. That is, the compositions of the present invention is effectively used to make it difficult to regenerate the tissue (brain, heart, etc.) that directly administers medicament from outside the body.

Cells mobilized into the tissue requiring regeneration differentiate into various cell types, thereby contributing to the functional regeneration, maintenance, and enhancement of the tissue requiring regeneration. In the present invention, tissues requiring regeneration include, but are not limited to, tissues damaged by various pathological conditions such as ischemia, chronic ischemia/hypoxia, trauma, burns, inflammation, autoimmunity, and genetic abnormalities.

As tissues in the present invention, as long as they are tissues that cells derived from bone marrow can differentiate into, there are no particular restrictions, and examples include: skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, myocardial tissue, nervous system tissue, lung tissue, digestive tract tissue, liver/gallbladder/pancreatic tissue, urinary/reproductive system, and all tissues in the body. In addition, by using the above-mentioned composition, not only can skin diseases such as refractory skin ulcers, skin wounds, bullous diseases, and alopecia be treated, but also functional tissue regeneration can be induced in tissues that require regeneration such as cerebral infarction, myocardial infarction, bone fractures, pulmonary infarction, gastric ulcers, and enteritis. As animal species to which the above-mentioned composition is administered, there are no particular restrictions, and examples include: mammals, birds, fish, and the like. As mammals, human or non-human animals can be exemplified, such as humans, mice, rats, apes, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, whales, etc., but are not limited thereto.

Examples of tissues other than the tissue to be regenerated include blood tissue, muscle tissue, subcutaneous tissue, intradermal tissue, and abdominal cavity.

Examples of neural tissue include, but are not limited to, central nervous system tissue. Furthermore, compositions for regenerating neural tissue can be used to treat, for example, cerebral infarction, cerebral hemorrhage, and cerebral contusion, but are not limited thereto. Furthermore, compositions for regenerating bone tissue can be used to treat, for example, fractures, but are not limited thereto. Furthermore, compositions for regenerating skin tissue can be used to treat, for example, skin ulcers, surgical wound suture defects, burns, cuts, contusions, skin erosions, and abrasions, but are not limited thereto.

In addition, in the present invention, as cells that are stimulated to migrate or mobilized from the bone marrow into the peripheral blood, undifferentiated cells and cells in various stages of differentiation can be listed, but are not limited to these. In addition, in the present invention, as cells that are stimulated to migrate or mobilized from the bone marrow into the peripheral blood, stem cells and non-stem cells can be listed, but are not limited to these. Stem cells include circulating stem cells and non-circulating stem cells. As non-circulating stem cells, tissue stem cells resident in tissues can be exemplified. As circulating stem cells, stem cells circulating in the blood can be exemplified.

In addition, as the cell that is stimulated to wander or the cell that is mobilized from the bone marrow to the peripheral blood, the cell of bone marrow origin or hematopoietic stem cell can be enumerated, but are not limited to this. In this specification sheets, " hematopoietic stem cell " is the stem cell that can be differentiated into the leukocytes such as neutrophil, eosinophil, basophil, lymphocyte, monocyte, macrophage and the blood cell system cells such as erythrocyte, platelet, mast cell, dendritic cell, as marker, it is known that CD34 is positive, CD133 is positive for people, is CD34 negative, c-Kit is positive, Sca-1 is positive, cell line marker is negative for mouse. In addition, the feature of hematopoietic stem cell is that, when cultivated in culture dish, it is difficult to cultivate alone, needs to be co-cultured with stromal cells.

In this specification, "marrow cells" refer to cells present in the bone marrow. On the other hand, "marrow-derived cells" refer to "marrow cells" mobilized from the bone marrow to the outside of the bone marrow. "Marrow cells" include cells comprising a group of tissue precursor cells present in the bone marrow. In addition, "marrow-derived cells" may include or exclude mesoangioblasts.

Tissue progenitor cells are defined as undifferentiated cells with the ability to differentiate into specific tissue cells other than the blood system, including undifferentiated cells with the ability to differentiate into the above-mentioned mesenchymal tissues, epithelial tissues, neural tissues, solid organs, and vascular endothelium.

"Marrow cells" and "marrow-derived cells" are stem cells represented by hematopoietic stem cells and differentiated cells such as white blood cells, red blood cells, platelets, osteoblasts, and fibroblasts derived from hematopoietic stem cells, or cells hitherto known as bone marrow mesenchymal stem cells or bone marrow mesenchymal multipotent stem cells or bone marrow multipotent stem cells. In this specification, "marrow stem cells" refer to stem cells existing in the bone marrow, and on the other hand, "marrow-derived stem cells" refer to "marrow stem cells" mobilized from the bone marrow to the outside of the bone marrow. In the present invention, "marrow-derived stem cells" can be listed as cells that are stimulated to migrate or mobilized from the bone marrow to the peripheral blood, but are not limited to these. "Marrow cells" and "marrow-derived cells" can be isolated by bone marrow collection (marrow cell collection) or peripheral blood collection. Hematopoietic stem cells are non-adherent cells, but a portion of "marrow cells" and "marrow-derived cells" can be obtained as adherent cells by culturing the mononuclear cell fraction in blood obtained by medullary collection (marrow cell collection) or peripheral blood collection.

Furthermore, "marrow cells" and "marrow-derived cells" include mesenchymal stem cells, preferably having the ability to differentiate into osteoblasts (which can be confirmed by confirming calcium deposition during differentiation induction), chondrocytes (which can be confirmed by positive staining with Alcian blue or Safranin-O), adipocytes (which can be confirmed by positive staining with Sudan III), and mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, and tendon cells, as well as neural cells, epithelial cells (e.g., epidermal keratinocytes and intestinal epithelial cells expressing the cytokeratin family), and vascular endothelial cells. The differentiated cells are not limited to the aforementioned cells and also include cells capable of differentiating into solid organ cells such as the liver, kidney, and pancreas.

In this specification, "bone marrow mesenchymal stem cells", "bone marrow mesenchymal multipotent cells", or "bone marrow pluripotent stem cells" are cells present in the bone marrow, and are cells with the following characteristics: they are collected directly from the bone marrow or indirectly from other tissues (blood or skin, fat, other tissues), can be cultured and proliferated as adherent cells attached to a culture dish (plastic or glass), and have the ability to differentiate into mesenchymal tissues such as bone, cartilage, and fat (mesenchymal stem cells) or skeletal muscle, myocardium, and neural tissue, epithelial tissue (pluripotent stem cells). They are cells that can be obtained by collecting bone marrow cells.

On the other hand, "marrow-derived mesenchymal stem cells," "marrow-derived mesenchymal multipotent cells," or "marrow-derived multipotent stem cells" mobilized from the marrow to the outside of the marrow are cells that can be obtained by sampling peripheral blood or collecting from mesenchymal tissues such as fat, epithelial tissues such as skin, and neural tissues such as the brain.

In addition, these cells have the following characteristics: they have the ability to differentiate into epithelial tissues such as keratinocytes that constitute the skin and neural tissues that constitute the brain, by directly administering them to the damaged part of the organism after collection or by administering cells temporarily attached to a culture dish to the damaged part of the organism.

Marrow mesenchymal stem cells, marrow mesenchymal multipotent stem cells, marrow multipotent stem cells, or these cells mobilized from the marrow to outside the marrow preferably have the ability to differentiate into osteoblasts (which can be determined by confirming calcium deposition when differentiation is induced), chondrocytes (which can be determined by positive staining with Alcian blue, positive staining with Safranin-O, etc.), and adipocytes (which can be determined by positive staining with Sudan III), as well as the ability to differentiate into mesenchymal cells such as fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, tendon cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (expressing the cytokeratin family, the hair keratin family, etc.), epithelial cells (for example, epidermal keratinocytes and intestinal epithelial cells express the cytokeratin family), endothelial cells, and solid organ cells such as the liver, kidney, and pancreas, but the differentiated cells are not limited to the above cells.

Examples of human bone marrow cells and cells derived from human bone marrow include, but are not limited to, cells obtained by bone marrow collection (marrow cell collection), peripheral blood collection, or adipose tissue collection, or cells that can be obtained as adherent cells by direct culture or culturing after separation of a mononuclear cell fraction. Examples of markers for human bone marrow cells and cells derived from human bone marrow include, but are not limited to, all or some of the following: PDGFRα-positive, Lin-negative, CD45-negative, CD44-positive, CD90-positive, CD29-positive, Flk-1-negative, CD105-positive, CD73-positive, CD90-positive, CD71-positive, Stro-1-positive, CD106-positive, CD166-positive, and CD31-negative.

In addition, examples of mouse medullary cells and mouse medullary-derived cells include, but are not limited to, cells obtained by medullary collection (marrow cell collection), peripheral blood collection, or adipose tissue collection, or cells that can be obtained as adherent cells by direct culture or culturing after separation of a mononuclear cell fraction. Examples of markers for mouse medullary cells and mouse medullary-derived cells include, but are not limited to, all or some of the following: CD44-positive, PDGFRα-positive, PDGFRβ-positive, CD45-negative, Lin-negative, Sca-1-positive, c-kit-negative, CD90-positive, CD29-positive, and Flk-1-negative.

In the present invention, PDGFRα-positive cells can be cited as cells that are stimulated to migrate or mobilized from the bone marrow into the peripheral blood, but are not limited thereto. In addition, as markers other than PDGFRα, all or part of CD29-positive, CD44-positive, CD90-positive, CD271-positive, CD11b-negative, and Flk-1-negative can be exemplified, but are not limited thereto. PDGFRα-positive cells include PDGFRα-positive bone marrow-derived cells, PDGFRα-positive bone marrow-derived bone marrow mesenchymal stem cells, tissue cells resident in PDGFRα-positive tissues (for example, fibroblasts, etc.), PDGFRα-positive bone marrow-derived cells, cells that can be obtained as adherent cells by culturing mononuclear cell fractions in blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection, etc., but are not limited thereto.

The composition of the present invention may contain other substances in addition to at least one of the substances described in (a) to (c) above. In the composition of the present invention, there is no particular limitation on substances other than at least one of the substances described in (a) to (c) above, as long as they do not inhibit cell migration stimulation activity, cell mobilization activity or tissue regeneration promotion activity. For example, in addition to containing at least one of the substances described in (a) to (c) above, the composition of the present invention may also contain related molecules (groups) that strengthen the functions of the substances described in (a) to (c), molecules (groups) that inhibit the effects other than the desired effects of the substances described in (a) to (c) above, factors that control cell proliferation and differentiation, and other factors that strengthen/maintain the functions of these factors or cells.

Examples of the HMGB1 protein in the present invention include, but are not limited to, a human-derived HMGB1 protein comprising the amino acid sequence shown in SEQ ID NO: 1 and a DNA encoding the protein comprising the base sequence shown in SEQ ID NO: 2.

Other examples include, but are not limited to, a mouse-derived HMGB1 protein comprising the amino acid sequence shown in SEQ ID NO: 3 and a DNA encoding the protein comprising the base sequence shown in SEQ ID NO: 4.

Other examples include, but are not limited to, a rat-derived HMGB1 protein comprising the amino acid sequence shown in SEQ ID NO: 5 and a DNA encoding the protein comprising the base sequence shown in SEQ ID NO: 6.

The composition of the present invention contains a peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein. The peptide consisting of a portion of the HMGB1 protein of the present invention is not particularly limited as long as it contains a domain having cell migration stimulating activity.

The cell migration stimulating activity of a peptide consisting of a portion of the HMGB1 protein can be confirmed by, for example, the methods described in the Examples and the following methods, but is not limited thereto and can also be determined by other in vitro or in vivo cell migration ability assays.

A method in which a silicone tube inserted with HMGB1 protein or peptide is implanted subcutaneously, removed after a certain period of time, and cells migrating into the tube are identified.

A method in which HMGB1 protein or peptide is bound to resin microbeads, etc., embedded in biological tissue, and then removed after a certain period of time to identify cells that have migrated to the microbeads.

A method in which HMGB1 protein or peptide is impregnated into a sustained-release polymer such as gelatin or hyaluronic acid, embedded in biological tissue, and then removed after a certain period of time to identify cells that have migrated to the polymer.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include, but are not limited to, the following peptides.

In the present invention, examples of the peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides having activity of mobilizing cells from the bone marrow into the peripheral blood or activity of promoting tissue regeneration.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides having cell migration stimulating activity comprising at least any of the following amino acid sequences: The following amino acid sequence is a portion of the amino acid sequence represented by any of SEQ ID NOs. 1, 3, and 5.

(1) Amino acid sequence from position 17 to position 25,

(2) amino acid sequence from position 45 to position 74,

(3) amino acid sequence from position 55 to position 84,

(4) Amino acid sequence from position 85 to position 169,

(5) amino acid sequence from position 89 to position 185, and

(6) Amino acid sequence from position 93 to position 215.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides consisting of all or part of the amino acid sequence from positions 1 to 195 or the amino acid sequence from positions 1 to 185 of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5, and peptides having cell migration stimulating activity comprising at least one of the following amino acid sequences. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5.

(1) a peptide comprising the amino acid sequence from position 17 to position 25,

(2) a peptide comprising the amino acid sequence from position 45 to position 74,

(3) a peptide comprising the amino acid sequence from position 55 to position 84,

(4) a peptide comprising the amino acid sequence from position 85 to position 169, and

(5) A peptide comprising the amino acid sequence from position 89 to position 185.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include: a peptide consisting of a portion of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity, and a peptide comprising at least the amino acid sequence from positions 17 to 25 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity. Examples of such peptides include, but are not limited to, a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5 (the number of amino acids in the peptide is a natural number selected from 195 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 185 (the number of amino acids in the peptide is a natural number selected from 185 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 170 (the number of amino acids in the peptide is a natural number selected from 170 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 84 (the number of amino acids in the peptide is a natural number selected from 84 or less), or a peptide consisting of all or part of the amino acid sequence from positions 1 to 44 (the number of amino acids in the peptide is a natural number selected from 44 or less), and a peptide having cell migration stimulating activity that includes at least the amino acid sequence from positions 17 to 25 of the amino acid sequence.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 17 to 25 of the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as a “peptide consisting of all or part of the amino acid sequence from positions 1 to 185 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 17 to 25 of the amino acid sequence and having cell migration stimulating activity” may also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 17 to 25 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 17 to 25 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5, and at least comprising the amino acid sequence from positions 17 to 25 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence of any one of the following (1) to (60), and at least comprising the amino acid sequence from positions 17 to 25 in the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5, and containing at least the amino acid sequence from positions 17 to 25 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5, and containing at least any one of the following amino acid sequences (1) to (57) and (59) to (61) in the amino acid sequence shown in any one of SEQ ID NOs. 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any one of SEQ ID NOs. 1, 3, and 5, and containing at least the amino acid sequence from positions 17 to 25 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of any one of the following amino acid sequences (1) to (61) and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any one of SEQ ID NOs. 1, 3, and 5.

(1) Amino acid sequence from position 1 to position 44,

(2) amino acid sequence from position 1 to position 25,

(3) amino acid sequence from position 1 to position 34,

(4) amino acid sequence from position 1 to position 42,

(5) amino acid sequence from position 1 to position 43,

(6) amino acid sequence from position 1 to position 45,

(7) amino acid sequence from position 1 to position 46,

(8) Amino acid sequence from position 1 to position 47,

(9) Amino acid sequence from position 1 to position 48,

(10) amino acid sequence from position 1 to position 49,

(11) amino acid sequence from position 1 to position 50,

(12) amino acid sequence from position 1 to position 51,

(13) amino acid sequence from position 1 to position 52,

(14) amino acid sequence from position 1 to position 62,

(15) amino acid sequence from position 1 to position 84,

(16) amino acid sequence from position 10 to position 25,

(17) amino acid sequence from position 11 to position 25,

(18) Amino acid sequence from position 11 to position 27,

(19) Amino acid sequence from position 11 to position 28,

(20) amino acid sequence from position 11 to position 29,

(21) amino acid sequence from position 11 to position 30,

(22) amino acid sequence from position 11 to position 34,

(23) amino acid sequence from position 11 to position 44,

(24) amino acid sequence from position 12 to position 25,

(25) amino acid sequence from position 12 to position 30,

(26) amino acid sequence from position 13 to position 25,

(27) Amino acid sequence from position 13 to position 30,

(28) Amino acid sequence from position 14 to position 25,

(29) Amino acid sequence from position 14 to position 30,

(30) amino acid sequence from position 15 to position 25,

(31) amino acid sequence from position 15 to position 30,

(32) amino acid sequence from position 16 to position 25,

(33) amino acid sequence from position 16 to position 30,

(34) amino acid sequence from position 17 to position 30,

(35) amino acid sequence from position 1 to position 70,

(36) amino acid sequence from position 1 to position 81,

(37) Amino acid sequence from position 1 to position 170,

(38) amino acid sequence from position 2 to position 25,

(39) amino acid sequence from position 2 to position 34,

(40) amino acid sequence from position 2 to position 42,

(41) amino acid sequence from position 2 to position 43,

(42) amino acid sequence from position 2 to position 44,

(43) amino acid sequence from position 2 to position 45,

(44) amino acid sequence from position 2 to position 46,

(45) amino acid sequence from position 2 to position 47,

(46) amino acid sequence from position 2 to position 48,

(47) amino acid sequence from position 2 to position 49,

(48) amino acid sequence from position 2 to position 50,

(49) amino acid sequence from position 2 to position 51,

(50) amino acid sequence from position 2 to position 52,

(51) amino acid sequence from position 2 to position 62,

(52) amino acid sequence from position 2 to position 70,

(53) amino acid sequence from position 2 to position 81,

(54) amino acid sequence from position 2 to position 84,

(55) amino acid sequence from position 2 to position 170,

(56) Amino acid sequence from position 17 to position 44,

(57) amino acid sequence from position 1 to position 185,

(58) amino acid sequence from position 1 to position 195,

(59) amino acid sequence from position 2 to position 185,

(60) amino acid sequence from position 2 to position 195, and

(61) Amino acid sequence from position 17 to position 25.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides having cell migration stimulating activity consisting of a portion of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, and peptides having cell migration stimulating activity comprising at least the amino acid sequence from positions 45 to 74 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5. Examples of such peptides include, but are not limited to, a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5 (the number of amino acids in the peptide is a natural number selected from 195 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 185 (the number of amino acids in the peptide is a natural number selected from 185 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 170 (the number of amino acids in the peptide is a natural number selected from 170 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 84 (the number of amino acids in the peptide is a natural number selected from 84 or less), or a peptide consisting of all or part of the amino acid sequence from positions 45 to 84 (the number of amino acids in the peptide is a natural number selected from 40 or less), and a peptide comprising at least the amino acid sequence from positions 45 to 74 of the amino acid sequence and having cell migration stimulating activity.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 45 to 74 of the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as a “peptide consisting of all or part of the amino acid sequence from positions 1 to 185 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 45 to 74 of the amino acid sequence and having cell migration stimulating activity” may also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 45 to 74 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 45 to 74 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 45 to 74 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence of any one of the following (a) to (k), and at least comprising the amino acid sequence from positions 45 to 74 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and containing at least the amino acid sequence from positions 45 to 74 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and containing at least any one of the following amino acid sequences (a) to (h) and (j) to (l) in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5, and comprising at least amino acids from positions 45 to 74 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of any of the amino acid sequences (a) to (l) below and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5.

(a) amino acid sequence from position 1 to position 84,

(b) amino acid sequence from position 45 to position 84,

(c) amino acid sequence from position 1 to position 81,

(d) amino acid sequence from position 1 to position 170,

(e) amino acid sequence from position 2 to position 81,

(f) amino acid sequence from position 2 to position 84,

(g) amino acid sequence from position 2 to position 170,

(h) amino acid sequence from position 1 to position 185,

(i) amino acid sequence from position 1 to position 195,

(j) amino acid sequence from position 2 to position 185,

(k) amino acid sequence from position 2 to position 195, and

(l) Amino acid sequence from position 45 to position 74.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include: peptides having cell migration stimulating activity consisting of a portion of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, and peptides having cell migration stimulating activity comprising at least the amino acid sequence from positions 55 to 84 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5.

Examples of such peptides include, but are not limited to, a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5 (the number of amino acids in the peptide is a natural number selected from 195 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 185 (the number of amino acids in the peptide is a natural number selected from 185 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 170 (the number of amino acids in the peptide is a natural number selected from 170 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 84 (the number of amino acids in the peptide is a natural number selected from 84 or less), or a peptide consisting of all or part of the amino acid sequence from positions 45 to 84 (the number of amino acids in the peptide is a natural number selected from 40 or less), and a peptide having cell migration stimulating activity that includes at least the amino acid sequence from positions 55 to 84 of the amino acid sequence.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 55 to 84 in the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as a “peptide consisting of all or part of the amino acid sequence from positions 1 to 185 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 55 to 84 in the amino acid sequence and having cell migration stimulating activity” may also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 55 to 84 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 55 to 84 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 55 to 84 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence of any one of the following (A) to (J), and at least comprising the amino acid sequence from positions 55 to 84 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 55 to 84 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least any one of the following amino acid sequences (A) to (G) and (I) to (K) in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of amino acids 1 to 195 of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5, and comprising at least amino acids 55 to 84 of the amino acid sequence, and having cell migration stimulating activity" includes a peptide consisting of any of the following amino acid sequences (A) to (K) and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5.

(A) amino acid sequence from position 1 to position 84,

(B) amino acid sequence from position 45 to position 84,

(C) amino acid sequence from position 1 to position 170,

(D) amino acid sequence from position 2 to position 84,

(F) a peptide comprising the amino acid sequence from position 2 to position 170,

(G) amino acid sequence from position 1 to position 185,

(H) amino acid sequence from position 1 to position 195,

(I) amino acid sequence from position 2 to position 185,

(J) amino acid sequence from position 2 to position 195, and

(K) Amino acid sequence from position 55 to position 84.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include: peptides having cell migration stimulating activity consisting of a portion of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, and peptides having cell migration stimulating activity comprising at least the amino acid sequence from positions 85 to 169 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5.

Examples of such peptides include, but are not limited to, a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, or 5 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 195 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 185 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 185 or less), a peptide consisting of all or part of the amino acid sequence from positions 1 to 170 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 170 or less), or a peptide consisting of all or part of the amino acid sequence from positions 89 to 185 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 101 or less), and a peptide comprising at least the amino acid sequence from positions 85 to 169 of the amino acid sequence and having cell migration stimulating activity.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 85 to 169 in the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as a “peptide consisting of all or part of the amino acid sequence from positions 1 to 185 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 85 to 169 in the amino acid sequence and having cell migration stimulating activity” may also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 85 to 169 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 85 to 169 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 85 to 169 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence of any one of the following (i) to (vi), and at least comprising the amino acid sequence from positions 85 to 169 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 85 to 169 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least any one of the following amino acid sequences (i) to (iii) and (v) to (vii) in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of amino acids 1 to 195 of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5, and comprising at least amino acids 85 to 169 of the amino acid sequence, and having cell migration stimulating activity" includes a peptide consisting of any of the following amino acid sequences (i) to (vii) and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5.

(i) amino acid sequence from position 1 to position 170,

(ii) amino acid sequence from position 2 to position 170,

(iii) amino acid sequence from position 1 to position 185,

(iv) amino acid sequence from position 1 to position 195,

(v) amino acid sequence from position 2 to position 185,

(vi) amino acid sequence from position 2 to position 195, and

(vii) Nos. 85 to 169.

In the present invention, peptides having cell migration stimulating activity and consisting of a portion of an HMGB1 protein include peptides consisting of a portion of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5 and having cell migration stimulating activity, and peptides having cell migration stimulating activity that contain at least the amino acid sequence from positions 89 to 185 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5. Examples of such peptides include, but are not limited to, peptides consisting of all or a portion of the amino acid sequence from positions 1 to 195 of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5 (the number of amino acids in the peptide is a natural number selected from 195 or less), peptides consisting of all or a portion of the amino acid sequence from positions 1 to 185 (the number of amino acids in the peptide is a natural number selected from 185 or less), and peptides having cell migration stimulating activity that contain at least the amino acid sequence from positions 89 to 185 of the amino acid sequence.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 89 to 185 in the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as “a peptide consisting of all or part of the amino acid sequence from positions 1 to 185 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 89 to 185 in the amino acid sequence and having cell migration stimulating activity” can also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 89 to 185 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 89 to 185 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 89 to 185 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence of any one of the following (i) to (vi), and at least comprising the amino acid sequence from positions 89 to 185 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 89 to 185 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 1 to 195 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least any one of the following amino acid sequences (I) and (III) to (V) in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of amino acids 1 to 195 of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5, and comprising at least amino acids 89 to 185 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of any of the following amino acid sequences (I) to (V) and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs. 1, 3, and 5.

(I) amino acid sequence from position 1 to position 185,

(II) amino acid sequence from position 1 to position 195,

(III) amino acid sequence from position 2 to position 185,

(IV) amino acid sequence from position 2 to position 195, and

(V) Amino acid sequence from position 89 to position 185.

In the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include peptides consisting of a portion of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity, and peptides comprising the amino acid sequence from positions 93 to 215 of the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5.

Examples of such peptides include: a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 171 or less), a peptide consisting of all or part of the amino acid sequence from positions 63 to 215 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 153 or less), or a peptide consisting of all or part of the amino acid sequence from positions 89 to 215 (the number of amino acids in the peptide is a number of amino acids selected from natural numbers 123 or less), and a peptide that contains at least the amino acid sequence from positions 93 to 215 in the amino acid sequence and has cell migration stimulating activity, but is not limited thereto.

Hereinafter, a “peptide consisting of all or part of the amino acid sequence from positions 45 to 215 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 93 to 215 of the amino acid sequence and having cell migration stimulating activity” will be described. Other peptides contained in the peptide, such as a “peptide consisting of all or part of the amino acid sequence from positions 63 to 215 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 89 to 215 of the amino acid sequence and having cell migration stimulating activity” may also be described in the same manner.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 93 to 215 in the amino acid sequence and having cell migration stimulating activity" can also be expressed as "a peptide consisting of a continuous amino acid sequence selected from the amino acid sequence from positions 45 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 93 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity".

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and at least comprising the amino acid sequence from positions 93 to 215 in the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of any one of the following amino acid sequences (W) to (Y), and at least comprising the amino acid sequence from positions 93 to 215 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least the amino acid sequence from positions 93 to 215 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5, and comprising at least any one of the following amino acid sequences (X) to (Z) in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5 and having cell migration stimulating activity.

In the present invention, "a peptide consisting of all or part of the amino acid sequence from positions 45 to 215 of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, or 5, and comprising at least the amino acid sequence from positions 93 to 215 of the amino acid sequence and having cell migration stimulating activity" includes a peptide consisting of any of the following amino acid sequences (W) to (Z) and having cell migration stimulating activity. The following amino acid sequence is a portion of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, or 5.

(W) a peptide comprising the amino acid sequence from position 45 to position 215,

(X) a peptide comprising the amino acid sequence from position 63 to position 215,

(Y) a peptide comprising the amino acid sequence from position 89 to position 215, and

(Z) A peptide comprising the amino acid sequence from position 93 to position 215.

Specifically, in the present invention, examples of peptides having cell migration stimulating activity and consisting of a portion of the HMGB1 protein include the following peptides, but are not limited thereto.

<1> A peptide comprising the amino acid sequence from position 1 to position 44,

<2> a peptide comprising the amino acid sequence from position 1 to position 25,

<3> a peptide comprising the amino acid sequence from position 1 to position 34,

<4> A peptide comprising the amino acid sequence from position 1 to position 42,

<5> A peptide comprising the amino acid sequence from position 1 to position 43,

<6> A peptide comprising the amino acid sequence from position 1 to position 45,

<7> A peptide comprising the amino acid sequence from position 1 to position 46,

<8> A peptide comprising the amino acid sequence from position 1 to position 47,

<9> A peptide comprising the amino acid sequence from position 1 to position 48,

<10> A peptide comprising the amino acid sequence from position 1 to position 49,

<11> A peptide comprising the amino acid sequence from position 1 to position 50,

<12> A peptide comprising the amino acid sequence from position 1 to position 51,

<13> A peptide comprising the amino acid sequence from position 1 to position 52,

<14> A peptide comprising the amino acid sequence from position 1 to position 62,

<15> A peptide comprising the amino acid sequence from position 1 to position 84,

<16> A peptide comprising the amino acid sequence from position 10 to position 25,

<17> A peptide comprising the amino acid sequence from positions 11 to 25,

<18> A peptide comprising the amino acid sequence from positions 11 to 27,

<19> A peptide comprising the amino acid sequence from positions 11 to 28,

<20> A peptide comprising the amino acid sequence from positions 11 to 29,

<21> A peptide comprising the amino acid sequence from positions 11 to 30,

<22> A peptide comprising the amino acid sequence from positions 11 to 34,

<23> A peptide comprising the amino acid sequence from position 11 to position 44,

<24> A peptide comprising the amino acid sequence from positions 12 to 25,

<25> A peptide comprising the amino acid sequence from positions 12 to 30,

<26> A peptide comprising the amino acid sequence from positions 13 to 25,

<27> A peptide comprising the amino acid sequence from positions 13 to 30,

<28> A peptide comprising the amino acid sequence from positions 14 to 25,

<29> A peptide comprising the amino acid sequence from positions 14 to 30,

<30> A peptide comprising the amino acid sequence from position 15 to position 25,

<31> A peptide comprising the amino acid sequence from positions 15 to 30,

<32> A peptide comprising the amino acid sequence from positions 16 to 25,

<33> A peptide comprising the amino acid sequence from positions 16 to 30,

<34> A peptide comprising the amino acid sequence from positions 17 to 25,

<35> A peptide comprising the amino acid sequence from positions 17 to 30,

<36> A peptide comprising the amino acid sequence from position 45 to position 74,

<37> A peptide comprising an amino acid sequence from position 45 to position 84,

<38> A peptide comprising an amino acid sequence from position 45 to position 215,

<39> A peptide comprising an amino acid sequence from position 55 to position 84,

<40> A peptide comprising an amino acid sequence from position 63 to position 215,

<41> A peptide comprising the amino acid sequence from position 1 to position 70,

<42> A peptide comprising the amino acid sequence from position 1 to position 81,

<43> A peptide comprising the amino acid sequence from position 1 to position 170,

<44> A peptide comprising the amino acid sequence from position 2 to position 25,

<45> A peptide comprising the amino acid sequence from position 2 to position 34,

<46> A peptide comprising the amino acid sequence from position 2 to position 42,

<47> A peptide comprising the amino acid sequence from position 2 to position 43,

<48> A peptide comprising the amino acid sequence from position 2 to position 44,

<49> A peptide comprising the amino acid sequence from position 2 to position 45,

<50> A peptide comprising the amino acid sequence from position 2 to position 46,

<51> A peptide comprising the amino acid sequence from position 2 to position 47,

<52> A peptide comprising the amino acid sequence from position 2 to position 48,

<53> A peptide comprising the amino acid sequence from position 2 to position 49,

<54> A peptide comprising the amino acid sequence from position 2 to position 50,

<55> A peptide comprising the amino acid sequence from position 2 to position 51,

<56> A peptide comprising the amino acid sequence from position 2 to position 52,

<57> A peptide comprising the amino acid sequence from position 2 to position 62,

<58> A peptide comprising the amino acid sequence from position 2 to position 70,

<59> A peptide comprising the amino acid sequence from position 2 to position 81,

<60> A peptide comprising the amino acid sequence from position 2 to position 84,

<61> A peptide comprising the amino acid sequence from position 2 to position 170,

<62> A peptide comprising an amino acid sequence from position 85 to position 169,

<63> A peptide comprising an amino acid sequence from position 89 to position 185,

<64> A peptide comprising an amino acid sequence from position 89 to position 195,

<65> A peptide comprising an amino acid sequence from position 89 to position 205,

(66) a peptide comprising the amino acid sequence from position 89 to position 215,

<67> A peptide comprising an amino acid sequence from position 93 to position 215,

<68> A peptide comprising the amino acid sequence from positions 17 to 44,

<69> A peptide comprising the amino acid sequence from position 1 to position 185,

<70> A peptide comprising the amino acid sequence from position 1 to position 195,

<71> A peptide comprising the amino acid sequence from position 1 to position 205,

<72> A peptide comprising the amino acid sequence from position 2 to position 185,

<73> A peptide comprising the amino acid sequence from position 2 to position 195, and

<74> A peptide comprising the amino acid sequence from positions 2 to 205.

Furthermore, in the present invention, the following peptides can be exemplified as peptides having cell migration stimulating activity:

A peptide that comprises a portion of the amino acid sequence shown in any one of sequence numbers 1, 3, and 5 and has cell migration stimulating activity and satisfies the following conditions: two peptides are selected from the above-mentioned group <1> to <74>, the shorter peptide is denoted as A and the longer peptide is denoted as B, and here, a peptide that contains at least A and is composed of all or part of B.

In addition, the present invention also provides peptides comprising at least any one of the following amino acid sequences and having cell migration stimulating activity and their uses. Such peptides include, for example, peptides having cell migration stimulating activity to which one or more (for example, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, 5 or less, 3 or less, 2 or less) amino acid sequences are added to any one of the following amino acid sequences. In addition, peptides comprising at least any one of the following amino acid sequences and having cell migration stimulating activity do not include peptides consisting of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5.

[1] the amino acid sequence from positions 17 to 25 in the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5,

[2] the amino acid sequence from position 45 to position 74 in the amino acid sequence of any one of SEQ ID NOs. 1, 3, and 5,

[3] the amino acid sequence from position 55 to position 84 in the amino acid sequence of any one of SEQ ID NOs. 1, 3, and 5,

[4] the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs. 1, 3, and 5, and

[5] The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5.

The amino acid sequences from positions 1 to 85 of mouse, rat, and human HMGB1 are known as the A-box, and the amino acid sequences from positions 86 to 169 are known as the B-box. The amino acid sequences from positions 1 to 169 in mouse, rat, and human are completely identical, sharing 100% homology. Furthermore, the amino acid sequences from positions 14 to 25 in mouse, rat, and human HMGB2 are also identical to those in HMGB1.

The present invention provides the above-mentioned peptides having cell migration stimulating activity. In addition, the present invention provides DNA encoding the peptide, a vector inserted with the DNA, and a transformed cell introduced with the vector. The peptide encoding DNA of the present invention, the vector inserted with the DNA, and the transformed cell introduced with the vector are prepared using known techniques. As long as the above-mentioned DNA encodes the peptide of the present invention, it can also be, for example, artificially synthesized DNA (e.g., a degenerate mutant).

The present invention also provides peptides that are peptides of the present invention and produced using cells, and peptides that are peptides of the present invention and artificially synthesized. The peptides of the present invention can be obtained in the form of recombinants by recombining DNA encoding the peptide into an appropriate expression system, or can also be artificially synthesized. To obtain the peptides of the present invention by genetic engineering methods, DNA encoding the peptide can be recombined into an appropriate expression system and expressed.

Therefore, the present invention provides a method for producing the peptide of the present invention, comprising the following steps (a) and (b):

(a) a step of introducing a DNA encoding the peptide of the present invention into cells and expressing the peptide,

(b) A step of recovering the peptide from the cells.

The present invention also provides a method for producing the peptide of the present invention having a higher cell migration stimulating activity than that of the HMGB1 protein, comprising the following steps (a) and (b):

(a) a step of introducing a DNA encoding the peptide of the present invention into cells and expressing the peptide, and

(b) A step of recovering the peptide from the cells.

The manufacturing method may further include the following steps.

(c) A step of selecting the peptide having a higher cell migration stimulating activity than that of the HMGB1 protein.

Examples of hosts applicable to the present invention include, but are not limited to, prokaryotic cells and eukaryotic cells. Examples of hosts applicable to the present invention include, but are not limited to, bacteria (e.g., Escherichia coli), yeast, animal cells (e.g., mammalian cells such as HEK293 cells and CHO cells, insect cells such as silkworm cells), and plant cells.

Examples of host/vector systems applicable to the present invention include the expression vector pGEX and Escherichia coli. pGEX can express foreign genes as fusion proteins with glutathione S-transferase (GST) (Gene, 67:31-40, 1988). Therefore, pGEX recombinant with DNA encoding the peptide of the present invention is introduced into E. coli strains such as BL21 by heat shock. After an appropriate incubation period, isopropylthio-β-D-galactopyranoside (IPTG) is added to induce expression of the GST fusion peptide. The GST of the present invention is adsorbed onto glutathione sepharose 4B, allowing the expression product to be easily isolated and purified by affinity chromatography.

In addition, the host/vector systems described below can be used as host/vector systems for obtaining recombinant peptides of the present invention. First, when bacteria are used as hosts, vectors for expressing fusion proteins using tags and the like are commercially available. Furthermore, the recombinant peptides of the present invention also include those in which a tag or a portion of a peptide is attached.

The tag attached to the peptide of the present invention is not particularly limited as long as it has no effect on the activity of the peptide of the present invention, and examples thereof include: histidine tag (e.g., 6×His, 10×His), HA tag, FLAG tag, GST tag, T7-tag, HSV-tag, E-tag, lck tag, B-tag, etc.

In yeast, it is well known that Pichia pastoris (Pichia) yeast is effectively expressed for the protein with sugar chains. In terms of the addition of sugar chains, it is also useful (Bio/Technology, 6:47-55,1988) to utilize the expression system of baculovirus vectors using insect cells as hosts. In addition, mammalian cells are used to transfect vectors using promoters such as CMV, RSV or SV40, and these host/vector systems can all be used as the expression system of the peptide of the present invention. In addition, viral vectors such as plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, Sendai virus vectors, Sendai virus envelope vectors, papillomavirus vectors can also be used to introduce genes, but are not limited to these. The vector can include promoter DNA sequences that efficiently induce gene expression, factors that control gene expression, and molecules required for maintaining the stability of DNA.

The obtained protein of the present invention can be separated from the host cell or extracellularly (culture medium, etc.) and purified into a substantially pure and uniform protein. The separation and purification of the protein can be carried out using the separation and purification methods commonly used in protein purification, without any limitation. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, etc. can be appropriately selected and combined to separate and purify the protein.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Cold Spring Harbor Laboratory Press, 1996). These chromatography methods can be performed using liquid chromatography such as HPLC and FPLC.

Furthermore, the peptides of the present invention are preferably substantially purified. Here, "substantially purified" means that the degree of purification of the peptide of the present invention (the proportion of the peptide of the present invention in the total protein component) is 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, 100%, or close to 100%. The upper limit close to 100% depends on the purification and analytical techniques of those skilled in the art, and examples include 99.999%, 99.99%, 99.9%, 99%, and the like.

Furthermore, as long as the degree of purification is as described above, proteins purified by any purification method are included in substantially purified proteins. For example, substantially purified proteins obtained by appropriately selecting or combining the above-mentioned chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, etc., are exemplified, but are not limited to these.

As cells that secrete the peptide of the present invention, the cells can be prepared by inserting a DNA encoding a secretion signal (e.g., ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG CTCTGG GTT CCA GGT TCC ACT GGT GAC; SEQ ID NO: 10) bound to the DNA encoding the peptide into a known expression vector or a gene therapy vector, and then introducing the vector into mammalian cells such as fibroblasts (e.g., normal skin fibroblasts and cell lines derived from the same), insect cells, and other cells. As DNA encoding a secretion signal, a DNA having the above-mentioned sequence can be exemplified, but is not limited thereto. In addition, the animal species from which these cells are derived is not particularly limited, and it is preferred to use cells of the animal species to which the vector is to be administered, cells of the subject itself, or cells derived from an animal that is related to the subject to which the vector is to be administered.

On the other hand, peptides composed of a portion of HMGB1 can also be artificially synthesized. In the peptide synthesis methods of the present invention, peptides can be chemically synthesized using either liquid-phase peptide synthesis or solid-phase peptide synthesis. In the present invention, peptides synthesized using solid-phase peptide synthesis are preferred. Solid-phase peptide synthesis is one of the commonly used methods for chemically synthesizing peptides. Polystyrene polymer gel beads with a diameter of approximately 0.1 mm, whose surfaces are modified with amino groups, are used as the solid phase, and amino acid chains are extended one amino acid at a time using a dehydration reaction. After the target peptide sequence is formed, it is excised from the solid phase surface to obtain the target substance. Solid-phase synthesis also enables the synthesis of ribosomal peptides, which are difficult to synthesize in bacteria, the introduction of unnatural amino acids such as D-isomers and heavy atom-substituted forms, and the modification of peptide and protein backbones. Using the solid-phase method, long peptide chains of 70 to over 100 amino acids can be synthesized by combining two peptide chains using natural chemical ligation.

The composition of the present invention can be administered orally or parenterally. Specific examples of parenterally administered methods include, but are not limited to, injection, nasal administration, transpulmonary administration, and transdermal administration. Examples of injection include, but are not limited to, intravenous, intramuscular, intraperitoneal, and subcutaneous injections, to administer the composition of the present invention systemically or locally (e.g., subcutaneously, intradermally, to the skin surface, to the eyeball or conjunctiva, to the nasal mucosa, to the oral and digestive tract mucosa, to the vaginal/uterine mucosa, or to a lesion).

Examples of methods for administering the composition of the present invention include, but are not limited to, intravascular administration (intraarterial administration, intravenous administration, etc.), intrablood administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

In addition, the administration site is not limited and can be exemplified by the site of the tissue to be regenerated or its vicinity, a site different from the tissue to be regenerated, or a site remote from and different from the tissue to be regenerated. Examples include, but are not limited to, blood (intra-arterial, intravenous, etc.), muscle, subcutaneous, intradermal, and intraperitoneal administration.

In addition, the method of administration can be appropriately selected according to the patient's age and symptoms. When administering the peptide of the present invention, the dosage can be selected within the range of 0.0000001 mg to 1000 mg per kg of body weight for a single administration, for example. Alternatively, the dosage can be selected within the range of 0.00001 to 100000 mg/body per patient, for example. When administering cells that secrete the peptide of the present invention or gene therapy vectors into which DNA encoding the peptide is inserted, the amount of the peptide can also be administered in a manner that is within the above-mentioned range. However, the pharmaceutical composition of the present invention is not limited to these dosages.

The pharmaceutical composition of the present invention can be prepared into a preparation (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.) according to a conventional method, and can contain pharmaceutically acceptable carriers and additives simultaneously. Examples thereof include surfactants, excipients, colorants, spices, preservatives, stabilizers, buffers, suspending agents, isotonic agents, adhesives, disintegrants, lubricants, flow promoters, flavoring agents, etc., but are not limited to these, and other commonly used carriers can be appropriately used. Specifically, examples thereof include light silicic anhydride, lactose, microcrystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc.

Furthermore, the present invention provides a kit comprising the substance described in any one of the following (a) to (c).

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

The kit can be used to stimulate cell migration, mobilize myeloid cells from the bone marrow into peripheral blood, or regenerate tissue. Examples of the kit include: a kit comprising (1) the above-mentioned substance dissolved in fibrinogen and (2) thrombin, or a kit comprising (1) the above-mentioned substance, (2) fibrinogen, and (3) thrombin. In the present invention, commercially available fibrinogen and thrombin can be used. Examples include: Fibrinogen HT-Wf (Benesis-Mitsubishi Pharma), Beriplast (ZLB Behring), Tisseel (Baxter), Bolheal (Kaikeken), TachoComb (ZLB Behring), but are not limited to these.

Furthermore, the uses of a peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein, cells secreting the peptide, and a vector inserted with a DNA encoding the peptide can also be expressed as the following items (1) to (9).

(1) A method for stimulating cell migration, comprising the step of administering an effective amount of a substance described in any one of the following (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(2) A method for mobilizing cells from bone marrow into peripheral blood, comprising the step of administering an effective amount of a substance described in any one of (a) to (c) below:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(3) A method for tissue regeneration, comprising the step of administering an effective amount of a substance described in any one of the following (a) to (c):

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(4) Use of a substance described in any one of the following (a) to (c) in the manufacture of a composition for stimulating cell migration:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(5) Use of a substance described in any one of the following (a) to (c) for producing a composition for mobilizing cells from bone marrow into peripheral blood:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(6) Use of any one of the following (a) to (c) in the manufacture of a composition for tissue regeneration:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(7) A substance according to any one of the following (a) to (c) for use in a method for stimulating cell migration:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(8) A substance according to any one of the following (a) to (c) for use in a method for mobilizing cells from bone marrow into peripheral blood:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

(9) A substance according to any one of the following (a) to (c) for use in a method for tissue regeneration:

(a) A peptide having cell migration stimulating activity and consisting of a portion of the HMGB1 protein;

(b) a cell secreting the peptide described in (a);

(c) A vector into which the DNA encoding the peptide described in (a) is inserted.

Furthermore, the use of a peptide comprising at least any one of the following amino acid sequences and having cell migration stimulating activity can also be replaced with the same expression as above.

[1] the amino acid sequence from positions 17 to 25 in the amino acid sequence of any one of SEQ ID NOs. 1, 3, and 5,

[2] the amino acid sequence from position 45 to position 74 in the amino acid sequence of any one of SEQ ID NOs. 1, 3, and 5,

[3] the amino acid sequence from position 55 to position 84 in the amino acid sequence of any one of SEQ ID NOs. 1, 3, and 5,

[4] the amino acid sequence from position 85 to position 169 of the amino acid sequence set forth in any one of SEQ ID NOs. 1, 3, and 5, and

[5] The amino acid sequence from position 89 to position 185 in the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, or 5.

In addition, all prior art documents cited in this specification are incorporated into this specification as reference.

The present invention is further illustrated by the following examples, but is not limited to the following examples.

Example 1

Purification of HMGB1 and HMGB1-derived peptides using HEK293

RNA was extracted from newborn mouse skin using Trizol (Invitrogen), and cDNA was synthesized using the SuperScript III cDNA Synthesis Kit (Invitrogen). Using this cDNA as a template, the HMGB1 cDNA was amplified using PCR (polymerase chain reaction). This cDNA was inserted into the plasmid vector pCAGGS (Figure 1), used for protein expression in mammalian cells, to express a protein with an IgGκ chain signal sequence attached as a secretion signal, and an HA tag, GST tag, and 6×His tag attached to the N-terminus of the amino acid sequence for purification. Furthermore, a sequence obtained by HRV3C cleavage was inserted between the His tag and the target protein and peptide. After HRV3C cleavage, a peptide fragment of GlyProGlyThyGln (SEQ ID NO: 7) was added to the N-terminus of the target protein and peptide. Furthermore, restriction enzyme sites were added to the full-length HMGB1 and peptide cDNAs using PCR and inserted into the KpnI and EcoRI portions of the vector.

The pCAGGS expression vector prepared above was transfected into the HEK293 cultured cell line derived from human embryonic kidney cells using polyethyleneimine (PEI), and the cells and culture supernatant were recovered after 48 hours. The cells and culture supernatant were centrifuged at 4400g × 5 minutes at 4 ° C, and the supernatant was separated from the cells and recovered separately. The recovered supernatant was further passed through a cellulose acetate filter with a pore diameter of 0.8 μm, and then passed through a nitrocellulose filter with a pore diameter of 0.45 μm to prepare a sample after removing the insoluble fraction. The sample was inserted into a 5 mL HisTrap FF (GE) equilibrated with 50 mM Tris HCl (50 mL) containing 50 mM NaCl at a pH of 8.0, and the adsorbed components were further washed with 50 mM NaCl 50 mM Tris HCl at a pH of 8.0 containing 10 mM imidazole to remove nonspecific adsorption components. The specifically adsorbed component was eluted from the column using 50 mM NaCl, 50 mM Tris-HCl, pH 8.0, containing 100 mM imidazole. The adsorbed fractions were fractionated into silica-coated plastic tubes at 500 μL/tube, and the protein-containing fractions were pooled. The imidazole was then removed using a PD10 (GE) desalting column, and the sample was eluted using 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. HRV3C (Novagen) was added to the eluted sample and incubated at 4°C for 8 hours. After the tag was removed, the sample was bound to a 1 mL HiTrap heparin column (GE) equilibrated with 50 mM Tris HCl, 150 mM NaCl, pH 7.5. The inside of the column was washed with 50 mM Tris HCl, 150 mM NaCl, pH 7.5, and the bound protein or peptide was eluted with 50 mM Tris HCl, 1000 mM NaCl, pH 7.5.

Migration analysis

Mouse bone marrow mesenchymal stem cell line (MSC-1 cells, produced by Tamai et al., Osaka University (PDGFRα-positive cells in bone marrow mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia. (Tamanai et al., Proc Natl Acad Sci USA, 2011 Apr 4.))) were recovered from the culture dish using trypsin by centrifugation at 4°C and 1200 rpm for 10 minutes. The cells were broken up and suspended in D-MEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum) to a concentration of 2.0 to 3.0 × ^10⁶ cells/ml. Recombinant proteins and peptides produced using HEK293 cells were diluted in D-MEM supplemented with 10% FBS. PBS (phosphate-buffered saline) was used as a negative control. Using an acrylic resin Boyden chamber, mouse bone marrow mesenchymal stem cell line adjusted to 3×10 ⁶ cells/ml was added to the upper layer, and diluted protein or peptide was added to the lower layer. In more detail, 28 μl of protein or peptide solution was added to each well of the bottom plate of the 48-well chemotaxis chamber (NEURO PROBE 48-well chemotaxis chamber), a polycarbonate membrane with 8 μm pores (Neuro Probe, Inc, catalog number: 866-417-0014) was placed on the bottom plate, and then the upper plate was placed and fixed tightly with screws. 50 μl of mouse bone marrow mesenchymal stem cell line after cell concentration adjustment was added to the upper plate wells. The chamber was placed in an incubator at 37°C and 5% CO _2. After 4 hours, the membrane of the chamber was recovered, and Diff-Quik (Sysmex, catalog number: 16920) was used to detect cells that migrated through the pores of the membrane to the lower layer by staining.

result

The migration activity of full-length mouse HMGB1 (1-215), a peptide from positions 1 to 84 (1-84), a peptide from positions 85 to 169 (85-169), a peptide from positions 1 to 44 (1-44), a peptide from positions 45 to 84 (45-84), and a negative control (PBS) were examined. The protein and peptides were used at a concentration of 50 μg/ml. While no migration activity was detected with 85-169, the other peptides, 1-215, 1-84, 1-44, and 45-84, showed migration activity (Figure 2).

Furthermore, the migration activity was confirmed using a peptide from position 45 to position 215 (45-215) and a peptide from position 63 to position 215 (63-215) at concentrations of 5, 15, and 25 μg/ml, respectively ( FIG. 3 ).

discuss

In mouse, rat and human HMGB1 (sequence numbers 3, 5 and 1, respectively), the amino acid sequence from position 1 to 85 is known as an A-box, and the amino acid sequence from position 86 to 169 is known as a B-box. The amino acid sequences from position 1 to 185 of mouse, rat and human are all identical, with 100% homology. The amino acid sequence from position 186 to 215 is a repeating sequence of glutamic acid and aspartic acid, which is 100% identical in mouse and rat, and differs from human by only two amino acids. No migration activity was detected in 85-169, and it is believed that the fragment has no activity or the activity is below the detection limit under the experimental conditions of this experiment. On the other hand, good migration activity was confirmed for 1-84, 1-44, and 45-84. Therefore, it is speculated that there are domains with migration activity in at least two places between the amino acid sequences from position 1 to 44 and between the amino acid sequences from position 45 to 84. HMGB1 is known to induce migration of cells such as dendritic cells, and this is believed to be induced by HMGB1 stimulating a receptor called RAGE. The RAGE-binding domain of HMGB1 is known to exist in the region corresponding to amino acids 150 to 181. Surprisingly, the domains that induce migration of bone marrow mesenchymal stem cells are located in at least two locations distinct from the RAGE-binding domain.

Both 45-215 and 63-215 demonstrated concentration-dependent migration activity. Furthermore, 45-215 demonstrated stronger activity than 63-215. Therefore, it is speculated that there is a domain with migration activity between at least amino acids 63 and 84. Furthermore, the following peptides produced using HEK293 also demonstrated migration activity against the bone marrow mesenchymal stem cell line MSC-1:

A peptide comprising the amino acid sequence from position 1 to position 42 (1-42),

A peptide comprising the amino acid sequence from position 1 to position 43 (1-43),

A peptide comprising the amino acid sequence from position 1 to position 45 (1-45),

A peptide comprising the amino acid sequence from position 1 to position 46 (1-46),

A peptide comprising the amino acid sequence from position 1 to position 47 (1-47),

A peptide comprising the amino acid sequence from position 1 to position 48 (1-48),

A peptide comprising the amino acid sequence from position 1 to position 49 (1-49),

A peptide comprising the amino acid sequence from position 1 to position 50 (1-50),

A peptide comprising the amino acid sequence from position 1 to position 51 (1-51),

A peptide (1-52) comprising the amino acid sequence from position 1 to position 52,

A peptide (1-62) comprising the amino acid sequence from position 1 to position 62.

Example 2

Isolation and confirmation of migration activity of primary cultured PDGFRα-positive bone marrow mesenchymal stem cells

Femurs and tibias were excised from donor mice (B6.129S4-Pdgfratm11(EGFP)Sor/J) (PDGFRα-GFP mice). Adhering muscle and other tissues were removed and the bones were finely broken and reacted in 0.2% collagenase (Roche, reference number: 10103586001) in DMEM and 2% FBS (filtrated) at 37°C for 40 minutes. Cells were passed through a 40μm nylon mesh to further remove cell clumps and muscle tissue. Cells were centrifuged at 1200 rpm for 10 minutes, suspended in αMEM containing 10% FBS and 1% P/S, and cultured in a 37°C, 5% _CO2 incubator until 100% confluence. Cells were recovered and the following experiments were performed according to the protocol provided with CD11b microbeads (Miltenyi Biotec, order number: 130-049-601). The cells were adjusted to 10 ⁷ cells/90 μl with PBS (-), and 10 μl CD11b microbeads were added to every 10 ⁷ cells, and the reaction was carried out at 4°C for 15 minutes. Then, the cells were washed twice and suspended in 500 μl PBS (-). The tube was set on an AutoMACS sorter, and the cells were recovered by the "Depletes" separation program. The recovered cells were inoculated into a culture dish for adherent cells, and the fluorescence of GFP was observed using a fluorescence microscope after adhesion. CD11b-negative cells were used to examine the migration activity of the peptide 1-44 produced using HEK293. The peptide was used at a concentration of 40 μg/ml.

result

PDGFRαGFP cells were almost absent among CD11b-positive cells, while many PDGFRαGFP cells were observed among CD11b-negative cells (Figure 4). In addition, peptide 1-44 produced using HEK293 cells showed strong migratory activity against CD11b-negative, PDGFRα-positive cells (Figure 5).

discuss

CD11b-negative, PDGFRα-positive cells are thought to contain a large number of bone marrow mesenchymal stem cells, a type of multipotent stem cell in the bone marrow. Peptide 1-44 is thought to have migratory activity not only on established bone marrow mesenchymal stem cell lines but also on primary cultured bone marrow mesenchymal stem cells.

Confirmation of PDGFRα protein expression in human bone marrow mesenchymal stem cells

method

Human mesenchymal stem cells (hMSC) (Takarabio, Inc., Product No. PT034) were cultured using the human mesenchymal stem cell-specific complete synthetic medium kit (MSCGM-CD(tm) Bullet Kit(tm)) (Takarabio, Inc., Product No. B0632) according to the product manual. Cells used in this experiment must have been passaged at least five times.

For western blotting, approximately 5 × ¹⁰⁷ cells were harvested and suspended in 1 mL of PBS. 200 μL of 6× SDS-PAGE loading buffer was added to the cell suspension and heated at 95°C for 5 minutes. As a positive control, a bacterial lysate of Escherichia coli (JM109) expressing rat PDGFRα was dissolved in loading buffer and used. In addition, 20 μL of the sample and an accurate molecular weight two-color prestained standard (Bio-Rad (Catalog No.: 161-0374) as a molecular weight marker were electrophoresed on a 7.5% SDS-PAGE gel. After the electrophoresis, the gel was recovered and transferred to a PVDF membrane according to the conventional method. The PVDF membrane with the sample transferred was blocked by soaking it in 2% skim milk containing 0.1% Tween20 (PBS-T) at room temperature for 1 hour. The excess skim milk attached to the membrane was washed with PBS-T. The PDGFRA rabbit anti-human polyclonal antibody (Lifespan Bioscience (Catalog No.: LS-C9640) as the primary antibody was diluted 3000 times and dissolved in 2% skim milk containing PBS-T, and the blocked membrane was soaked for 1 hour. The membrane was then soaked in PBS-T for 10 minutes and washed 3 times in total. Anti-rabbit IgG and HRP-Linked Whole Ab as secondary antibodies were Donkey (donkey HRP-conjugated full antibody, GE healthcare, catalog number: NA934) was diluted 15,000-fold in 2% skim milk containing PBS-T, and the membrane was soaked for 1 hour. The membrane was then soaked in PBS-T for 10 minutes, for a total of three washes. PDGFRα bands were detected using ECL Prime (GE healthcare (catalog number: RPN2232) according to the product manual.

result

As a positive control, rat PDGFRα produced in E. coli showed a band at approximately 170 kDa, while PDGFRα from human bone marrow mesenchymal stem cells was detected at a slightly higher molecular weight ( FIG2B ).

discuss

Western blotting revealed PDGFRα protein expression not only in mice but also in human bone marrow mesenchymal stem cells. Compared to rat PDGFRα produced in E. coli, it appears slightly larger, possibly due to modifications such as sugar chains. PDGFRα expression has also been confirmed in human bone marrow mesenchymal stem cells.

Example 3

<Confirmation of the multipotency of primary cultured PDGFRα-positive bone marrow mesenchymal stem cells>

FACS sorting of PDGFRα-positive, lineage-negative, and c-kit-negative cells

C57Bl6 mice (6 weeks old, male) were deeply anesthetized using isoflurane and euthanized by inhalation of carbon dioxide. The femur and tibia were removed, the fat and muscle tissue were excised, and the tissue attached to the bone was fully removed by immersing in EtOH. The bone marrow tissue was removed using a syringe with a 26G needle. DMEM containing 0.2% collagenase A was added to the removed bone marrow cells and incubated at 37°C for 40 minutes. 10% FBS DMEM was added and centrifuged at 1500 rpm for 10 minutes using a centrifuge. The supernatant was discarded and the precipitated bone marrow cells were recovered.

Inoculate into a 10 cm diameter culture dish and culture in DMEM containing 10% FBS and 1× streptomycin-penicillin using a 5% CO ₂ , 37°C incubator. Replace the culture medium every 3 days. Discard the culture medium of the attached cells and add 10 ml of PBS to the cells to wash twice. Add 5 ml of 0.25% trypsin and incubate at 37°C for 10 minutes. Recover the cells detached from the culture dish and add DMEM containing 10% FBS to terminate the trypsin reaction. Centrifuge the cells at 1200 rpm for 3 minutes and recover the precipitated cells. Dilute 1×10 ⁶ recovered cells into 100 μl of PBS containing 2% FBS and dispense into each well of a round-bottom 96-well plate. 10 μl of the primary antibody APC-mouse cell line antibody cocktail (BD Phamingen, catalog number 558074) was added to each well. 1 μl each of PE-mouse CD140a (PDGFRa) (BD Bioscience, catalog number 12-1401-81) and FITC-mouse c-kit (BD Bioscience) were added to each well and incubated in the dark at 4°C for 20 minutes. 200 μl of PBS containing 2% FBS was added to each well and the cells were centrifuged at 1500 rpm for 10 minutes, after which the supernatant was discarded. The cells were then washed twice in the same manner. Cells were sorted using a BD FACSAria cell sorter.

Bone differentiation induction

When cells reached 70% confluence, the osteogenic differentiation medium (R&D, conditioned with SC010) was replaced every 2-3 days. Culture was continued in a 37°C, 5% _CO2 incubator for approximately 3 weeks.

ALP staining

After induction of osteogenic differentiation, cells were fixed for 10 seconds using the kit's fixative solution (previously prepared in the fixative preparation solution). Staining was performed at 37°C for 3 minutes using a substrate solution (Muto Chemical, catalog number 1568-2) prepared from Fast Blue RR Salt and substrate stock solution. ALP-positive cells were stained bluish-purple.

Adipogenic differentiation induction

When cells reached 100% confluence, adipogenic differentiation medium (R&D, medium adjusted with SC010) was replaced every 3-4 days. Culture was continued in a 37°C, 5% _CO2 incubator for approximately 2 weeks.

Oil Red staining

After adipocyte differentiation was induced, the cells were fixed with the propylene glycol fixative provided with the kit, and then adipocytes were stained using oil red O solution (DBS, item number KT 025).

result

After osteogenic differentiation induction, cells stained blue-purple were observed, indicating osteoblast differentiation ( FIG6A ). Under adipogenic differentiation induction, adipocyte oil droplets stained red were observed, indicating adipocyte differentiation ( FIG6B ).

discuss

It is believed that PDGFRα-positive cells in the bone marrow contain at least bone marrow mesenchymal stem cells that are capable of osteodifferentiation and adipogenesis.

Example 4

Confirmation of migration activity of synthetic peptides

The following peptides were synthesized by solid phase method at MBL (Medical Biology Research Institute, Inc.). Separately, peptides were synthesized based on the mouse HMGB1 sequence (SEQ ID NO: 3). The synthetic peptides in the following examples were also prepared based on the mouse HMGB1 sequence.

A synthetic peptide (1-10) consisting of the amino acid sequence from position 1 to position 10 of HMGB1,

A synthetic peptide consisting of amino acid sequences from position 1 to position 34 (1-34),

A synthetic peptide consisting of the amino acid sequence from position 37 to position 62 (37-62),

A synthetic peptide consisting of the amino acid sequence from position 27 to position 62 (27-62),

A synthetic peptide consisting of the amino acid sequence from position 56 to position 72 (56-72),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 20 (11-20),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 25 (11-25),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 30 (11-30),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 34 (11-34),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 44 (11-44),

A synthetic peptide consisting of the amino acid sequence from position 17 to position 44 (17-44),

A synthetic peptide (1-25) comprising amino acids 1 to 25, and

As a positive control, full-length mouse HMGB1 (1-215 (HEK)) produced in HEK293 was adjusted to 100 μg/ml and added to the lower layer of the chemotaxis chamber to examine its migratory activity on a bone marrow mesenchymal stem cell line (MSC-1).

result

At least the synthetic peptides (11-34), (1-34), (11-44), (1-44), and (11-30) showed activity comparable to or greater than that of the positive control ( FIG7 ). Furthermore, activity was also confirmed for the synthetic peptides (11-25) and (1-25) ( FIG7 ).

discuss

Based on the results of Example 1, it is speculated that there is at least one migration-active portion in each of the amino acid sequences from positions 1 to 44 and from positions 45 to 84. The results of Example 4 indicate that the synthetic peptide (11-34) also exhibits strong migration activity, suggesting that an active center exists at least in the amino acid sequence from positions 11 to 34. Furthermore, although the activity of the synthetic peptide (11-25) is slightly weaker, activity is confirmed, suggesting that the active center exists in the amino acid sequence from positions 11 to 25, and that the amino acid sequences preceding and following it enhance activity.

Example 5

method

To further identify the active center portion, a short peptide was synthesized as follows.

A synthetic peptide consisting of the amino acid sequence from position 11 to position 27 of HMGB1 (11-27),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 28 (11-28),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 29 (11-29),

A synthetic peptide consisting of the amino acid sequence from position 12 to position 30 (12-30),

A synthetic peptide consisting of the amino acid sequence from position 13 to position 30 (13-30),

A synthetic peptide consisting of the amino acid sequence from position 14 to position 30 (14-30),

A synthetic peptide consisting of the amino acid sequence from position 15 to position 30 (15-30),

A synthetic peptide consisting of the amino acid sequence from position 16 to position 30 (16-30),

A synthetic peptide consisting of the amino acid sequence from position 17 to position 30 (17-30),

A synthetic peptide consisting of the amino acid sequence from position 18 to position 30 (18-30),

A synthetic peptide consisting of the amino acid sequence from position 19 to position 30 (19-30),

A synthetic peptide consisting of the amino acid sequence from position 20 to position 30 (20-30),

A synthetic peptide consisting of the amino acid sequence from position 21 to position 30 (21-30),

A synthetic peptide consisting of the amino acid sequence from position 10 to position 25 (10-25),

A synthetic peptide consisting of the amino acid sequence from position 11 to position 25 (11-25),

A synthetic peptide consisting of the amino acid sequence from position 12 to position 25 (12-25),

A synthetic peptide consisting of the amino acid sequence from position 13 to position 25 (13-25),

A synthetic peptide consisting of the amino acid sequence from position 14 to position 25 (14-25),

A synthetic peptide consisting of the amino acid sequence from position 15 to position 25 (15-25),

A synthetic peptide consisting of the amino acid sequence from position 16 to position 25 (16-25),

A synthetic peptide consisting of the amino acid sequence from position 17 to position 25 (17-25),

A synthetic peptide (186-215) consisting of the amino acid sequence from position 186 to position 215.

As positive controls, the supernatant (skin extract) of one-day-old mouse skin (one mouse) immersed in PBS and incubated at 4°C for 12 hours and the full-length mouse HMGB1 (HMGB1 (HEK_1-215) produced using HEK293 were used. A bone marrow mesenchymal stem cell line (MSC-1) was added to the upper layer of the chemotaxis chamber, and these proteins and synthetic peptides were inserted into the lower layer of the chemotaxis chamber at concentrations of 5 μM or 10 μM. Migration analysis was performed using the same method as in Example 1.

result

Strong migration activity was observed for synthetic peptides (11-27), (11-28), (11-29), (12-30), (13-30), (14-30), and (10-25) at a concentration of at least 5 μM. Weak activity was also observed for synthetic peptides (11-25), (12-25), (13-25), (14-25), (15-25), (15-30), (16-25), (16-30), (17-25), and (17-30) ( Figures 8A and 8B ).

discuss

Based on the results of Example 4, it was speculated that a domain with migration activity existed in the amino acid sequence from position 11 to position 25. Therefore, it was considered that one of the domains with migration activity existed between amino acids 17 and 25 (9 amino acids).

Comparison of the migration activity of various HMGB1 fragments on bone marrow mesenchymal stem cells

method

The intensity of the migration activity of synthetic peptides 15-30, 16-30, 17-30, 17-44, 45-74, and 55-84, each composed of a fragment of HMGB1, on bone marrow mesenchymal stem cells (MSC-1) was compared with that of a negative control (PBS). The Boyden chamber method was performed in the same manner as above. The peptides were inserted into the lower layer of the chamber at 10 μM. 1.5×10 ⁶ cells were spread in 1 mL of DMEM containing 10% FBS and inserted into the upper layer of the chamber. A polycarbonate membrane with pores of 8 μm in diameter was inserted between the upper and lower layers. After incubation for 4 hours in an incubator at 37°C and 5% CO ₂ , the membrane was recovered and stained using Diff-Quikstain (trademark) to stain only the cells that had migrated to the lower layer. After staining, the membrane was air-dried, and the number of cells that had migrated to the lower layer was counted using a microscope and the average value was calculated.

result

Strong migration activity was confirmed for all peptides compared with the negative control.

discuss

Compared with the manufacture method using the bacterium such as HEK293, Escherichia coli, synthetic peptide is comparatively cheap and can ensure stable output, in addition, it is very good manufacture method in the medicine such as the pollution that can prevent the toxin etc. of biological origin in manufacture.On the other hand, owing to different from generation in organism, post-translational modification, folding can not be carried out accurately, therefore, the low molecular peptide consisting of the amino acid with high hydrophobicity often becomes extremely insoluble to aqueous solution.In the present embodiment, also for the peptide that migration activity is relatively weak, therefore, use microscope to accurately detect the strength of activity relative to negative control.Any peptide all detects strong migration activity (Fig. 8 C) compared with negative control.

Example 6

Confirmation of migration activity of synthetic peptides

Migration assays were performed using a chemotaxis chamber in the same manner as in Example 5 for the synthetic peptides (1-44), (1-34), a peptide consisting of amino acids 45 to 74 (45-74), and a peptide consisting of amino acids 55 to 84 (55-84) used in Example 4. The assays were performed simultaneously at two concentrations, 10 μM and 5 μM.

result

Although the migration activity was weaker than that of the synthetic peptides (1-44) and (1-34), the migration activity was also confirmed for the synthetic peptides (45-74) and (55-84) ( FIG. 9 ).

discuss

In the results of Example 1, peptides (1-84), (1-44), and (45-84) produced using HEK293 cells demonstrated strong migratory activity against PDGFRα-positive mesenchymal stem cells. The results of Example 4 indicate that synthetic peptides (1-44) and (1-34) also exhibited strong activity. Meanwhile, in the results of this Example 6, although the migratory activity of synthetic peptides (45-74) and (55-84) was slightly reduced, migratory activity was also observed.

It is known that peptides and proteins synthesized in eukaryotic cells such as HEK293 undergo modifications such as sugar chain modifications. However, the synthesized peptides are not modified. The migration activity of the HEK293-produced peptide (45-84) is stronger than that of the synthetic peptides (45-74) and (55-84) containing amino acids 45 to 84, suggesting the possibility of some kind of modification.

In addition, previous experiments using angioblast stem cells (Palumbo et al., J. Cell Biol., 164:441-449, 2004) showed that the cell migration activity of HMGB1 (full length 215 amino acids) was retained in a sequence consisting of amino acids 1 to 187 after cleavage of the C-terminus, but was almost completely lost in a sequence consisting of amino acids 1 to 89, a sequence consisting of amino acids 90 to 176, and a sequence consisting of amino acids 1 to 176. Furthermore, the putative ligand site for RAGE, one of the known receptors for HMGB1, is a sequence consisting of amino acids 150 to 181, and the aforementioned literature shows that dominant-negative RAGE also inhibits migration activity. In addition, another report (Yang et al., J Leukoc Biol. Jan; 81(1): 59-66, 2007) showed that HMGB1 also utilizes the RAGE receptor when inducing dendritic cell migration. So far, the C-terminal peptide of HMGB1, which is a ligand for RAGE, has attracted attention regarding the migration activity of HMGB1.

In this example, two migration-active sites were successfully identified within an N-terminal peptide previously thought to lack cell migration activity. Excessive inflammation is known to act as an inhibitory factor in tissue regeneration. The newly discovered active site is completely distinct from the RAGE ligand, thus enabling the simultaneous mobilization of PDGFRα-positive stem cells while mobilizing inflammatory cells such as dendritic cells. This is expected to lead to the development of pharmaceuticals with fewer side effects.

Example 7

Quantitative comparison of the migration activity of peptide (1-44) and full-length HMGB1

As in Example 1, the mouse HMGB1 (1-44) expression vector was transfected into HEK293 cells using polyethyleneimine. HMGB1 secreted into the cell supernatant was recovered and purified (HEK293 transient transfection). Furthermore, following the same transfection, 2 μg/ml of puromycin was added to the culture medium, and drug screening was performed on cells stably secreting HMGB1 (1-44). HMGB1 secreted into the cell supernatant was purified (HMGB1 stable transfection).

Production of HMGB1-derived peptides using Escherichia coli

To produce a peptide from amino acids 1 to 44 using E. coli, a cDNA expressing a chimeric peptide was inserted into the pENTR vector (Invitrogen) attached to the N-terminus of a cDNA encoding amino acids 1 to 44 of mouse HMGB1 after cleavage with HRV3C. An LR reaction was performed to reconstitute the pDEST17 vector. This expression vector has a T7 promoter and can express proteins in E. coli. In addition, a 6×His tag was attached to the N-terminus. After HRV3C cleavage, the 6×His tag was removed, and a peptide fragment consisting of Gly Pro Gly Thy Gln (SEQ ID NO: 7) was attached to the N-terminus of the peptide.

The above expression vector was transformed into Escherichia coli BL21 (DE3) by electroporation. SOC medium was added and cultured at 37°C using a shaker for 60 minutes. Inoculate onto LB agar medium containing carbenicillin and incubate at 37°C for 18 hours. Collect a single colony and add LB medium containing carbenicillin, and culture at 37°C using a shaker. After O.D.600 reaches 0.4-0.5, add IPTG at a final concentration of 0.1mM, and then shake at 30°C. After 6 hours, recover E. coli, centrifuge at 3500rpm for 30 minutes, and recover the precipitated E. coli. Add 50mM Tris HCl containing 6M urea and 50mM NaCl at a pH of 8.0 to the E. coli, and perform aspiration and lysis. The column was inserted into a 5mL HisTrap FF (GE) equilibrated with 50mM Tris HCl (pH 8.0) containing 6M urea and 50mM NaCl. The adsorbed components were then washed with 50mM NaCl/50mM Tris HCl (pH 8.0) containing 6M urea and 10mM imidazole to remove nonspecific adsorbed components. Specific adsorbed components were eluted from the column with 50mM NaCl/50mM Tris HCl (pH 8.0) containing 6M urea and 300mM imidazole. The adsorbed fractions were fractionated into silica-coated plastic tubes at 500μL/tube, and the protein-containing fractions were pooled. The imidazole was then removed using a desalting column PD10 (GE), and eluted with 50mM Tris HCl (pH 7.5) and 150mM NaCl. HRV3C (Novagen) was added to the eluted sample and reacted at 4°C for 8 hours. After the tag was cleaved, the sample was applied to a 1 mL HisTrap FF column equilibrated with 50 mM Tris HCl, 150 mM NaCl, pH 7.5, to recover the peptide as the non-bound fraction.

Synthetic peptide (1-44) was prepared as described above. These peptides and proteins were used at a concentration of 2 μM to analyze the migration of bone marrow mesenchymal stem cells (MSC-1). The area of the chemotaxis chamber wells and the area of the migrated cells were measured using image analysis software.

result

At equimolar levels, both the HEK293-produced peptide (1-44) and the E. coli-produced peptide (1-44) exhibited approximately 1.6-fold higher migration activity than full-length HMGB1. At equal mass levels, both the HEK293-produced peptide (1-44) and the E. coli-produced peptide (1-44) exhibited approximately 8-fold higher migration activity than full-length HMGB1. The synthetic peptide (1-44) exhibited 0.57-fold higher migration activity than full-length HMGB1 at equimolar levels and 2.86-fold higher migration activity at equal mass levels (Figure 10).

discuss

From Examples 1 and 6, it can be considered that the center of migration activity exists in at least one location in the amino acid sequence from positions 1 to 44 and in the amino acid sequence from positions 45 to 84, respectively, and there are at least two active sites as a whole. In addition, the results of Example 7 show that, under the condition of equal mass, the peptide (1-44) produced using HEK293 has nearly 8 times the migration activity compared to the full-length HMGB1 (about 1.6 times when equimolar). In addition, the peptide (45-84) produced using HEK293 also has the same migration activity (Figure 2). From the above, it can be seen that in HMGB1, there are at least two sites in the amino acid sequence from positions 1 to 44 and the amino acid sequence from positions 45 to 84 that have migration activity exceeding the equimolar amount of the full-length HMGB1, but the activity of the full-length HMGB1 is extremely weak compared to the sum of the activities of these two sites. Results from crystallographic analysis of full-length HMGB1 suggest that the peptides at positions 1 to 44 and 45 to 84 are adjacent to each other and may inhibit each other's activity. It is thought that the peptides release the inhibition and enhance the activity of each.

Example 8

Expression of PDGFRα, cell lineage markers, and CD44 in bone marrow mesenchymal stem cell line (MSC-1) FACS analysis

Mouse bone marrow mesenchymal stem cell line MSC-1 was seeded into a 10 cm diameter culture dish and cultured in DMEM containing 10% FBS and 1× streptomycin-penicillin in a 5% CO ₂ incubator at 37°C. After proliferation to 80-90% confluence, the culture medium was discarded and 10 ml of PBS was added to the cells, washing twice. 5 ml of 0.25% trypsin was added and incubated at 37°C for 10 minutes. The cells detached from the culture dish were recovered and DMEM containing 10% FBS was added to terminate the trypsin reaction. The cells were centrifuged at 1200 rpm for 3 minutes to recover the precipitated cells. 1×10 ⁶ of the recovered cells were diluted in 100 μl of PBS containing 2% FBS and dispensed into each well of a round-bottom 96-well plate. 10 μl of the primary antibody APC-mouse cell line antibody cocktail (APC-mouse Lineage antibody cocktail, BD Phamingen, catalog number 558074) was added to each well. 1 μl each of PE-mouse CD140a (PDGFRa) (BD Bioscience, catalog number 12-1401-81) and FITC-mouse CD44 (BD Bioscience, catalog number 553-133) was added to each well and incubated in the dark at 4°C for 20 minutes. 200 μl of PBS containing 2% FBS was added to each well, and the cells were centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded. The cells were washed twice in the same manner. The cells were suspended in 100 μl of PBS and analyzed using BD FACSCantTMII.

result

The bone marrow mesenchymal stem cell line (MSC-1) is PDGFRα-positive, Lineage-negative, and CD44-positive ( FIG11 ).

discuss

The cells used in the migration activity retain the properties of PDGFRα-positive bone marrow mesenchymal stem cells.

Example 9

Migration activity of synthetic peptide (1-34) on mouse keratinocytes

After euthanizing C57/Bl6 newborn mice using isoflurane and carbon dioxide inhalation, they were thoroughly washed with EtOH and PBS. The skin was peeled off along with the dermis, and the blood was rinsed with PBS. The peeled skin was left to stand at 4°C in Dispase I (Sanko Pure Chemical Industries, Ltd., catalog number: GD81060) for 16 hours. The epidermis and dermis were peeled off with tweezers, and the epidermis was left to stand at 37°C in trypsin (Nacalaitesque, catalog number: 3554-64) for 10 minutes. After the skin began to become turbid, the reaction was terminated with S-MEM (GIBCO, catalog number: 11380) 15% FBS (Ca-) P/S and centrifuged at 160×G for 5 minutes. The cells were suspended in CnT-07 culture medium (CELLnTEC, catalog number: CnT-07BM) and inoculated into 10 cm culture dishes. Culture the cells at 37°C under 5% CO ₂ , changing the medium every three days. Subculture the cells when they are 80-90% confluent. Retrieve the cells from the culture dish using trypsin and inactivate the trypsin in D-MEM supplemented with 10% FBS. The migration activity of a synthetic peptide (1-34) consisting of amino acids 1 to 34 was then assessed using the migration assay described above.

Expression of PDGFRα in mouse keratinocytes

B6.129S4-Pdgfratm11(EGFP)Sor/J newborn mice were perfused and fixed with 4% PFA. 1.0×1.0 cm ² of newborn mouse skin was cut and further infiltrated and fixed with 4% PFA for 12 hours + 30% sucrose for 12 hours at 4°C. Washed with PBS(-) and cryoembedded in OTC compound. 8 μm frozen sections were cut using a freezing microtome. Washed twice with PBS, rinsed with compound, and blocked with 10% goat serum PBS at room temperature for 1 hour. The primary antibody used was rabbit anti-keratin 5 (Covance, catalog number: PRB-160P) or rabbit anti-vimentin (Abcam, catalog number: ab7783-500) diluted 500 times with 10% goat serum PBS and incubated at 4°C for 5 hours. Then, washed twice with PBS. The secondary antibody used was Alexa Fluor 546 goat anti-rabbit IgG (H+L) (Invitrogen, catalog number: A11035) diluted 500-fold in 10% goat serum in PBS. The cells were incubated at room temperature for 45 minutes. The cells were then washed twice with PBS. The cells were incubated with 2 μg/ml of DAPI (4',6-diamidino-2-phenylindole) at room temperature for 3 minutes. The cells were washed twice with PBS and mounted with a mounting medium containing a fluorescence antifade.

result

The synthetic peptide (1-34) showed no migration activity on mouse keratinocytes ( FIG. 12A ). In addition, no GFP fluorescence was observed in cells positive for keratin 5, a mouse keratinocyte marker ( FIG. 12B ).

discuss

Keratinocytes do not express PDGFRα. In addition, the synthetic peptide (1-34) has no migration activity on keratinocytes.

Example 10

Migration activity of synthetic peptide (1-34) on mouse skin fibroblasts

After euthanizing C57/Bl6 neonatal mice using isoflurane and carbon dioxide inhalation, wash thoroughly with EtOH and PBS. The skin, along with the dermis, is excised and blood is rinsed with PBS. The excised skin is thoroughly minced with scissors. The minced skin is placed in 0.2% collagenase (Roche, reference number: 10103586001) in DMEM (Nacalai tesque, catalog number: 08458-45) and shaken at 37°C for 30 minutes. The reaction is terminated with DMEM 30% FBS/P/S and centrifuged at 160×g for 5 minutes. The cells are seeded into 10 cm culture dishes. Culture is maintained at 37°C under 5% _CO₂ , with the medium changed every 3 days. Cells are passaged when the cells are 80–90% confluent. The cells were recovered from the culture dish using trypsin, and after inactivating trypsin with D-MEM containing 10% FBS, the migration activity of a synthetic peptide (1-34) consisting of amino acids 1 to 34 was examined according to the above-mentioned migration assay.

B6.129S4-Pdgfratm11(EGFP)Sor/J newborn mice were perfused and fixed with 4% PFA. 1.0×1.0 cm ² of newborn mouse skin was cut and further infiltrated and fixed with 4% PFA, 30% sucrose at 4°C for 12 hours. Washed with PBS(-) and cryo-embedded in OTC compound. 8 μm frozen sections were cut using a freezing microtome. Washed twice with PBS, rinsed with compound, and blocked with 10% goat serum PBS at room temperature for 1 hour. The primary antibody used rabbit anti-vimentin (Abcam, catalog number: ab7783-500) diluted 500 times with 10% goat serum PBS and incubated at 4°C for 5 hours. Then, washed twice with PBS. The secondary antibody used Alexa Fluor 546 goat anti-rabbit IgG (H+L) (Invitrogen, catalog number: A11035) diluted 500 times with 10% goat serum PBS and incubated at room temperature for 45 minutes. Then, the sections were washed twice with PBS, incubated with 2 μg/ml DAPI (4',6-diamidino-2-phenylindole) at room temperature for 3 minutes, washed again with PBS twice, and sealed with a mounting medium containing a fluorescent quencher.

result

The synthetic peptide (1-34) showed migration activity on skin fibroblasts ( FIG. 13A ). In addition, cells positive for GFP fluorescence were observed among cells positive for vimentin, a fibroblast marker ( FIG. 13B ).

discuss

Skin fibroblasts express PDGFRα. A synthetic peptide (1-34) has migratory activity against skin fibroblasts. Both bone marrow mesenchymal stem cells and newborn skin fibroblasts are PDGFRα-positive, and peptide (1-34) has migratory activity against both. However, keratinocytes, which are PDGFRα-negative, show no migratory activity. It is speculated that PDGFRα is useful as a marker for cells that exhibit migratory activity, containing the amino acid sequence of peptide (1-34).

Example 11

Confirmation of PDGFRα expression in mouse skin fibroblasts using FACS

Newborn B6.129S4-Pdgfratm11(EGFP)Sor/J mice were washed thoroughly with EtOH and PBS. The skin was peeled off from the muscle and cut into 3 mm wide slices. The slices were transferred to 5% FBS-DMEM containing 500 units/ml of dispase and incubated at 4°C for 18 hours. The dermis and epidermis were peeled off and the dermis was thoroughly minced with scissors. The finely minced dermis was placed in DMEM containing 0.2% collagenase and shaken in a 37°C incubator for 30 minutes. DMEM containing 30% FBS was added and centrifuged at 160×G for 5 minutes. The precipitated cells were seeded into a 10 cm culture dish. The cells were cultured at 5% _CO2 and 37°C, with the culture medium changed every 3 days. The cells were passaged when they were 80-90% confluent. The cells were recovered from the culture dish using trypsin, and after adding D-MEM containing 10% FBS, the cells were recovered by centrifugation. BD FACSCantTMII was used to detect GFP fluorescence of the cells and analyze the results.

result

More than 98% of fibroblasts in the skin of newborn mice were PDGFRα-positive ( FIG. 14 ).

discuss

The number of PDGFRα-positive cells was quantified using FACS. Similar to the immunohistochemical results, the majority of fibroblasts were PDGFRα-positive.

Example 12

method

10 μg of synthetic peptide (amino acid 1 to amino acid 44) was diluted into 200 μl of PBS and administered from the tail vein of C57Bl6 mice (8 weeks old, female) using a syringe with a 30G needle. The same amount of PBS was administered to the negative control. Peripheral blood was collected from the left ventricle under general anesthesia using isoflurane 12 hours later. After adding 3ml of PBS (Nacalai tesque, catalog number 14249-95), 3ml of Ficoll-Paque Plus (GE Healthcare, catalog number 17-1440-02) was stacked. Centrifuge was used to centrifuge for 45 minutes at 25°C with 400G. The serum on the upper layer was discarded and only the cells that looked like white bands in the middle layer were recovered. 45ml of PBS was added to the recovered cells and centrifuged for 20 minutes at 25°C with 800G. Discard the supernatant, add 10 ml of PBS, and centrifuge at 1500 rpm at 25°C for 10 minutes. Discard the supernatant, add 1 ml of HLB (hemolysis buffer) (manufactured by Immuno-Biological Research Institute), pipette and let stand for 5 minutes. Add 10 ml of PBS and centrifuge at 1500 rpm at 25°C for 10 minutes. Collect the precipitated monocytes. Adjust the recovered monocytes to 1×10 ⁶ cells/100 μl (PBS containing 2% FBS) in a round-bottom 96-well plate. Add 1 μl of PE-mouse CD140a (PDGFRα) (BD Bioscience, catalog number 12-1401-81) or FITC-mouse CD44 (BD Bioscience, catalog number 553-133) to each well containing monocytes and incubate at 4°C for 20 minutes in the dark. Add 200 μl of PBS to each tube and centrifuge at 1500 rpm at 4°C for 10 minutes. Discard the supernatant, add 200 μl of PBS to each tube again, and centrifuge at 1500 rpm at 4°C for 10 minutes. Resuspend the cells in 100 μl of PBS and add 300 μl of 1% formaldehyde. Also, prepare a control using an isotype control antibody. Analyze the cells prepared above using a FACSCant™ II.

result

In the negative control group (PBS administration group), the proportion of PDGFRα-positive and CD44-positive cells in peripheral blood was an average of 1.33%, whereas it increased to an average of 4.33% in the peptide (1-44) administration group ( FIG. 15 ).

discuss

A peptide synthesized from amino acids 1 to 44 of HMGB1 was administered intravenously to mice. Twelve hours later, the number of PDGFRα-positive and CD44-positive cells increased. In Example 8, in vitro migration activity of PDGFRα-positive and CD44-positive bone marrow mesenchymal stem cells was confirmed. This example demonstrates that PDGFRα-positive and CD44-positive cells are also mobilized into peripheral blood in vivo. Both PDGFRα-positive and CD44-positive are markers for bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cells are known to be effective in regenerative medicine, and intravenous administration of the peptide of this invention is expected to be effective in treating damaged tissues.

Example 13

Preparation of the middle cerebral artery occlusion model

Male Wister rats aged 8-10 weeks were used. The rats were anesthetized by inhalation using isoflurane while being kept warm with a heating pad equipped with a temperature monitor. After confirming that the anesthetic effect was sufficient, the hair on the neck was removed to expose the skin, and the surgical site was disinfected with alcohol. The skin on the midline of the neck was incised with a scalpel. The right external carotid artery was ligated, and after pressurizing the right common carotid artery to temporarily stop the blood flow, a thread plug obtained by coating the end of a No. 4 nylon monofilament with silicon was inserted from the right external carotid artery to the right internal carotid artery. The pressure on the common carotid artery was relaxed to allow blood to flow through, and the thread plug advanced from the internal carotid artery to the branch of the middle cerebral artery to block the blood flow. In addition, the thread tied to the right common carotid artery was ligated to completely block the blood flow for 50 minutes. After pulling out the thread plug and loosening the thread of the common carotid artery, the skin was sutured to end the operation.

Administration of therapeutic drugs

50 μg of the synthetic peptide (1-44) was administered via the tail vein. The first administration was 6 hours after the infarction, and thereafter, the administration was repeated 5 times at 24-hour intervals (5 days in total).

Confirmation of cerebral infarction size

Fourteen days after the final dose, the rats were fully anesthetized and then confirmed to have stopped cardiac and respiratory activity in a container filled with carbon dioxide. The brains were removed and quickly immersed in 10% buffered formalin for fixation. After paraffin embedding, thin sections were cut and stained with hematoxylin-eosin. Four sections were made for each brain at 1.92 mm (1.92), 0.60 mm (0.60) anterior to the bregma, and 1.56 mm (-1.56), and 3.24 mm (-3.24) posterior to the bregma, and the areas were compared.

result

In the group (N=10) administered with the synthetic peptide (1-44), only one animal had an infarct extending into the cortex; the remaining nine animals had infarcts limited to the basal ganglia (Figure 16A1 (1.92 mm in front of the bregma), B1 (0.60 mm in front of the bregma), C1 (1.56 mm posterior to the bregma), and D1 (3.24 mm posterior to the bregma)). In contrast, in the negative control group (N=11), eight animals had infarcts extending from the basal ganglia to the cortex (Figure 16A2 (1.92 mm in front of the bregma), B2 (0.60 mm in front of the bregma), C2 (1.56 mm posterior to the bregma), and D2 (3.24 mm posterior to the bregma)). Furthermore, the area of the infarcted portion of the right brain was measured for each of the four sections prepared, and the percentage relative to the normal brain area of the right brain was calculated. In any section, the infarct area in the synthetic peptide-administered group was significantly reduced compared with that in the negative control group ( FIG. 17 ).

discuss

In recent years, there have been reports of improved prognosis in patients with cerebral infarction after intravenous administration of their own bone marrow mesenchymal stem cells, demonstrating that the therapeutic effects of cells within the bone marrow on cerebral infarction are becoming increasingly clear. Furthermore, studies in rodents have shown that bone marrow mesenchymal stem cells differentiate into osteoblasts, chondrocytes, adipocytes, and other cells, and have also been shown to differentiate into various other cell types, including epithelial cells and neurons. Furthermore, since bone marrow cells produce various growth factors and cell proliferation factors, there is also hope that substances secreted by bone marrow cells that migrate to the infarct site may have neuroprotective effects.

Furthermore, in rats, it is known that the size of a cerebral infarct is approximately 80% to 90% of the infarct size by 48 hours after ischemia, and gradually expands over the next 7 days. Furthermore, once ischemia occurs, it is known that there is a central zone, which inevitably undergoes necrosis regardless of treatment, and a penumbra, which can be protected from necrosis with treatment. The goal of cerebral infarction treatment is to prevent necrosis in the penumbra before the infarct expands.

The brain is mainly divided into the basal ganglia and the cerebral cortex. In particular, the basal ganglia are more intolerant to hypoxia than the cerebral cortex and are therefore most susceptible to damage during cerebral infarction. In the present results, the reduction of the infarct focus caused by the synthetic peptide (1-44) was also mainly seen in the cortex, and necrosis almost occurred in the basal ganglia. The cerebral cortex is the center of sensation and movement, and improving these functions is extremely important for social rehabilitation after the treatment of cerebral infarction. Considering the current situation of few effective therapeutic drugs for cerebral infarction, there is a high expectation for the necessity of the peptide of the present invention as a medicine.

In this experiment, the peptide was administered 6 hours after infarction, and a reduction in infarct size was observed 19 days later. The therapeutic effect is hypothesized to be due to the neuroprotective effects of bone marrow cells and tissue regeneration caused by differentiation into neural tissue. This experiment strongly suggests that peptides composed of a portion of HMGB1 can mobilize cells to the injured site not only when administered to or near the injured site, but also when administered intravenously at a site distant from and different from the injured site. Furthermore, some of the peptides in Examples 4, 5, and 6 have weak migration activity, resulting in seemingly undetectable activity. It is believed that the activity of some of these peptides falls below the detection limit of this analytical method. By optimizing the solvent used to dissolve the peptide, the time for measuring migration activity, and the number of cells inserted into the upper chamber, it may be possible to detect activity. Currently, the drug tPA, used in the treatment of cerebral infarction, has strict dosing criteria, including administration within 4 hours of onset and the need for imaging diagnostic assessment, to prevent side effects such as post-infarction bleeding. Because cerebral infarction occurs suddenly, it is difficult to predict its onset. Therefore, most people who suffer from cerebral infarction have exceeded the time limit when they go to medical institutions for treatment, and most of them are not suitable for the application of tPA. In this embodiment, the therapeutic effect was obtained by administering it 6 hours after the cerebral infarction was made. It is believed that the peptide of the present invention does not have anticoagulant activity, so it can be administered after 6 hours, and it is speculated that it can be applied to most people who suffer from cerebral infarction. In addition, in this embodiment, 1 rat (about 250g/individual) was administered at 50μg/time, which is equivalent to 200μg per kilogram of body weight. It is believed that the amount for intravenous administration to patients with cerebral infarction is an appropriate amount.

Example 14

Preparation of expression vectors for HMGB1 fragments, protein/peptide expression, and bone marrow mesenchymal stem cell migration analysis

The methionine (M) at the N-terminal end of human HMGB1 was deleted and replaced with MKHHHHHHENLYFQ (SEQ ID NO. 11). HHHHHH (SEQ ID NO. 12) is a tag (6×His tag) used to purify the expressed protein or peptide using a nickel column, and ENLYFQG (SEQ ID NO. 13) is a sequence recognized by TEV protease (Figure 18A). In addition, a vector encoding the target protein or peptide (2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81, 2-170, 93-215, 85-169) was constructed downstream of the T7 promoter and lac operator. In addition, a kanamycin resistance gene was used as the drug resistance gene, and pBR322ori or f1ori was used as the replication origin. When the protein or peptide produced using this expression vector is excised with TEV protease, a protein or peptide of human HMGB1 starting from the second amino acid can be produced. The prepared plasmid was used to transform BL-21 (DE3). 5 ml of the bacterial solution cultured overnight at 37°C in LB medium containing kanamycin was transferred to 100 ml of LB medium and cultured at 37°C at 140 rpm. The turbidity was measured using a turbidimeter, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added after reaching OD 0.5-0.7 to a final concentration of 1 mM. For 2-215, 2-84, 2-70, 2-81, 2-170, 93-215, and 85-169, the bacteria were collected after cultured at 37°C for 5 hours, and for 2-44, 45-84, and 2-62, the bacteria were collected after cultured overnight at 15°C. After SDS-PAGE, the expressed proteins and peptides were confirmed by protein staining and protein immunoblotting using a tag or anti-HMGB1 antibody.

Purification of various HMGB1 fragments (2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81, 2-170, 93-215)

To the collected cells, 3 ml of equilibration buffer (PBS (137 mM NaCl, 8.1 _{mM Na₂HPO₄} , 2.68 mM KCl, 1.47 mM _KH₂PO₄ ), 10 mM imidazole; pH 7.4) was added, and ultrasonic lysis _was performed. Centrifugation was performed at 15,000 rpm for 10 minutes at 4°C, and the supernatant was recovered. A Micro Bio-Spin column (Bio-Rad) was filled with 1 ml of His-Pur (trademark) Ni-NTA resin (Thermo Scientific) and equilibrated with equilibration buffer. The protein solution was added, and after centrifugation at 2,000 rpm for 2 minutes, the resin was washed with wash buffer (PBS, 25 mM imidazole; pH 7.4). Elution was performed in a stepwise manner using elution buffer (PBS, 250 mM or 500 mM imidazole), and each fraction was subjected to SDS-PAGE (e-PAGEL (registered trademark) 15% (ATTO)) to confirm the eluted protein. After affinity purification using a nickel column, 2-215 was subjected to ion exchange chromatography using Q sepharose (trademark) Fast Flow (GE Healthcare), 93-215 was subjected to ion exchange chromatography using Q sepharose (trademark) Fast Flow (GE Healthcare) and SP sepharose (trademark) Fast Flow (GE Healthcare), and the remaining fractions were subjected to ion exchange chromatography using SP sepharose (trademark) Fast Flow (GE Healthcare).

Human HMGB1 fragments (2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81, 2-170)

A Micro Bio-Spin column was filled with 1 ml of each agarose gel and equilibrated with PBS. The column was filled with the affinity-purified protein solution and washed with PBS. Elution was performed with elution buffer (20 mM HEPES, 1 M NaCl; pH 7.5), and each fraction was analyzed by SDS-PAGE.

Human HMGB1 fragment (93-215)

The protein solution after affinity purification was subjected to anion exchange and cation exchange, respectively, using Q and SP sepharoses. Furthermore, the fractions passed through SP sepharose (trademark) Fast Flow were loaded onto Q sepharose (trademark) Fast Flow for anion exchange. Each fraction was confirmed by SDS-PAGE.

Human HMGB1 fragment (85-169)

To every 0.1g of collected bacteria, add 1ml of buffer (PBS, 10mM imidazole; pH 7.4) and perform ultrasonic lysis. Centrifuge at 20,000rpm for 1 hour at 4°C and recover the supernatant. Purify by column chromatography using BioLogic DuoFlow (Bio-Rad). First, use 5ml of HisTrap (trademark) FF (GE healthcare), bacterial lysis solution (PBS, 10mM imidazole (pH 7.4)) for buffer A, and PBS, 500mM imidazole (pH 7.4) for buffer B for affinity purification. After balancing the column with buffer A, fill it with protein solution and wash and purify according to the following procedure. The procedure is as follows:

Isocratic mobile phase (buffer A: 97%, buffer B: 3%, 20 ml) → linear gradient (buffer A: 97% → 0%, buffer B: 3% → 100%, 20 ml) → isocratic mobile phase (buffer B: 100%, 20 ml) → fraction collection (20-40 ml, 2 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Next, ion exchange purification was performed. 85-169 used a 5 ml HiTrap (trademark) SP HP (GE Healthcare) column, with PBS (pH 7.4) as buffer A and 20 mM HEPES, 1 M NaCl (pH 7.5) as buffer B. After equilibration with an appropriate amount of buffer A, the column was filled with the affinity-purified protein solution and washed and purified according to the following procedure. The procedure was performed as follows:

Isocratic mobile phase (buffer A: 100%, buffer B: 0%, 10 ml) → Isocratic mobile phase (buffer A: 50%, buffer B: 50%, 2 ml) → Isocratic mobile phase (buffer A: 0%, buffer B: 100%, 20 ml) → Fraction collection (10-32 ml, 1 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Concentration determination

The concentration of each fragment was measured by BSA conversion using the Bradford method (Bio-Rad Protein Assay).

Migration analysis

The migration activity of each of the above peptides on the bone marrow mesenchymal stem cell line MSC-1 was confirmed. A phosphate buffered saline solution containing 500 mM NaCl containing each fragment was diluted with 2 volumes of DMEM to a final concentration of 2 μM. This was then inserted into the lower layer of the chamber, sandwiched between a polycarbonate membrane with 8 μm pores, and MSC-1 cells diffused in DMEM containing 10% FBS were inserted into the upper layer. After incubation for 4 hours in a 37°C, 5% _CO2 incubator, cells migrating from the upper layer to the lower layer were detected using Diff-Quik stain (trademark).

result

2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81, 2-170, and 93-215 all demonstrated migration activity for bone marrow mesenchymal stem cells ( Figure 18B ). When the migration activity of 2-215 was set to 1, 2-84 demonstrated 2.37-fold, 2-44 demonstrated 1.82-fold, and 45-84 demonstrated 2.04-fold greater activity on a molar basis. When converted to equal mass, these activity levels were 5.5-fold, 7.1-fold, and 8.1-fold, respectively ( Figures 18C and D ).

discuss

By fragmenting the N-terminal side of human HMGB1 (2-215) produced by Escherichia coli, an enhancement of migration activity was confirmed. In addition, migration activity was confirmed at least at 2-44 and 45-84 on the N-terminal side. This is the same result as the HMGB1 fragment produced in the cultured cells of eukaryotic organisms (HEK293 cells). It is speculated that fragmentation may expose the epitope of the receptor for MSC-1 and make it easy to bind to the receptor. Depending on the protein, there are proteins that lose their activity by fragmentation, but the protein of the present invention enhances its activity by fragmentation. In the expression of known proteins, post-translational modifications such as sugar chain attachment are performed in eukaryotic cells such as HEK293. For receptor ligands, the presence or absence of these modifications sometimes affects activity. Therefore, the fact that not only the protein produced by Escherichia coli without the same post-translational modification as in eukaryotic cells maintains activity, but also the fragments produced by Escherichia coli maintain activity indicates that post-translational modifications are not necessary for the activity of these fragments. In summary, by fragmenting HMGB1, a drug for mobilizing bone marrow mesenchymal stem cells with higher activity can be developed. In addition, since post-translational modification is not necessary, a production method using Escherichia coli or chemical synthesis can be used, and a preparation with stable quality can be produced more cheaply. In addition, a comparison of the peptide described in this example with the peptides described in other examples (for example, a comparison of 1-44 with 2-44 or a comparison of 1-84 with 2-84) shows that the presence or absence of the first methionine of the HMGB1 protein has no effect on migration activity. Therefore, in the case where a certain peptide has migration activity, it is believed that the peptide after removing the first methionine from the peptide also has migration activity. In addition, in the case where the peptide after removing the first methionine has migration activity, it is believed that the peptide after adding the first methionine to the peptide also has migration activity.

Example 15

method

The methionine (M) on the N-terminal side of human HMGB1 was deleted and replaced with MKHHHHHHENLYFQ (sequence number 11). HHHHHH (sequence number 12) is a tag (6×His tag) used to purify the expressed protein or peptide using a nickel column, and ENLYFQG (sequence number 13) is a sequence recognized by TEV protease (Figure 18A). In addition, a vector encoding the target protein or peptide (89-215, 89-205, 89-195, 89-185) was constructed, and a kanamycin resistance gene was used as the drug resistance gene, and pBR322ori and f1ori were used as the replication origin. When the protein or peptide produced using this expression vector is removed with TEV protease, a protein or peptide of human HMGB1 starting from the second amino acid can be produced.

The prepared plasmid was used to transform BL-21 (DE3). 5 ml of the bacterial solution cultured overnight at 37°C in LB medium containing kanamycin was transferred to 100 ml of LB medium and cultured at 37°C at 140 rpm. The turbidity was measured using a turbidimeter, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM after reaching OD 0.5-0.7. For human HMGB1 fragments (89-215, 89-205, 89-195, 89-185), the bacteria were collected after cultured overnight at 15°C. After SDS-PAGE, the expressed proteins and peptides were confirmed by protein staining and protein immunoblotting using a tag or anti-HMGB1 antibody.

Purification of each HMGB1 fragment (89-215, 89-205, 89-195, 89-185)

2 ml of buffer (PBS, 10 mM imidazole; pH 7.4) was added to each 0.1 g of collected cells and ultrasonically lysed. Centrifugation was performed at 20,000 rpm for 1 hour at 4°C, and the supernatant was recovered. Purification was performed by column chromatography using BioLogic DuoFlow (Bio-Rad).

Human HMGB1 fragment (89-215)

To every 0.1g of collected bacteria, add 1ml of buffer (PBS, 10mM imidazole; pH 7.4) and perform ultrasonic lysis. Centrifuge at 20,000rpm for 1 hour at 4°C and recover the supernatant. Purify by column chromatography using BioLogic DuoFlow (Bio-Rad). First, use 5ml of HisTrap (trademark) FF (GE healthcare), bacterial lysis solution (PBS, 10mM imidazole (pH 7.4)) for buffer A, and PBS, 500mM imidazole (pH 7.4) for buffer B for affinity purification. After balancing the column with buffer A, fill it with protein solution and wash and purify according to the following procedure. The procedure is as follows:

Isocratic mobile phase (buffer A: 97%, buffer B: 3%, 20 ml) → linear gradient (buffer A: 97% → 0%, buffer B: 3% → 100%, 20 ml) → isocratic mobile phase (buffer B: 100%, 20 ml) → fraction collection (20-40 ml, 2 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Next, ion exchange purification was performed. 89-215 was purified using 5 ml of HiTrap (trademark) Q HP (GE Healthcare), with PBS (pH 7.4) as buffer A and 20 mM HEPES, 1 M NaCl (pH 7.5) as buffer B. After equilibration with an appropriate amount of buffer A, the column was filled with the affinity-purified protein solution and washed and purified according to the following procedure. The procedure was performed as follows:

Isocratic mobile phase (buffer A: 100%, buffer B: 0%, 10 ml) → Isocratic mobile phase (buffer A: 50%, buffer B: 50%, 2 ml) → Isocratic mobile phase (buffer A: 0%, buffer B: 100%, 20 ml) → Fraction collection (10-32 ml, 1 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Human HMGB1 fragments (89-205, 89-195, 89-185)

A soluble protein solution was prepared in the same manner as for the human HMGB1 fragment (89-215), and then gradient elution was performed using each column chromatography as follows.

First, affinity purification was performed using 5 ml of HisTrap (trademark) FF, buffer A (PBS, 10 mM imidazole (pH 7.4)) and buffer B (PBS, 500 mM imidazole (pH 7.4)). After equilibration with buffer A, the column was filled with the protein solution and washed and purified according to the following procedure. The procedure was as follows:

→ Isocratic mobile phase (Buffer A: 97%, Buffer B: 3%, 50 ml) → Linear gradient (Buffer A: 97% → 0%, Buffer B: 3% → 100%, 120 ml) → Fraction collection (50-170 ml, 5 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Next, ion exchange purification was performed. Human HMGB1 fragments (89-215) and (89-205) were purified using a 5 ml HiTrap (trademark) Q HP column, and other fragments were purified using a 5 ml HiTrap (trademark) SP HP column. Buffer A used PBS (pH 7.4), and buffer B used 7×PBS (pH 7.4). After equilibration of the column with an appropriate amount of buffer A, the protein solution after affinity purification was filled, and washing and purification were performed according to the following procedure. The procedure was carried out as follows:

Isocratic mobile phase (buffer A: 100%, buffer B: 0%, 50 ml) → linear gradient (buffer A: 100% → 0%, buffer B: 0% → 100%, 50 ml) → isocratic mobile phase (buffer A: 0%, buffer B: 100%, 5 ml) → fraction collection (50-105 ml, 3 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Concentration determination

The concentration of each fragment was measured by BSA conversion using the Bradford method (Bio-Rad Protein Assay).

Migration analysis

The migration activity of each of the peptides on the bone marrow mesenchymal stem cell line MSC-1 was confirmed. A phosphate buffered saline solution containing 500 mM NaCl containing each fragment was diluted with 2 volumes of DMEM to a final concentration of 2 μM. This was then inserted into the lower chamber, sandwiched with a polycarbonate membrane with 8 μm pores, and MSC-1 cells diffused in DMEM containing 10% FBS were inserted into the upper chamber. After incubation for 4 hours in a 37°C, 5% CO2 incubator, cells migrating from the upper chamber to the lower chamber were detected using Diff-Quik stain (trademark).

result

After affinity purification of the human HMGB1 fragment (89-215) using a nickel column, gradient elution was performed using ion exchange chromatography (Q column) with gradually increasing salt concentrations. A 15.5 kDa peptide was isolated from fractions 6 and 7, peptides of 15.5 kDa, 16 kDa, and 17 kDa were isolated from fractions 8 and 9, and peptides of 16 kDa and 17 kDa were isolated from fractions 10 and 11 ( Figure 19A ).

The migration activity of the sample obtained by mixing fractions 6 and 7 (6+7) and fractions 9 and 10 on bone marrow mesenchymal stem cells (MSC-1) was examined. The results showed that the sample obtained by mixing fractions 6 and 7 (6+7) showed strong activity, while the activities of fractions 9 and 10 were weak ( Figure 19B ).

After affinity purification of the human HMGB1 fragment (89-205) using a nickel column, gradient elution was performed using ion exchange chromatography (Q column) with gradually increasing salt concentrations. The shortest fragment (*4) eluted first in fraction 6, followed by the second shortest fragment (*5) and then the longest fragment (*6) in fraction 7. Meanwhile, 89-195 and 89-185 could be purified as a single fragment by affinity purification on a nickel column (Figure 19C).

Fractions 6, 7, and 8 were examined for their migration activity on bone marrow mesenchymal stem cells (MSC-1). Strong activity was observed in fraction 6, while the activity gradually decreased in fractions 7 and 8. Meanwhile, 89-195 and 89-185 exhibited stronger activity than any of the 89-215 fragments ( FIG19D ).

discuss

Human HMGBN1 fragments (89-215, 89-205, 89-195, 89-185) were produced using Escherichia coli. 89-215 and 89-205 showed weak bone marrow mesenchymal stem cell migration activity, but cleavage occurred, believed to be caused by a protease derived from E. coli (Figures 19A and C), and the short cleaved fragments showed strong activity (Figures 19B and D). On the other hand, 89-195 and 89-185 showed strong bone marrow mesenchymal stem cell migration activity (Figures 19C and D). There is a repeating sequence of glutamine and aspartic acid at amino acids 186 to 215 at the C-terminus of HMGB1. These sequences are said to contribute to protein stabilization. Through this study, it was clarified for the first time that this part inhibits the migration activity of the HMGB1 fragment (89-215), and by removing this sequence, the migration activity of the HMGB1 fragment (89-215) can be enhanced. Furthermore, it is believed that the repeating sequence of glutamic acid and aspartic acid at the C-terminus of HMGB1 (amino acid sequence from positions 186 to 215), known as the acidic tail, is essential for binding to RAGE. Furthermore, RAGE is the receptor for HMGB1-induced migration in dendritic cells and other cells, and it was hypothesized that the C-terminus and the RAGE ligand are essential for migratory activity. However, surprisingly, it was discovered that the absence of the C-terminus is more conducive to migratory activity in bone marrow mesenchymal stem cells. This was first clarified based on the following phenomenon: when an HMGB1 fragment containing the C-terminus was produced using E. coli, the degradation product, believed to be caused by E. coli-derived proteases, exhibited greater migratory activity than the undegraded HMGB1 fragment. Furthermore, when an HMGB1 fragment lacking the C-terminus was produced, it exhibited even stronger activity than the HMGB1 fragment containing the C-terminus. Typically, a protein has a single site contributing to a specific activity, but surprisingly, HMGB1 has multiple sites contributing to the migration activity of bone marrow mesenchymal stem cells. Even more surprising, the activity of each site is approximately twice that of full-length HMGB1 at an equivalent molecular weight. Furthermore, while physiologically active peptides are generally less stable and less active as they shorten, surprisingly, there are peptides where shorter fragments exhibit stronger activity than longer fragments.

Example 16

method

The methionine (M) on the N-terminal side of human HMGB1 was deleted and replaced with MKHHHHHHENLYFQ (sequence number 11). HHHHHH (sequence number 12) is a tag (6×His tag) used to purify the expressed protein or peptide using a nickel column, and ENLYFQG (sequence number 13) is a sequence recognized by TEV protease (Figure 18A). In addition, a vector encoding the target protein or peptide (85-169, 2-215) was constructed by inserting a cDNA encoding the target protein or peptide (85-169, 2-215) downstream of the T7 promoter and lac operon, using a kanamycin resistance gene as a drug resistance gene, and using pBR322ori and f1ori as a replication origin. When the protein or peptide produced using this expression vector is removed with TEV protease, a protein or peptide of human HMGB1 starting from the second amino acid can be produced.

The prepared plasmid was used to transform BL-21 (DE3). 5 ml of the bacterial solution cultured overnight at 37°C in LB medium containing kanamycin was transferred to 100 ml of LB medium and cultured at 37°C at 140 rpm. The turbidity was measured using a turbidimeter, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM after reaching OD 0.5-0.7. For the human HMGB1 fragment (85-169), the bacteria were collected after cultured overnight at 15°C. After SDS-PAGE, the expressed proteins and peptides were confirmed by protein staining and protein immunoblotting using a tag or anti-HMGB1 antibody.

To every 0.1g of collected bacteria, add 1ml of buffer (PBS, 10mM imidazole; pH 7.4) and perform ultrasonic lysis. Centrifuge at 20,000rpm for 1 hour at 4°C and recover the supernatant. Purify by column chromatography using BioLogic DuoFlow (Bio-Rad). First, use 5ml of HisTrap (trademark) FF (GE healthcare), bacterial lysis solution (PBS, 10mM imidazole (pH 7.4)) for buffer A, and PBS, 500mM imidazole (pH 7.4) for buffer B for affinity purification. After balancing the column with buffer A, fill it with protein solution and wash and purify according to the following procedure. The procedure is as follows:

Isocratic mobile phase (buffer A: 97%, buffer B: 3%, 20 ml) → linear gradient (buffer A: 97% → 0%, buffer B: 3% → 100%, 20 ml) → isocratic mobile phase (buffer B: 100%, 20 ml) → fraction collection (20-40 ml, 2 ml/fraction)

Each fraction was confirmed by SDS-PAGE.

Concentration determination

The concentration of the recombinant protein was measured by BSA conversion using the Bradford method (Bio-Rad Protein Assay).

Migration analysis

The migration activity of each of the above peptides on the bone marrow mesenchymal stem cell line MSC-1 was confirmed. A phosphate buffered saline solution containing 500 mM NaCl containing each fragment was diluted with 2 volumes of DMEM to a final concentration of 2 μM. This was then inserted into the lower layer of the chamber, sandwiched between a polycarbonate membrane with 8 μm pores, and MSC-1 cells diffused in DMEM containing 10% FBS were inserted into the upper layer. After incubation for 4 hours in a 37°C, 5% _CO2 incubator, cells migrating from the upper layer to the lower layer were detected using Diff-Quik stain (trademark).

result

The human HMGB1 fragment (85-169) demonstrated stronger bone marrow mesenchymal stem cell migration activity than the human HMGB1 fragment (2-215) ( Figure 20A ). The migration activity was 1, which was 1.59 times greater at equal concentrations and 3.6 times greater at equal masses ( Figures 20B and C ).

discuss

Human HMGB1 fragment (85-169) was also confirmed to have stronger bone marrow mesenchymal stem cell migration activity than (2-215), similar to (89-195) and (89-185). Based on the above, it is speculated that there is at least one sequence with bone marrow mesenchymal stem cell mobilization activity in the amino acid sequence of positions 85 to 185. In Example 1, no migration activity was confirmed for HMGB1 fragment 85-169 produced using HEK293. In this example, migration activity was confirmed for HMGB1 fragment 85-169 produced using Escherichia coli. It is speculated that this difference is due to the extremely weakened or absent migration activity due to the different production methods. There are differences in post-translational modification and folding between eukaryotic organisms such as HEK293 and prokaryotes such as Escherichia coli. Therefore, even if the same protein or peptide is produced, the properties are often different.

This study revealed that HMGB1 contains at least three sites within its amino acid sequence that are active in mobilizing bone marrow mesenchymal stem cells, and that these sites are inhibited by a repeating sequence of glutamic acid and aspartic acid at the C-terminus. By creating HMGB1 fragments that remove these inhibitory sequences at the C-terminus, it is possible to create active preparations with potent bone marrow stem cell mobilization effects.

Example 17

Mouse mesenchymal stem cell migration activity 1

method

Purification of HMGB1 fragments

Inverse PCR was performed using KOD-Plus-ver.2 (Toyobo). The expression vector for the HMGB1 fragment containing amino acids 2 to 215 of human HMGB1 was used as a template plasmid. The cDNAs encoding amino acids 2 to 205, 2 to 195, and 2 to 185, respectively, were amplified using PCR, along with a histidine tag located at the N-terminus, a TEV protease recognition sequence, and a plasmid backbone. The gene product generated from this PCR product is a protein containing a histidine tag, a TEV protease recognition sequence, and a human HMGB1 fragment randomly arranged from the N-terminus. The restriction enzyme DpnI (Toyobo) was added to the PCR product to digest the template plasmid. Subsequently, phosphate groups were added to the PCR product using T4 polynucleotide kinase (NEB), and self-ligation was performed using a ligase (2× Quick Ligase, NEB or Ligation Convenience Kit, Nippongene). This was transformed into Escherichia coli JM109, and colonies were obtained by selection with kanamycin. Plasmids were extracted using the GenElute Plasmid Miniprep Kit (SIGMA-ALDRICH), and after confirming the base sequence by sequence analysis, the plasmids were transformed into Escherichia coli BL21 (DE3) to obtain colonies.

Expression induction

After shaking and culturing each colony overnight at 37°C in LB medium containing kanamycin at a final concentration of 50 mg/L, 5 ml of the bacterial suspension was transferred to 100 ml of LB medium and cultured at 37°C with shaking at 140 rpm. Turbidity was measured using a turbidimeter. When the OD reached 0.5-0.7, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. The culture was then shaken and cultured overnight at 15°C, and the cells were harvested.

Recombinant protein purification

To the collected bacterial cells, 12 ml (25-50 mg/ml) of equilibration buffer (PBS (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ), 10 mM imidazole; pH 7.4) was added, and leupeptin hydrochloride was added to a final concentration of 5 μg/ml, followed by ultrasonic lysis. Centrifugation was performed at 15,000 rpm for 60 minutes at 4°C, and the supernatant was recovered. A portion of the supernatant was taken and subjected to western blotting using an anti-human HMGB1 antibody to confirm the expression of the target protein. The remaining supernatant was sterilized by passing through a 0.45 μm filter. The target protein was purified by column chromatography using BioLogic DuoFlow (Bio-Rad).

First, affinity purification was performed using 5 ml of HisTrap (trademark) FF, buffer A (PBS, 10 mM imidazole (pH 7.4)) and buffer B (PBS, 500 mM imidazole (pH 7.4)). After equilibration with buffer A, the column was filled with the protein solution, and washing and purification were performed according to the following procedure.

program:

Isocratic mobile phase (buffer A: 97%, buffer B: 3%, 50 ml)

Linear gradient (Buffer A: 97% → 0%, Buffer B: 3% → 100%, 120 ml)

Fraction collection (50-170 ml, 5 ml/fraction)

Each fraction was confirmed by SDS-PAGE (e-PAGEL (registered trademark) 5-20% (ATTO)).

Next, ion exchange purification was performed. A 5 ml HiTrap (trademark) Q HP column was used for GNX-E-022 only, and a 5 ml HiTrap (trademark) SP HP column was used for all other columns. PBS (pH 7.4) was used as buffer A, and 7×PBS (pH 7.4) was used as buffer B. After equilibration of the column with an appropriate amount of buffer A, the protein solution after affinity purification was filled, and washing and purification were performed according to the following procedure.

program:

Isocratic mobile phase (buffer A: 100%, buffer B: 0%, 50 ml, 4 ml/min)

Linear gradient (buffer A: 100% → 0%, buffer B: 0% → 100%, 50 ml, 4 ml/min)

Isocratic mobile phase (buffer A: 0%, buffer B: 100%, 5 ml, 4 ml/min)

Fraction collection (50-105 ml, 3 ml/fraction)

Each fraction was subjected to SDS-PAGE, and the purified protein was confirmed by protein staining of the gel.

Concentration determination

The concentration of the recombinant protein was measured by BSA conversion using the Bradford method (Bio-Rad Protein Assay).

Migration analysis

The migration activity of each of the peptides on the bone marrow mesenchymal stem cell line MSC-1 was confirmed. A phosphate buffered saline solution containing 500 mM NaCl containing each fragment was diluted with 2 volumes of DMEM to a final concentration of 2 μM. This was then inserted into the lower chamber, sandwiched with a polycarbonate membrane with 8 μm pores, and MSC-1 cells diffused in DMEM containing 10% FBS were inserted into the upper chamber. After incubation for 4 hours in a 37°C, 5% CO2 incubator, cells migrating from the upper chamber to the lower chamber were detected using Diff-Quik stain (trademark).

result

Compared with the 2-215 fragment and the 2-205 fragment, the 2-195 fragment and the 2-185 fragment showed stronger migration activity ( FIG. 21A ).

discuss

The fragment from positions 186 to 215 consists of a 30-amino acid repeating sequence of aspartic acid and glutamic acid, known as the acidic tail. These data suggest that the acidic tail strongly inhibits the migration activity of HMGB1 in bone marrow mesenchymal stem cells, with a particularly strong inhibitory effect on the 20 amino acids at the C-terminus. Based on the data to date, multiple active domains of HMGB1 have been identified that contribute to the migration activity of bone marrow mesenchymal stem cells. Whether this domain inhibits the activity of all of these domains requires further detailed experiments.

Human mesenchymal stem cell migration activity 2

method

Human HMGB1 fragments (2-215, 2-84, 2-44, 45-84, 85-169, 89-185, 89-195, and 89-205) were produced in E. coli and purified using appropriate columns in the same manner as in Examples 14, 15, and 16. However, 89-215 was purified using the same method as for 89-205 in Example 15.

Concentration determination

The concentration of each fragment was measured by BSA conversion using the Bradford method (Bio-Rad Protein Assay).

Migration analysis

For each fragment, the migration analysis of human-derived bone marrow mesenchymal stem cells was performed in the same manner as the migration analysis of mouse-derived bone marrow mesenchymal stem cells (MSC-1) described above. Fragments with a final concentration equivalent to 2 μM were used. In addition, human-derived bone marrow mesenchymal stem cells used hMSC (human mesenchymal stem cells, manufactured by Takara) of the 4th passage. The culture medium for proliferation used mesenchymal stem cell proliferation medium (MF medium, manufactured by TOYOBO) and was cultured in a 37°C, 5% CO ₂ incubator. Fresh culture medium was replaced every 2-4 days, and passage was performed when the cells reached 80% confluence.

result

Regarding human HMGB1 fragments (2-215, 2-84, 2-44, 45-84), HMGB1 fragments (2-84, 2-44, 45-84) were confirmed to have stronger activity than human HMGB1 fragment (2-215) as in Example 14. Regarding human HMGB1 fragments (89-185, 89-195, 89-205, 89-215), active human HMGB1 fragments (89-185, 89-195) obtained by shortening the C-terminal acidic tail were confirmed to have strong activity as in Example 15. Regarding human HMGB1 fragment (85-169), human HMGB1 fragment (2-215) was confirmed to have strong activity as in Example 16 ( FIG. 21B ).

discuss

In human bone marrow mesenchymal stem cells, each fragment demonstrated the same migratory activity as mouse-derived bone marrow mesenchymal stem cells. At least one site within the human HMGB1 fragments (2-44, 45-84, and 85-169) independently exhibited migratory activity against human bone marrow mesenchymal stem cells. Proteins typically have a single site with specific activity, so the presence of multiple active sites is surprising. Furthermore, the activity of each fragment was even stronger than that of the nearly full-length sequence (2-215). Meanwhile, the RAGE binding site is located between amino acids 150 and 183, but activity also occurs between amino acids 89 and 169, suggesting that RAGE may not be required for migratory activity against human bone marrow mesenchymal stem cells. As with mouse-derived cells, the human HMGB1 fragments (89-185, 89-195, 89-205, and 89-215) exhibited strong activity due to fragments lacking the C-terminal acidic tail. The C-terminus is believed to also play an inhibitory role in the migration of human bone marrow mesenchymal stem cells. By shortening or deleting the C-terminal acidic tail, a more active HMGB1 fragment can be produced.

Changes in migration activity of fusion fragments obtained by adding human HMGB1 acidic tail fragments 186-215, 186-205, and 186-195 to human HMGB1 fragment 2-84

method

A cDNA was prepared by similarly attaching human HMGB1 fragments 186-215, 186-205, or 186-195 to the C-terminus of human HMG1 fragment 2-84. Furthermore, as described above, an expression vector was designed by deleting the methionine (M) on the N-terminal side of human HMGB1 in the fragment expressed in E. coli and attaching MKHHHHHHENLYFQ (SEQ ID NO. 11) instead. Furthermore, HHHHHH (SEQ ID NO. 12) is a tag (6×His tag) for purifying the expressed protein or peptide using a nickel column, and ENLYFQG (SEQ ID NO. 13) is a sequence recognized by TEV protease ( FIG. 18 ). Furthermore, a vector was constructed in which the cDNA described above was inserted downstream of the T7 promoter and lac operator, a kanamycin resistance gene was used as a drug resistance gene, and pBR322ori and f1ori were used as replication origins. When a protein or peptide produced using this expression vector is cleaved with TEV protease, a protein or peptide of human HMGB1 starting from the second amino acid can be produced.

Use the prepared plasmid to transform BL-21 (DE3). After shaking overnight in LB medium containing kanamycin at 37°C, transfer 5 ml of the bacterial suspension to 100 ml of LB medium and shake-incubate at 37°C at 140 rpm. Measure the turbidity using a turbidimeter. Once the OD reaches 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Shake and incubate overnight at 15°C, then harvest the cells.

Purification of each HMGB1 fragment (2-84+186-215, 2-84+186-205, 2-84+186-195)

Equilibration buffer (PBS (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ), 10 mM imidazole; pH 7.4) was added to the collected bacterial cells to a final concentration of 5 μg/ml, and ultrasonic lysis was performed. Centrifugation was performed at 15,000 rpm for 60 minutes at 4°C, and the supernatant was recovered. The remaining supernatant was sterilized by passing through a 0.45 μm filter. The target protein was purified by column chromatography using a BioLogic DuoFlow (Bio-Rad).

Next, ion exchange purification was performed. For 2-84+186-215, 2-84+186-205, and 2-84+186-195, 5 ml of HiTrap (trademark) Q HP (GE Healthcare) was used, PBS (pH 7.4) was used as buffer A, and 7×PBS (pH 7.4) was used as buffer B. After equilibration of the column with an appropriate amount of buffer A, the protein solution after affinity purification was filled, and washing and purification were performed according to the following procedure. The procedure was carried out as follows:

Isocratic mobile phase (buffer A: 100%, buffer B: 0%, 50 ml, 4 ml/min)

Linear gradient (buffer A: 100% → 0%, buffer B: 0% → 100%, 50 ml, 4 ml/min)

Isocratic mobile phase (buffer A: 0%, buffer B: 100%, 5 ml, 4 ml/min)

Fraction collection (50-105 ml, 3 ml/fraction)

Each fraction was subjected to SDS-PAGE, and the purified protein was confirmed by protein staining of the gel.

Furthermore, each fragment was subjected to Western blotting using an antibody that recognizes human HMGB1 to confirm that it was the target fragment.

Migration assay using HMGB1 fragment (2-84) and HMGB1 fusion fragments (2-84+186-215, 2-84+186-205, 2-84+186-195) of MSC-1

Using the fragments prepared by the above method, migration analysis was performed using the bone marrow mesenchymal stem cell line MSC-1. The migration analysis was performed in the same manner as described above.

result

Fragment 2-84 showed migration activity, but human HMGB1 fusion fragments of 10 amino acids, 20 amino acids, and 30 amino acids to which an acidic tail sequence was added showed no migration activity ( FIG. 21C ).

discuss

The above examples demonstrate that human HMGB1 fragment 89-215 has extremely weak migration activity for mesenchymal stem cells, but when the acidic tail at the C-terminus is removed, migration activity is enhanced. This example clearly demonstrates that the acidic tail functions to attenuate the migration activity of fragments 2-84 and 89-185. Furthermore, while the 2-84 fragment contains multiple core regions of migration activity, fusion of the 186-215 fragment to the 2-84 fragment virtually eliminates migration activity, suggesting that all core regions within the 2-84 fragment are inhibited. The discovery that a single HMGB1 molecule contains at least three core sequences that contribute to the migration activity of bone marrow mesenchymal stem cells is a surprising finding. Furthermore, the surprisingly finding that the acidic tail, consisting of only 30 amino acids at the C-terminus, almost completely inhibited the migration activity of the core sequence present in at least two locations within the 2-84 fragment on the N-terminal side of HMGB1, and also inhibited the migration activity of the core sequence present in the 85-185 fragment, thereby weakening the migration activity of the entire HMGB1 was also found. The 1-85 fragment of HMGB1 is believed to have an anti-inflammatory effect on inflammation caused by LPS (lipopolysaccharide), and Wei Gong et al. reported that a fragment obtained by fusing the 186-215 fragment to the 1-85 fragment reduced the mortality rate caused by LPS administration compared to the 1-85 fragment (Journal of Biomedicine and Biotechnology Volume 2010, Article ID 915234, doi:10.1155/2010/915234). A paper by Wei Gong et al. showed that the acidic tail is required to enhance the anti-inflammatory effects of 1-85. However, the present invention demonstrates that the acidic tail actually inhibits the migration of bone marrow mesenchymal stem cells. According to the present invention, shortening or completely eliminating the acidic tail is expected to further improve the therapeutic effects of bone marrow mesenchymal stem cells in diseases for which treatment has been shown.

Example 18

method

Experimental animal uses SD rat (male, 8 week old).After fully anesthetizing by utilizing the inhalation anesthesia of isoflurane, make horizontal 3cm, vertical 7cm long strip skin incision on the back.But one side of head side is not excised, and skin is fully free from subcutaneous tissue.Use No. 4 silk thread by 3 sides of incision and surrounding skin suture, use Tegaderm transparent dressing (3M company manufacture) to protect to prevent bacterial infection.

Mouse full-length HMGB1 (100 μg/time/day) produced using HEK293 and a chemically synthesized HMGB1 peptide (1-44 amino acids, 50 μg/time/day) were diluted to 200 μl with phosphate buffer and administered to rats via the tail vein. The initial administration was 6 hours after surgery, and then every 24 hours, for a total of 5 doses. A negative control was administered with phosphate-buffered saline. After 1 week, Tegaderm was removed, and the wound site was observed weekly. The area of necrosis and ulceration was measured.

result

One week after surgery, skin necrosis occurred in 4 of 5 rats in the negative control group. In the full-length HMGB1 administration group, skin necrosis occurred in 3 of 5 rats. In the HMGB1 peptide (1-44 amino acids) group, skin necrosis occurred in 1 of 5 rats. Seven weeks after surgery, strong skin contracture occurred in 4 of 5 rats in the negative control group. In contrast, contracture was confirmed in 3 of 5 rats in the full-length HMGB1 administration group and in 2 of 5 rats in the HMGB1 peptide (1-44 amino acids) group (Figure 24).

discuss

In the groups administered with full-length HMGB1 produced in HEK293 cells and the group administered with HMGB1 peptide (1-44 amino acids), a reduction effect on necrotic tissue was observed after one week, with the HMGB1 peptide (1-44 amino acids) group demonstrating a further reduction effect. The wound area was reduced to half that of the negative control group after one, two, and three weeks. After three weeks, the wound area in the HMGB1 peptide (1-44 amino acids) group was further reduced compared to the other two groups. During the seven-week healing process, the HMGB1 peptide (1-44 amino acids) group also showed a trend toward smaller wound area. Contracture at the wound site after seven weeks was also the mildest in the HMGB1 peptide (1-44 amino acids) group. Bone marrow mesenchymal stem cells are known to promote the proliferation of skin cells under hypoxic conditions, and it is hypothesized that HMGB1-mobilized bone marrow mesenchymal stem cells inhibit the expansion of skin necrosis caused by the lack of nutrition and oxygen during flap creation, thereby promoting wound healing. This suggests that such an effect suppresses the expansion of damage caused by skin ischemia, trauma, or surgery, and also has an excellent effect on the beauty of the face after healing.

Example 19

method

Experimental animal uses 15 SD rats (male, 8 week old) of every group.After fully anesthetizing by utilizing the inhalation anesthesia of isoflurane, make horizontal 3cm, vertical 7cm long strip skin incision on the back.But one side of head side is not excised, and skin is fully free from subcutaneous tissue.Use No. 4 silk thread by 3 sides of incision and surrounding skin suture, use Tegaderm transparent dressing (3M company manufacture) to protect to prevent bacterial infection.

Chemically synthesized HMGB1 peptide (1-44 amino acids, 50 μg/time/day) or HMGB1 peptide (17-25 amino acids, 50 μg/time/day) was diluted to 200 μl with phosphate buffer and administered to rats with wounds through the tail vein. The initial administration was 6 hours after surgery, and then every 24 hours, for a total of 5 doses. Phosphate-buffered saline was administered as a negative control. After 1 week, Tegaderm was removed, and the wound site was observed 2 weeks after the wound was created. The area of the unhealed area (ulcer, necrosis) was measured.

result

The ratio (%) of the area of the unhealed portion relative to the total flap area was calculated 2 weeks after flap creation. In the HMGB1 peptide (1-44 amino acids) group, the unhealed portion averaged 14.1%, while in the HMGB1 peptide (17-25 amino acids, 50 μg/time/day) group, it averaged 9.1%. Furthermore, the ratio (%) of the area of the unhealed portion relative to the total flap area was calculated 6 weeks after flap creation. The results showed that in the HMGB1 peptide (1-44 amino acids) group, the unhealed portion averaged 4.1%, while in the HMGB1 peptide (17-25 amino acids, 50 μg/time/day) group, it averaged 3.5% (Figure 26).

discuss

In the experiment of the skin injury model of Example 19, the group administered with chemically synthesized HMGB1 peptide (1-44 amino acids) showed a tendency to have a smaller wound area during the healing process from one week after injury to seven weeks, compared with the group administered with full-length HMGB1 produced by HEK293. In addition, according to this experiment of chemical synthesis, the HMGB1 peptide (17-25 amino acids) group was confirmed to have an effect on improving skin injuries that was equal to or higher than that of the HMGB1 peptide (1-44 amino acids, 50 μg/time/day) group. There are multiple bone marrow mesenchymal stem cell mobilization active sites (core sequences) in vitro, and the shortest sequence currently identified is a sequence consisting of 9 amino acids from amino acids 17 to 25. The length of the core sequence of another mesenchymal stem cell mobilization active site needs to be 30 amino acids in length or 85 amino acids in length. This study not only confirmed the improvement effect on skin damage in vitro, but also confirmed the improvement effect in vivo in a group of HMGB1 peptides (17 to 25 amino acids) composed of only 9 amino acids. Compared with protein preparations made from cultured cells of eukaryotic origin such as HEK293, peptides containing 9 amino acids as the core domain can be expected to be more cheaply and stably produced.

Industrial applicability

The present invention provides peptides that maintain PDGFRα-positive cell-mobilizing activity, with a molecular weight, for example, less than 1/10 that of the full-length HMGB1 protein, which consists of approximately 200 amino acids. These peptides can be produced not only by methods utilizing cultured cells derived from Escherichia coli or eukaryotic organisms, but also by chemical synthesis using a peptide synthesizer. Therefore, when manufacturing the peptides as pharmaceuticals, improved purification purity, stable production, and cost reductions are expected.

Furthermore, when the recombinant peptide is produced in E. coli or cultured cells, an approximately 2-fold increase in molar activity and a 6-fold increase in mass activity compared to full-length HMGB1 have been observed. Therefore, in clinical applications of the drug, the dosage can be reduced, leading to cost reductions and the suppression of side effects.

Full-length HMGB1 is known to have binding activity to LPS (lipopolysaccharide), a type of endotoxin. Furthermore, it has been reported that HMGB1 fragments containing amino acids 1 to 79 or 88 to 162 lose their LPS binding activity (Youn et al., J Immunol 2008; 180; 5067-5074).

Even small amounts of LPS in pharmaceuticals can cause fever and other serious side effects, often leading to severe side effects. Therefore, the inclusion of LPS in pharmaceuticals is strictly controlled. Because HMGB1 has an affinity for LPS, it is difficult to completely remove LPS from pharmaceuticals. However, by reducing its affinity for LPS through peptidylserine hydrolysis, it is speculated that the inclusion of LPS in pharmaceuticals can be reduced. Therefore, the use of peptides comprising the PDGFRα-positive cell-mobilizing moiety identified in this invention will enable the development of safer pharmaceuticals.

By directly applying the peptide of the present invention to the tissue or its vicinity that needs to be regenerated, it is possible to induce or promote the regeneration of the tissue. In addition, by using methods such as intravenous administration, the peptide of the present invention is applied to tissues different from the tissue that needs to be regenerated, it is possible to induce or promote the regeneration of the tissue that needs to be regenerated. For example, in the case of diseases of deep organs in the body such as cerebral infarction, it is difficult to directly apply therapeutic drugs to the damaged part (brain). On the other hand, in the present invention, it is possible to treat by intravenous administration widely implemented in general diagnosis and treatment, therefore, it is possible to safely and simply administer therapeutic drugs with any number of times and concentrations, which is an effect superior to previous methods of treatment.

A recently developed treatment for cerebral infarction using bone marrow cells is known to be effective in collecting cells from the patient's own bone marrow and re-administering them into the bloodstream. However, collecting bone marrow cells requires inserting a thick needle deep into the body's bone marrow for aspiration, which inevitably involves considerable invasiveness. In contrast, the present invention utilizes intravenous administration of a drug, enabling direct mobilization of bone marrow cells into the bloodstream. Therefore, even if administered multiple times to a patient with cerebral infarction, it does not involve significant invasiveness.

Bone marrow-derived pluripotent stem cells have the potential to differentiate into a variety of cells, including mesenchymal, epithelial, and neural systems. It is believed that after moving to the site of injury, they differentiate according to the surrounding microenvironment and induce tissue repair. In regenerative medicine or cell therapy, rare bone marrow pluripotent stem cells are cultured in vitro to proliferate and then used for treatment. However, unlike previous drugs, there is a risk of cell deterioration (cancer or contamination by bacteria, viruses, etc.) during the culture process, so sufficient safety management is required. In contrast, the present invention does not remove the cells from the body and perform artificial operations, so it is safer.

Sequence Table

<110> Genomex Co., Ltd.

National University Corporation Osaka University

<120> Peptides for inducing tissue regeneration and their applications

<130> G6-X1001Y1P

<150> JP 2011-098270

<151> 2011-04-26

<150> JP 2011-219454

<151> 2011-10-03

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225

Claims (12)

1. A peptide with PDGFRα-positive cell migration stimulatory activity, comprising any one of the following amino acid sequences: (1) amino acid sequence from position 1 to position 34 of any one of the sequence numbers 1, 3, and 5; (2) amino acid sequence from position 1 to position 44 of any one of the sequence numbers 1, 3, and 5; (3) amino acid sequence from position 1 to position 70 of any one of the sequence numbers 1, 3, and 5; (4) amino acid sequence from position 1 to position 81 of any one of the sequence numbers 1, 3, and 5; (5) amino acid sequence from position 2 to position 34 of any one of the sequence numbers 1, 3, and 5; (6) amino acid sequence from position 2 to position 44 of any one of the sequence numbers 1, 3, and 5; (7) amino acid sequence from position 1 to position 44 of any one of the sequence numbers 1, 3, and 5. (8) The amino acid sequence from position 2 to position 62 in the amino acid sequence; (9) The amino acid sequence from position 2 to position 70 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (10) The amino acid sequence from position 10 to position 25 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (11) The amino acid sequence from position 11 to position 25 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (12) The amino acid sequence from position 11 to position 27 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (13) The amino acid sequence from position 11 to position 30 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (14) The amino acid sequence from position 11 to position 62 in the amino acid sequence shown in any of the sequence numbers 1, 3, and 5. (15) The amino acid sequence from position 11 to position 44 in any of the sequence numbers 1, 3, and 5; (16) The amino acid sequence from position 12 to position 25 in any of the sequence numbers 1, 3, and 5; (17) The amino acid sequence from position 12 to position 30 in any of the sequence numbers 1, 3, and 5; (18) The amino acid sequence from position 13 to position 25 in any of the sequence numbers 1, 3, and 5; (19) The amino acid sequence from position 13 to position 30 in any of the sequence numbers 1, 3, and 5; (20) The amino acid sequence from position 14 to position 25 in any of the sequence numbers 1, 3, and 5; (21) The amino acid sequence from position 14 to position 30 in any of the sequence numbers 1, 3, and 5. (22) The amino acid sequence from position 15 to position 25 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (23) The amino acid sequence from position 15 to position 30 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (24) The amino acid sequence from position 16 to position 25 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (25) The amino acid sequence from position 16 to position 30 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (26) The amino acid sequence from position 17 to position 25 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (27) The amino acid sequence from position 17 to position 30 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5; (28) The amino acid sequence from position 17 to position 44 of the amino acid sequence shown in any of the sequence numbers 1, 3, and 5. 2. The peptide according to claim 1, wherein the peptide is composed of amino acid sequences from position 17 to position 25 of the amino acid sequence indicated by any one of sequence numbers 1, 3, and 5. 3. The peptide according to claim 1, wherein the peptide is composed of amino acid sequences from position 1 to position 44 of the amino acid sequence indicated by any one of sequence numbers 1, 3, and 5. 4. The peptide according to claim 1, comprising any one of the following amino acid sequences: (1) the amino acid sequence from position 17 to position 25 of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5; (2) the amino acid sequence from position 1 to position 44 of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5; (3) the amino acid sequence from position 1 to position 70 of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5; (4) the amino acid sequence from position 2 to position 44 of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5; and (5) the amino acid sequence from position 2 to position 70 of the amino acid sequence shown in any one of the sequence numbers 1, 3, and 5. 5. The peptide according to any one of claims 1 to 4, wherein it is a synthetic peptide. 6. The peptide according to any one of claims 1 to 4, wherein it is a peptide manufactured using cells. 7. The peptide according to any one of claims 1 to 4, wherein it is a tagged peptide. 8. The peptide according to any one of claims 1 to 4, wherein the peptide is a peptide with a peptide fragment derived from a tag attached. 9. A DNA that encodes the peptide according to any one of claims 1 to 4. 10. A vector containing the DNA of claim 9. 11. A transformed cell containing the DNA of claim 9, wherein the transformed cell is not a plant cell. 12. A transformed cell comprising the carrier of claim 10, wherein the transformed cell is not a plant cell.