WO2011052668A1 - 骨髄間葉系および/または多能性幹細胞の血中動員による組織再生促進剤 - Google Patents
骨髄間葉系および/または多能性幹細胞の血中動員による組織再生促進剤 Download PDFInfo
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- WO2011052668A1 WO2011052668A1 PCT/JP2010/069133 JP2010069133W WO2011052668A1 WO 2011052668 A1 WO2011052668 A1 WO 2011052668A1 JP 2010069133 W JP2010069133 W JP 2010069133W WO 2011052668 A1 WO2011052668 A1 WO 2011052668A1
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Definitions
- the present invention relates to a tissue regeneration promoter that is administered to a tissue different from a tissue that requires regeneration.
- Regenerative medicine is medical treatment aimed at functional and organic regeneration of damaged organs, and utilizes cells or tissues cultured and processed outside the body.
- a skin culture sheet or the like is obtained by culturing skin cells collected from the person or another person outside the body and transplanting the processed product into a sheet into damaged skin.
- Cell culture may cause cell degradation (aging, oncogenesis, contamination with bacteria, viruses, etc.), so manufacturing in a facility with GMP standards guaranteed to maintain safety is essential. It is expected that increasing the price will be an issue.
- the living body is equipped with a mechanism for repairing and repairing a damaged site for organ damage, but it is known that when the damaged site is large, the damaged site is blocked by a non-functional scar tissue.
- Such wound healing by scar tissue is an inhibitor of nerve regeneration in cerebral infarction and spinal cord injury, a heart rupture factor in myocardial infarction, and keloid in extensive burns and surgical wounds. This is a cause of significant loss of quality of life. If it becomes possible to activate the repair and regeneration mechanism of damaged tissue in a living body, it can be expected to induce regeneration of damaged tissue (organ) by functional tissue without healing the scar.
- [2] The drug according to [1], which is administered parenterally. [3] The drug according to [2], which is administered by injection. [4] The drug according to [1], which is administered intravascularly, intramuscularly, subcutaneously, intradermally or intraperitoneally. [5] The drug according to any one of [1] to [4], wherein the cell or tissue extract is produced by a method comprising a step of immersing the cell or tissue in a solvent.
- [6] The drug according to any one of [1] to [4], wherein the heparin-binding fraction of the cell or tissue extract is produced by a method comprising the following steps; (A) a step of immersing cells or tissues in a solvent; (B) a step of bringing the extract obtained in step (a) into contact with immobilized heparin; and (c) a step of eluting a heparin-binding fraction from the immobilized heparin. [7] The drug according to any one of [1] to [6], which is used for promoting regeneration of nerve tissue, bone tissue, or skin tissue.
- a tissue regeneration promotion kit comprising a composition containing the substance according to any one of (a) to (q) below, wherein the composition is applied to a tissue different from a tissue requiring regeneration.
- (A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Cell or tissue extract ( q) A hepar
- HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Cell or tissue extract ( q) A heparin-binding fraction of cell or tissue extracts.
- [14] The method of [13], which is parenteral administration.
- [17] The method according to any one of [13] to [16], wherein regeneration of nerve tissue, bone tissue, or skin tissue is promoted.
- tissue regeneration promoter is administered parenterally.
- tissue regeneration promoter is administered by injection.
- tissue regeneration promoter is administered intravascularly, intramuscularly, subcutaneously, intradermally or intraperitoneally.
- tissue regeneration accelerator is a regeneration promoter for nerve tissue, bone tissue, or skin tissue.
- composition of [23] which is administered parenterally.
- composition of [24] which is administered by injection.
- composition of [23] which is administered intravascularly, intramuscularly, subcutaneously, intradermally or intraperitoneally.
- composition according to any one of [23] to [26], wherein the method for promoting tissue regeneration is a method for promoting regeneration of nerve tissue, bone tissue, or skin tissue.
- HMGB1, HMGB2, HMGB3, S100A8, and S100A9 have the activity of mobilizing bone marrow pluripotent stem cells.
- Bone marrow pluripotent stem cells can differentiate into epithelial and nervous cells as well as mesenchymal cells. If it is possible to mobilize bone marrow pluripotent stem cells to the damaged site through the bloodstream during extensive tissue damage, it is expected to promote repair and regeneration of physiologically damaged tissue by bone marrow pluripotent stem cells it can.
- the bone marrow pluripotent stem cell mobilization factor, HMGB1, HMGB2, HMGB3, S100A8, S100A9 is administered to the distal part from the damaged site by a method such as intravenous administration, so that Methods were provided to mobilize pluripotent stem cells and promote repair of damaged tissue.
- a method such as intravenous administration
- Methods were provided to mobilize pluripotent stem cells and promote repair of damaged tissue.
- a method such as intravenous administration, so that Methods were provided to mobilize pluripotent stem cells and promote repair of damaged tissue.
- Bone marrow-derived pluripotent stem cells have the potential to differentiate into various cells such as mesenchymal, epithelial, and nervous systems, but after moving to the damaged site, they differentiate according to the surrounding niche environment and repair tissue It is thought to induce. In regenerative medicine and cell therapy, rare bone marrow pluripotent stem cells are cultured in vitro and then used for treatment. Therefore, sufficient safety management is necessary. In the present invention, by administering HMGB1, HMGB2, HMGB3, S100A8, S100A9, the bone marrow pluripotent stem cells are mobilized into the peripheral circulation blood, but the cells are not taken out of the body and not subjected to artificial manipulations, so it is safe. Is a high treatment.
- the fraction bound to the column was subjected to SDS-PAGE and then subjected to Coomassie staining. It is a photograph which shows the signal of GFP of the skin after an ulcer is closed, and a sliced piece of skin. A skin ulcer was created on the back of a GFP bone marrow transplanted mouse, and HMGB1 or S100A8 was administered intravenously. In the skin of mice intravenously administered with HMGB1 and S100A8, many GFP-positive bone marrow-derived cells were detected as compared to controls. It is a graph which shows the skin ulcer area measured with time.
- a skin ulcer was created on the back of the mouse and HMGB1 or S100A8 was administered intravenously.
- the skin ulcer reduction effect was observed in the HMGB1 administration group compared to the control group.
- skin ulcer reduction effect was observed in the S100A8 administration group compared to the control group (vertical axis; ulcer area ⁇ area at the time of skin ulcer preparation X100, horizontal axis; after skin ulcer preparation) Days).
- staining (MT) to the thin section of the skin after skin ulcer closure.
- the cell of the arrow in the right photograph is a cell positive for GFP and ⁇ III tubulin. Bone marrow-derived cells were confirmed to express neural markers. It is a photograph which shows the detection result of an infarcted part. Cerebral infarction disease model mice were prepared and HMGB1 was administered intravenously. After the treatment was completed, a thin slice of the brain was prepared and stained with Nissl. In the case of PBS administration (control), necrotic tissue was observed in the cortex, but in the HMGB1-treated case, no necrotic tissue was observed in the cortex. It is a graph which shows the survival rate for 7 days after cerebral infarction preparation.
- HMGB1 was treated by intravenous administration. Improvement in survival rate was confirmed by treatment with HMGB1 in both 45-minute and 60-minute ischemia.
- mouth it is the photograph which shows that the bone marrow cell of the GFP bone marrow chimera mouse moved to the fracture site
- a GFP bone marrow transplanted mouse and a wild-type mouse were combined with the skin to produce a fracture in the wild-type mouse.
- the lower left photo shows the accumulation of GFP fluorescence in the skin graft
- the lower middle photo shows the enlarged image of the skin graft
- the lower right photo shows the accumulation of GFP fluorescence in the enlarged skin.
- the first row from the left is DAPI staining (nuclear staining).
- the upper photograph shows a weakly magnified image (100x) of the skin skin
- the middle photograph shows its epidermal / dermis boundary with a strongly magnified image (200x).
- the lower row shows the hair follicle with a strong magnified image (200x).
- the second row from the left is a GFP fluorescence image of each region in the first row
- the third row from the left is an immunostained image of the keratin 5 (K5)
- the fourth row from the left is an overlay of the respective fluorescence.
- Indicates (Merge) Many GFP positive epidermal cells and dermal fibroblasts have been observed. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber.
- the upper left photo shows a bone marrow mesenchymal system that migrates from the upper tank of the Boyden chamber to the skin extract side (lower tank side) through a microscopic hole on the silicon film and adheres to the lower side of the silicon film.
- the upper right photo shows a strong magnified image at 0h
- the lower left photo shows a strong magnified image at 12h
- the lower right photo shows a strong magnified image at 24h.
- a stained image of bone marrow-derived mesenchymal stem cells migrated to a solution neutralized by adding an anti-HMGB1 polyclonal antibody to the purified HMGB1 sample used (mostly migratory activity disappeared). It is a photograph which shows the in vivo bone marrow origin mesenchymal stem cell mobilization activity by HMGB1.
- the HMGB1 fraction (final purified fraction number 4) showed about three times the mobilization activity of the control (final purified fraction number 1).
- the left photograph shows a bright field image of migrating cells seeded in the medium, and the right photograph shows a GFP fluorescence image in the dark field. It is the photograph 24 hours after the culture
- the photo on the left shows a bright field image of fibroblast-like cells and epithelial cell-like cells growing on a cultured plastic petri dish, and the photo on the right shows a GFP fluorescence image in the dark field. It is the photograph two weeks after the culture
- the left and right photos have the same field of view, the left photo is bright field, the right photo is through a fluorescent filter (B and D detect GFP fluorescence, F detects keratin 5 fluorescence) .
- a linear hair-like morphology is observed on the left side of the bone marrow-derived GFP-positive cell population that forms colonies in a circular shape in a cultured plastic petri dish (indicated by ⁇ ).
- F indicates that bone marrow-derived cells exhibit a hair-like morphological change and express keratin 5 (indicated by ⁇ (arrow)). It is the photograph which detected the HMGB family in the newborn mouse skin extract using the Western * blot * method.
- HMGB family in a mammalian cell. It has cytomegalovirus enhancer and chicken ⁇ -actin promoter, and synthesizes a large amount of mRNA HM encoded by cDNA of HMGB family (complementary DNA) located downstream of the promoter. It is a photograph showing the results of Western blot of purified recombinant Flag ⁇ tag-HMGB family fusion protein expressed in HEK293 cells. It is a figure which shows the bone marrow mesenchymal stem cell migration activity of recombinant HMGB1, HMGB2, and HMGB3 using the Boyden chamber. All the recombinant proteins showed migration activity compared to the control group.
- A is a photograph in which a mouse was fixed to a stereotaxic apparatus, a midline incision was made in the head with a scalpel, and burr was made using a drill.
- B is a photograph in which a negative pressure was applied to the brain using a syringe and a portion of the brain tissue was aspirated.
- C is a photograph after injecting 5 ⁇ l of the purified heparin column fraction of the skin extract dissolved in fibrinogen of the fibrin glue preparation and then injecting 5 ⁇ l of the fibrin glue preparation thrombin.
- D and E are photographs two weeks after the brain injury model treatment. Compared with control D, accumulation of GFP-positive cells was observed in E of the treatment group by the purified fraction of the skin extract heparin column.
- F and G are photographs 6 weeks after the brain injury model treatment. Compared with control F, accumulation of GFP positive cells was observed in G of the treatment group using the purified fraction of the skin extract heparin column. It is a figure which administers a skin extract (SE) from a mouse tail vein, and collects peripheral blood.
- SE skin extract
- the part surrounded by the blue gland shows the CD44-positive and PDGFR ⁇ -positive cell group, which is increased in the skin extract administration group (SE) compared to the PBS group.
- SE skin extract administration group
- the left is a negative control PBS-administered mouse, and the right is a HMGB1-administered mouse.
- the vertical axis represents the expression level of CD44
- the horizontal axis represents the expression level of PDGFR ⁇ .
- the part surrounded by the blue line shows a CD44-positive and PDGFR ⁇ -positive cell group, which is increased in HMGB1-administered mice compared to PBS-administered mice.
- A is a figure which shows the result of the flow cytometry showing the existence frequency of the cell which has CD44 and PDGFR (alpha).
- Cell groups of both PDGFR ⁇ -positive and CD44-positive cells and PDGFR ⁇ -positive and CD44-negative cells in peripheral blood are increased by HMGB1 administration.
- B shows PDGFR ⁇ -positive and CD44-positive cells
- C shows PDGFR ⁇ -positive and CD44-negative cells in the PBS administration group and the HMGB1 administration group, respectively.
- any cell group there is a statistically significant increase in the HMGB1 administration group.
- It is a photograph which shows GFP fluorescence accumulation with respect to the transplanted skin piece observed after skin transplantation to the back of a GFP bone marrow transplanted mouse.
- the left photograph (A) stains the nucleus with DAPI.
- the middle photo (B) shows GFP-positive bone marrow-derived cells accumulated in the skin graft in green fluorescent color.
- the right photograph (C) is a view in which the photograph (A) and the photograph (B) are superimposed.
- Bone marrow-derived cells reconstruct skin tissue. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber.
- Bone marrow mesenchymal stem cells that have migrated from the inside of the upper tank of the Boyden chamber to the lower tank side containing the skin extract and passed through the fine holes in the polycarbonate membrane membrane with 8 ⁇ m holes. Is an image dyed with a blue pigment.
- the lower layer contained skin extracts collected from 2-day-old mice and 6-week-old mice. It is the photograph which confirmed the presence of the protein of S100A8 and S100A9 in a skin extract by Western blot method. It is a photograph which shows the result of having eluted Heparin (TM) binding protein in a skin extract from Heparin (affinity) column (s) by NaCl concentration gradient.
- Proteins of each fraction were fractionated by SDS-PAGE and detected by silver staining. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber. Bone marrow mesenchymal stem cells that migrate from the upper tank of the Boyden chamber through the micropores on the membrane to the heparin-bound fractions of the skin extract (lower tank side) and adhere to the submembrane tank side Is an image dyed with a blue pigment. It is a photograph showing the results of detecting the presence of S100A8 and S100A9 proteins in each heparin-bound fraction of the skin extract using the Western Blot method. It is a figure of S100A8, S100A9 expression vector.
- B is a CD45 negative cell group
- the left diagram shows the percentage of CD44 positive and PDGFR ⁇ positive cell groups
- the right diagram shows the CD44 positive and PDGFR ⁇ positive cell groups in a bar graph. It is a photograph showing cells after sorting adherent cells derived from PDGF receptor ⁇ -GFP knock-in mouse bone marrow using anti-CD11b11MACS beads. CD11b-positive cells hardly express GFP. GFP expression is observed in almost all CD11b negative cells. CD11b positive cells are negative for PDGF receptor ⁇ , and CD11b negative cells are positive for PDGF receptor ⁇ .
- HMGB1 has almost no migration activity for macrophages that are CD11b positive cells and has migration activity for mesenchymal stem cells that are CD11b negative cells. It is a photograph which shows the result of having observed the accumulation
- the present invention provides a tissue regeneration-promoting agent containing a substance according to any one of the following (a) to (q), which is administered to a tissue different from a tissue that requires regeneration.
- HMGB1 protein b) Cell that secretes HMGB1 protein
- HMGB2 protein e
- e Cell that secretes HMGB2 protein
- Vector inserted with DNA g) HMGB3 protein (h) Cells secreting HMGB3 protein
- i Vector inserted with DNA encoding HMGB3 protein
- j S100A8 protein
- k Cells secreting S100A8 protein
- l Vector inserted with DNA encoding S100A8 protein
- m) S100A9 protein n
- Cell secreting S100A9 protein o
- Vector inserted with DNA encoding S100A9 protein p) Cell or tissue extract
- the tissue regeneration-promoting agent is administered to a tissue different from a tissue that requires regeneration, thereby mobilizing bone marrow cells from the bone marrow to a tissue that requires regeneration via peripheral circulation (also expressed as induction or local induction). It is characterized by doing.
- peripheral circulation also expressed as induction or local induction.
- peripheral circulation also referred to as “blood circulation” and “peripheral circulation blood flow”.
- the tissue regeneration promoter of the present invention preferably inhibits scar healing and induces non-scar healing.
- Scar healing is a state in which functional tissue is replaced with fibrous collagen.
- non-scarring healing means a state in which a damaged site regenerates a functional tissue composed of cellular components, and is superior to scar healing in terms of functionality and aesthetics.
- the tissue regeneration promoting agent of the present invention includes such a non-scarring tissue regeneration promoting agent.
- the agent of the present invention is a tissue regeneration promoter, and is administered to a tissue different from the tissue that requires regeneration, mobilizes bone marrow cells from the bone marrow into the peripheral blood, and is regenerated via the peripheral circulation system.
- a drug characterized by promoting tissue regeneration by mobilizing bone marrow-derived cells to the required tissue A non-scarring tissue regeneration-promoting agent that is administered to a tissue different from the tissue in need of regeneration, A non-scarring tissue regeneration-promoting agent that is administered to a tissue different from the tissue that requires regeneration, mobilizes bone marrow cells from the bone marrow into the peripheral blood, and passes through the peripheral circulatory system to the tissue that requires regeneration. It can also be expressed as a drug characterized by promoting tissue regeneration by mobilizing derived cells.
- tissues that need to be regenerated include damaged tissue, necrotic tissue, post-operative tissue, tissue with reduced function, fibrosis, aged tissue, diseased tissue, etc.
- living skin tissue, body biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testis, blood, etc.) can be exemplified.
- administration to a tissue different from a tissue requiring regeneration means administration to a site other than the site requiring regeneration (a site different from the site requiring regeneration).
- tissue different from the tissue that needs to be regenerated refers to a site that is different from the tissue that needs to be regenerated, a site that is different from the site that needs to be regenerated, a site away from the tissue that needs to be regenerated, or a site that needs to be regenerated. It can also be expressed as a site far from the site, a site distal to the site that needs regeneration, a tissue distal to the tissue that needs to be regenerated, a distal portion, and a distal tissue.
- the drug of the present invention is effectively used to regenerate tissues (brain, heart, etc.) where it is difficult to administer the drug directly from outside the body.
- Bone marrow-derived cells mobilized to tissues that need regeneration differentiate into various cells and contribute to functional regeneration, functional maintenance, and function enhancement of tissues that need regeneration.
- the tissues that need to be regenerated include tissues damaged by various pathologies caused by ischemia, ischemia / hypoxia, trauma, burns, inflammation, autoimmunity, gene abnormalities, etc. It is not limited to the cause.
- the tissue in the present invention is not particularly limited as long as bone marrow-derived cells can be differentiated.
- skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, myocardial tissue, nervous system tissue, lung tissue examples include all tissues in the living body, such as gastrointestinal tissue, liver / bile / pancreatic tissue, and urinary / genital organs.
- tissue regeneration promoter regeneration of cerebral infarction, myocardial infarction, fracture, lung infarction, gastric ulcer, enteritis, etc.
- tissue in need of treatment a treatment that induces functional tissue regeneration becomes possible.
- regeneration promoter A mammal, birds, fish, etc. are mentioned.
- mammals include humans and non-human animals, and examples include, but are not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales. is not.
- tissue different from the tissue that needs to be regenerated include blood tissue, muscle tissue, subcutaneous tissue, intradermal tissue, and abdominal cavity.
- the drug of the present invention includes a drug for promoting the regeneration of the tissue.
- the medicament of the present invention preferably includes, but is not limited to, regeneration promoting agents such as nerve tissue, bone tissue, skin tissue and the like.
- the nerve tissue regeneration promoter include, but are not limited to, a central nerve tissue regeneration promoter.
- the regeneration promoter for nerve tissue can be used for the treatment of cerebral infarction, cerebral hemorrhage, cerebral contusion, etc., but is not limited thereto.
- the bone tissue regeneration promoter can be used, for example, for the treatment of fractures, but is not limited thereto.
- the skin tissue regeneration promoter can be used for the treatment of, for example, skin ulcers, suture defects of surgical wounds, burns, cuts, bruises, skin erosions, and abrasions, but is not limited thereto.
- bone marrow cells and “bone marrow-derived cells” are cells other than hematopoietic stem cells and leukocytes, erythrocytes, and platelets derived therefrom, and so far bone marrow mesenchymal stem cells or bone marrow stromal pluripotency Stem cells represented by cells called stem cells or bone marrow pluripotent stem cells.
- bone marrow cells also include cells that contain a tissue precursor cell population present in the bone marrow.
- bone marrow cells and “bone marrow-derived cells” can be isolated by bone marrow collection (bone marrow cell collection) or peripheral blood collection.
- Hematopoietic stem cells are non-adherent cells, but some "bone marrow cells” and “bone marrow-derived cells” are mononuclear cell fractions in blood obtained by bone marrow collection (bone marrow cell collection) and peripheral blood collection. It is obtained as an adherent cell by culture.
- Bone marrow cells and “bone marrow-derived cells” include mesenchymal stem cells, osteoblasts (can be identified by calcium deposition when differentiation is induced), chondrocytes (positive for Alcian blue staining, safranin -O staining positive), fat cells (identified by Zudan III staining positive), mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, tendon cells, nerve cells, It preferably has an ability to differentiate into epithelial cells (for example, epidermal keratinocytes, intestinal epithelial cells express cytokeratin family) and vascular endothelial cells.
- epithelial cells for example, epidermal keratinocytes, intestinal epithelial cells express cytokeratin family
- vascular endothelial cells for example, epidermal keratinocytes, intestinal epithelial cells express cytokeratin family
- the cells after differentiation are not limited to the above-mentioned cells, but also include the ability to differentiate into parenchymal organ cells such as liver, kidney and pancreas.
- bone marrow cell means a cell existing in the bone marrow
- bone marrow-derived cell means “bone marrow cell” mobilized outside the bone marrow.
- bone marrow mesenchymal stem cell is a cell that is present in the bone marrow and directly from the bone marrow or other It is indirectly collected from tissues (blood, skin, fat, other tissues) and can be cultured and proliferated as adherent cells on culture dishes (plastic or glass).
- Mesenchymal tissues such as bone, cartilage, and fat (Mesenchymal stem cells), or cells that have the characteristic of differentiating into skeletal muscle, myocardium, or even neural tissue or epithelial tissue (pluripotent stem cells), and can be obtained by collecting bone marrow cells It is.
- bone marrow mesenchymal stem cells In addition, “bone marrow mesenchymal stem cells”, “bone marrow stromal pluripotent cells” or “bone marrow pluripotent stem cells” mobilized from the bone marrow are collected from peripheral blood, as well as mesenchymal tissues such as fat, skin, etc. It is a cell that can be obtained by collecting from epithelial tissue, nerve tissue such as brain.
- bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or these cells mobilized from the bone marrow are directly damaged after collection or once adhered to the culture dish.
- bone marrow mesenchymal stem cells bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or cells mobilized from bone marrow include, but are not limited to, cells having CD11b-negative properties. It is not a thing.
- Bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or these cells mobilized from the bone marrow are osteoblasts (which can be identified by the presence of calcium deposition when differentiation is induced),
- osteoblasts which can be identified by the presence of calcium deposition when differentiation is induced
- chondrocytes identifiable by Alcian Blue staining positive, Safranin-O staining positive, etc.
- adipocytes identifiable by positive Sudan III staining, etc.
- fibroblasts smooth muscle cells, skeletal muscle cells, stromal Cells, tendon cells, mesenchymal cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (expressing cytokeratin family, hair keratin family, etc.), epithelial cells (eg epidermal keratinocytes, intestinal epithelial cells)
- endothelial cells Preferably expresses
- human bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or those mobilized from the bone marrow are obtained by bone marrow collection (bone marrow cell collection), peripheral blood collection, fat collection Examples of cells that can be obtained as adherent cells by culturing directly or after separating the mononuclear cell fraction are not limited thereto.
- Human bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or markers of these cells mobilized from bone marrow are all Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive Or a part can be illustrated, but it is not limited to these.
- mouse bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or cells mobilized from the bone marrow include cells that can be obtained by the methods described in the Examples. However, it is not limited to this.
- Mouse bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or markers of these cells recruited from the bone marrow include CD44 positive, PDGFR ⁇ positive, PDGFR ⁇ positive, CD45 negative, Lin negative, Sca Examples can include, but are not limited to, -1 positive, c-kit negative, CD90 positive, and CD29 positive in whole or in part.
- Tissue progenitor cells are defined as undifferentiated cells that have the ability to unidirectionally differentiate into specific tissue cells other than the blood system, and differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organ, and vascular endothelium as described above. Including undifferentiated cells having the ability.
- the substance other than at least one of the substances described in (a) to (q) above is not particularly limited as long as it does not inhibit the induction of bone marrow-derived cells or the promotion of tissue regeneration. There is no.
- the tissue regeneration promoter of the present invention in addition to at least one of the substances described in (a) to (q) above, the functional tissue regeneration of the substance described in (a) to (q) above is included.
- Examples of the animal species that is the source of the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, the extract, or the heparin-binding fraction in the tissue regeneration promoter of the present invention include humans and non-human animals. Examples include mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, etc., but the animal species is preferably the same as the animal species to which the substance is administered.
- HMGB1 protein in the present invention examples include, but are not limited to, proteins containing the amino acid sequence set forth in SEQ ID NO: 1, 3 or 5.
- the HMGB1 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3 or 5.
- proteins functionally equivalent to the protein examples include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 1, 3 or 5.
- HMGB2 protein in the present invention examples include, but are not limited to, a protein containing the amino acid sequence set forth in SEQ ID NO: 7, 9, or 11.
- the HMGB2 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 7, 9 or 11.
- proteins functionally equivalent to the protein examples include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 7, 9 or 11.
- Isolated protein functionally equivalent to the protein comprising the amino acid sequence described in 7, 9 or 11; and 2) stringent with the DNA comprising the base sequence described in SEQ ID NO: 8, 10 or 12
- HMGB3 protein in the present invention examples include, but are not limited to, a protein containing the amino acid sequence set forth in SEQ ID NO: 13 or 15.
- the HMGB3 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 13 or 15.
- proteins functionally equivalent to the protein examples include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 13 or 15.
- an isolated protein functionally equivalent to the protein comprising the amino acid sequence described in 15, and 2) hybridizing under stringent conditions with the DNA comprising the base sequence described in SEQ ID NO: 14 or 16
- An isolated protein which is a protein encoded by DNA and which is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 13 or 15.
- S100A8 protein in the present invention examples include, but are not limited to, proteins containing the amino acid sequence described in SEQ ID NO: 17, 19, or 21.
- the S100A8 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 17, 19, or 21.
- proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 17, 19, or 21.
- Examples of such a protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 17, 19, or 21.
- Isolated protein functionally equivalent to the protein comprising the amino acid sequence described in 17, 19 or 21; and 2) stringent with the DNA comprising the base sequence described in SEQ ID NO: 18, 20 or 22
- S100A9 protein in the present invention examples include, but are not limited to, a protein containing the amino acid sequence described in SEQ ID NO: 23, 25, or 27.
- the S100A9 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 23, 25 or 27.
- proteins functionally equivalent to the protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 23, 25, or 27.
- Isolated protein functionally equivalent to the protein comprising the amino acid sequence described in 23, 25 or 27, and 2) stringent with DNA comprising the base sequence described in SEQ ID NO: 24, 26 or 28
- a protein encoded by DNA that hybridizes under conditions and an isolated protein that is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 23, 25, or 27.
- a protein functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is obtained by those skilled in the art. It can be isolated by a known method (experimental medicine separate volume, genetic engineering handbook, pp246-251, Yochisha, 1991).
- protein functionally equivalent to the protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, derived from bone marrow
- examples thereof include a protein having an activity of mobilizing a cell to a tissue in need of regeneration, or a protein having an activity of migrating bone marrow-derived cells.
- one or more amino acids are substituted, deleted, inserted, and And / or a protein comprising the added amino acid sequence and comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 and functional Proteins equivalent to include naturally occurring proteins.
- eukaryotic genes have a polymorphism, as is known for interferon genes and the like.
- One or more amino acids may be substituted, deleted, inserted, and / or added due to a change in the base sequence caused by this polymorphism.
- a naturally occurring protein and 1 in the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 a plurality of amino acids have an amino acid sequence substituted, deleted, inserted, and / or added, and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 , 25 or 27 are functionally equivalent to the protein consisting of the amino acid sequence described in HMGB1, HMGB2, HMGB3, S100A8 or S100A9.
- a gapped duplex method or the like can be used as a technique for bringing a target mutation to an arbitrary place (Kramer W. and Fritz HJ., Methods in Enzymol., 154: 350-367, 1987).
- a circular double-stranded vector in which a gene to be introduced with a mutation is cloned is made into a single strand, and a synthetic oligonucleotide having a mutation at the target site is hybridized.
- a complementary single-stranded DNA derived from a vector that has been linearized by cutting with a restriction enzyme is annealed to the circular single-stranded vector, and a gap between the synthetic nucleotide is filled with DNA polymerase, and further ligated.
- the number of amino acids to be modified is typically within 50 amino acids, preferably within 30 amino acids, and more preferably within 5 amino acids (eg, 1 amino acid).
- the protein of the present invention is a protein in which a conservative substitution is added in the above amino acid substitution, and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, A protein functionally equivalent to a protein comprising the amino acid sequence described in 25 or 27 is included. Conservative substitution is considered to be important, for example, when substituting amino acids of domains important for protein activity. Such amino acid conservative substitutions are well known to those skilled in the art.
- amino acid groups corresponding to conservative substitutions include basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), nonpolar amino acids (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine) , Tryptophan, histidine) and the like. It is also conceivable to increase the activity of the protein by non-conservative substitution (for example, including a constant activation protein).
- non-conservative substitution for example, including a constant activation protein.
- a protein functionally equivalent to the protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is obtained.
- Examples of the method include a method using hybridization. That is, the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein according to the present invention as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28 is obtained. Using the encoded DNA or a fragment thereof as a probe, DNA that can hybridize with this is isolated.
- a highly homologous base sequence can show, for example, 70% or more, desirably 90% or more identity.
- the stringent conditions can be specifically exemplified by conditions such as 6 ⁇ SSC, 40% formamide, hybridization at 25 ° C. and washing at 1 ⁇ SSC, 55 ° C.
- Stringency depends on conditions such as salt concentration, formamide concentration, and temperature, but those skilled in the art will readily set these conditions to obtain the required stringency.
- hybridization for example, HMGB1 other than a protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 DNA encoding a homologue of HMGB2, HMGB3, S100A8 or S100A9 protein can be isolated.
- a protein functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is usually a sequence. No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, and has high homology. High homology refers to sequence identity of at least 30% or more, preferably 50% or more, more preferably 80% or more (eg, 95% or more).
- the identity of base sequences and amino acid sequences can be performed using a homology search site using the Internet [for example, homology such as FASTA, BLAST, PSI-BLAST, and SSEARCH in Japan DNA Data Bank (DDBJ).
- search is available [For example, the homology search (Search ⁇ and Analysis) page of the Japan DNA Data Bank (DDBJ) website ;; http://www.ddbj.nig.ac.jp/E-mail/homology-j. html].
- search using BLAST can be performed in National Center for Biotechnology Information (NCBI) (for example, the BLAST page on the NCBI homepage; http://www.ncbi.nlm.nih.gov/BLAST /; Altschul, SF et al., J. Mol. Biol., 1990, 215 (3): 403-10; Altschul, SF & Gish, W., Meth. Enzymol., 1996, 266: 460-480; Altschul , SF et al., Nucleic Acids Res., 1997, 25: 3389-3402). ].
- the amino acid sequence identity in Advanced BLAST 2.1 is calculated using blastp as the program, Expect value is 10, Filter is all OFF, BLOSUM62 is used as Matrix, Gap existence cost, Per residue gap cost, and Lambda ratio Can be set to 11, 1, and 0.85 (default values), respectively, to obtain the identity value (%) (Karlin, S. and S. F. Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin, S. and S. F. Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7).
- a protein functionally equivalent to the protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 It may be a fragment of the amino acid sequence described in No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27.
- the protein according to the present invention or a functionally equivalent protein thereof is a protein to which various modifications such as physiological modifications such as sugar chains, labels such as fluorescence and radioactive substances, or fusion with other proteins are added. be able to. In particular, in the recombinants described later, there may be a difference in modification with sugar chains depending on the host to be expressed.
- any HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein disclosed in the present specification can be used as long as they have the same properties as the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, or a functionally equivalent protein.
- HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein can be obtained not only as a biomaterial but also as a recombinant by incorporating a gene encoding it into an appropriate expression system.
- HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein by genetic engineering techniques, the DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein described above is incorporated into an appropriate expression system and expressed. Just do it.
- Examples of host / vector systems applicable to the present invention include expression vector pGEX and E. coli.
- pGEX can express a foreign gene as a fusion protein with glutathione S-transferase (GST) (Gene, 67: 31-40, 1988), so the gene encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein
- GST glutathione S-transferase
- the incorporated pGEX is introduced into an E. coli strain such as BL21 by heat shock, and after appropriate incubation time, isopropylthio- ⁇ -D-galactoside (IPTG) is added to add GST fusion HMGB1, GST fusion HMGB2, GST fusion HMGB3, GST Induces expression of fusion S100A8 or GST fusion S100A9 protein. Since GST according to the present invention is adsorbed to glutathione sepharose 4B, the expression product can be easily separated and purified by affinity chromatography.
- HMGB1, HMGB2, HMGB3, S100A8 or S100A9 proteins include the following.
- a bacterium when used as a host, a fusion protein expression vector using a histidine tag, an HA tag, a FLAG tag or the like is commercially available.
- the gene recombinant of the present invention includes those to which a tag or a part of the peptide is added.
- yeast it is known that yeasts of the genus Pichia are effective for the expression of proteins with sugar chains.
- an expression system using a baculovirus vector hosted by insect cells is also useful (Bio / Technology, 6: 47-55, 1988).
- transfection of vectors using promoters such as CMV, RSV, or SV40 has been carried out using mammalian cells.
- These host / vector systems are all HMGB1, HMGB2, HMGB3, S100A8.
- it can be used as an expression system for S100A9 protein.
- the gene can be introduced using a viral vector such as a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.
- the obtained protein of the present invention can be isolated from the inside of the host cell or outside the cell (medium etc.) and purified as a substantially pure and homogeneous protein.
- the separation and purification of the protein may be performed using the separation and purification methods used in normal protein purification, and is not limited at all. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, proteins can be separated and purified.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Cold Spring Harbor Laboratory Press, 1996). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.
- the protein of the present invention is preferably a substantially purified protein.
- substantially purified means that the degree of purification of the protein of the present invention (ratio of the protein of the present invention in the whole protein component) is 50% or more, 60% or more, 70% or more, 80% or more, It means 90% or more, 95% or more, 100% or close to 100%. The upper limit close to 100% depends on the purification technique and analysis technique of those skilled in the art, and is, for example, 99.999%, 99.99%, 99.9%, 99%, etc.
- the protein purified by any purification method is included in the substantially purified protein.
- substantially purified protein can be exemplified by selection or combination, it is not limited thereto.
- the cells that release or secrete HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein in the present invention basically correspond to all tissue-derived cells in vivo.
- Examples of cells that can be easily collected and cultured include fibroblasts (for example, normal skin fibroblasts and cell lines derived therefrom), but are not limited thereto.
- cells that secrete HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein can be converted into DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, or DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein.
- a known expression vector or a gene therapy vector is a DNA that binds a DNA encoding a secretion signal (ATG-CAG-ACA-GAC-ACA-CTC-CTG-CTA-TGG-GTA-CTG-CTG-CTG-TGG-GTT-CCA-GGT-TCC-ACT-GGT-GAC; It can also be prepared by introducing a vector prepared by inserting into a mammalian cell such as a fibroblast (for example, normal skin fibroblast and a cell line derived therefrom), an insect cell, or other cells. it can.
- a mammalian cell such as a fibroblast (for example, normal skin fibroblast and a cell line derived therefrom), an insect cell, or other cells.
- a mammalian cell such as a fibroblast (for example, normal skin fibroblast and a cell line derived therefrom), an insect cell, or other cells.
- a mammalian cell such as a fibroblast (
- the animal species from which these cells are derived is not particularly limited, but the cells of the subject animal species to which the vector is administered, the subject's own cells, or cells derived from the relatives of the subject to whom the vector is administered are used. It is preferable.
- DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein in the present invention may be cDNA or genomic DNA as long as it encodes HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, It may be natural DNA or artificially synthesized DNA.
- DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein is usually administered in a state of being inserted into a vector.
- vectors in the present invention include, but are not limited to, plasmid vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, adenoassociate virus vectors, Sendai virus vectors, Sendai virus envelope vectors, papilloma virus vectors, and the like. It is not something.
- the vector may include a promoter DNA sequence that effectively induces gene expression, a factor that controls gene expression, and a molecule necessary to maintain the stability of the DNA.
- a partial peptide of HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein which has a bone marrow-derived cell mobilization activity, a cell that secretes the partial peptide, or a DNA encoding the partial peptide It is also possible to use a vector into which is inserted.
- the cell or tissue extract in the present invention can be produced by a method including a step of immersing the cells or tissue in a solvent.
- the cell or tissue immersed in the solvent is not particularly limited, but is tissue-derived cell, a cell line established from the tissue-derived cell (for example, HeLa, HEK293 can be exemplified, but is not limited thereto), isolation Examples thereof include cells that have been isolated, cells that have not been isolated (for example, cells that are present in an isolated tissue), cells into which DNA encoding HMGB1, HMGB2, HMGB3, S100A8, or S100A9 protein has been introduced.
- the tissue may be any tissue, for example, living skin tissue, in vivo biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, Testis, blood, etc.) can be exemplified, but not limited thereto.
- in vivo biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, Testis, blood, etc.) can be exemplified, but not limited thereto.
- the solvent examples include physiological saline, PBS (Phosphate-buffered saline), and TBS (Tris-buffered saline), but are not limited thereto.
- the time for immersing cells and tissues in a solvent is necessary and sufficient to induce cell necrosis, that is, 1 to 48 hours (for example, 6 to 48 hours), preferably 12 to 24 hours. There is, but is not limited to this time. Therefore, “the step of immersing the cells in the solvent” can be rephrased as “the step of immersing the cells in the solvent for a necessary and sufficient time to induce necrosis” and “the step of necrosing the cells”.
- the temperature at which cells and tissues are immersed in a solvent include 4 ° C.
- Examples of the pH for immersing cells and tissues in a solvent include pH 7 to 8, preferably pH 7.5, but are not limited thereto.
- Examples of the buffer component include a phosphate buffer solution having a concentration of 10 to 50 mM, preferably 10 to 20 mM, but is not limited thereto.
- the cells and tissues can be removed from the solvent containing the cells and tissues after the cells and tissues are immersed in the solvent.
- the method for removing cells and tissues from the solvent is not particularly limited as long as it is a method well known to those skilled in the art.
- cells and tissues can be removed from the solvent by centrifuging at 4 ° C. to 25 ° C. (eg 4 ° C.) and with a gravitational acceleration of 10 G to 100000 G (eg 440 G) and separating the supernatant.
- the supernatant can be used as a cell or tissue extract.
- Examples of the cell or tissue extract in the present invention include skin extract and peripheral blood mononuclear cell extract (peripheral blood extract), but are not limited thereto.
- the peripheral blood extract is prepared by collecting blood using a syringe or the like, then freezing the cells in a freezer, liquid nitrogen, dry ice, etc., and then re-thawing at a temperature of 0 ° C. or higher. Further, in order to remove insoluble components of cells, for example, centrifugation is performed at 4 ° C. to 25 ° C. (for example, 4 ° C.), gravitational acceleration of 10 G to 100000 G (for example, 440 G), and the supernatant is separated to remove cells from the solvent.
- Insoluble components can be removed, but the present invention is not limited to this.
- the supernatant can be used as a cell or tissue extract.
- insoluble components can be removed by passing through a nitrocellulose filter having a minute pore of 0.45 ⁇ m instead of centrifugation.
- centrifugation is performed at a gravitational acceleration of 10 G to 100,000 G (for example, 440 G), and the supernatant is separated to remove the insoluble components of the cells from the solvent, but it is not limited thereto.
- the supernatant can be used as a cell or tissue extract.
- insoluble components can be removed by passing through a nitrocellulose filter having a minute pore of 0.45 ⁇ m instead of centrifugation.
- the method for preparing cell extract from peripheral blood mononuclear cells is to collect peripheral blood whole blood using a syringe, etc., then dilute the whole volume to 4 mL with PBS, and add Ficoll-Paque® Plus (GE) solution to a centrifuge tube. After 3mL is inserted, the diluted blood is overlaid on it. Centrifuge at 400 G (18 ° C.) for 40 minutes, collect the intermediate layer containing mononuclear cells in a new centrifuge tube, add 45 ⁇ mL of PBS, and centrifuge at 800 G (18 ° C.) for 5 minutes to remove the supernatant.
- GE Ficoll-Paque® Plus
- centrifugation is performed at a gravitational acceleration of 440G to 1000000G, preferably 100000G to 20000G, and the supernatant is collected to obtain a cell extract.
- a cell extract can be obtained by removing insoluble components by passing a nitrocellulose filter or cellulose acetate having a minute pore of 0.45 ⁇ m.
- the heparin-binding fraction of the cell or tissue extract in the present invention can be produced by a method comprising the following steps.
- a heparin-binding fraction from the immobilized heparin (which can also be expressed as a heparin purified fraction or a heparin column purified fraction).
- Step of eluting Immobilized heparin is obtained by covalently binding heparin to an insoluble carrier.
- the insoluble carrier examples include Sepharose beads (Sepharose 4B, Sepharose 6B, etc .: GE Healthcare), but are not limited thereto.
- commercially available immobilized heparin Hitrap Hepalin HP column: GE Healthcare
- Examples of contact conditions between cell and tissue extracts and immobilized heparin are about pH 7-8 (preferably pH 7.5), and salt concentration is 0-200 mM, preferably about 100-200 mM.
- the time for which the extract and the immobilized heparin are in contact is not particularly limited, it is preferably maintained for 5 minutes or more from the viewpoint of sufficiently adsorbing the heparin-binding fraction to the immobilized heparin.
- the temperature includes 4 to 8 ° C., preferably 4 ° C., but is not limited thereto.
- the elution conditions of the heparin-binding fraction adsorbed on the immobilized heparin are exemplified by pH 7 to 8 and a salt concentration of 200 to 1000 mM (preferably about 1000 mM), but are not limited thereto.
- the administration method of the tissue regeneration accelerator of the present invention includes parenteral administration, and specific examples of such administration methods include injection administration, but are not limited to this method.
- the administration method of the tissue regeneration promoter of the present invention is not particularly limited as long as the tissue regeneration promoter does not stay at the administration site but enters the blood circulation.
- Examples of the method for administering the tissue regeneration promoter of the present invention include intravascular administration (intraarterial administration, intravenous administration, etc.), intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration. However, it is not limited to these.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose can be selected in the range of 0.0000001 mg to 1000 mg per kg of body weight per administration.
- the dose can be selected within the range of 0.00001 to 100,000 mg / body per patient.
- Tissues that need to be regenerated, even when administering cells that secrete HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins or gene therapy vectors inserted with DNA encoding HMGB1, HMGB2, HMGB3, S100A8, or S100A9 proteins , HMGB1, HMGB2, HMGB3, S100A8, or S100A9 protein can be administered so that the amount falls within the above range.
- the tissue regeneration promoter of the present invention is not limited to these doses.
- the tissue regeneration-promoting agent of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing, Company, Easton, USA) and includes pharmaceutically acceptable carriers and additives. It may be a thing.
- pharmaceutically acceptable carriers and additives for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents
- surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents can be used as appropriate.
- examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.
- the present invention is also a tissue regeneration promotion kit comprising a composition containing the substance according to any one of the following (a) to (q), wherein the composition is different from a tissue that requires regeneration.
- the kit is provided for administration to a tissue.
- HMGB1 protein b) Cell that secretes HMGB1 protein
- d) HMGB2 protein e
- f Encodes HMGB2 protein
- Vector inserted with DNA g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Cell or tissue
- the tissue regeneration-promoting kit is characterized in that bone marrow cells are mobilized from bone marrow to a tissue that requires regeneration via peripheral circulation by being administered to a tissue different from the tissue that requires regeneration.
- the kit of the present invention includes a kit for treating the above-mentioned tissue that needs to be regenerated.
- the kit of the present invention includes a kit administered parenterally, more preferably a kit administered by injection.
- the kit of the present invention preferably includes a kit administered intravascularly, intramuscularly, subcutaneously, intradermally or intraperitoneally.
- the kit of the present invention includes a kit used for promoting regeneration of nerve tissue, bone tissue, or skin tissue.
- the kit for promoting tissue regeneration includes (1) the above-mentioned substance dissolved in fibrinogen, and (2) a kit for promoting tissue regeneration containing thrombin, or (1) the above-mentioned substance, (2) fibrinogen, and (3) thrombin.
- the kit for promoting tissue regeneration include: In the present invention, commercially available fibrinogen and thrombin can be used. Examples include, but are not limited to, fibrinogen HT-Wf (Benesys Mitsubishi Mitsubishi Pharma), Veriplast (ZLB Behring), Tissir (Baxter), Bolheel (Kakekenken), and Taco Kombu (ZLB Behring).
- the use of the vector into which the encoding DNA is inserted can also be expressed as (1) to (3) below.
- the composition is administered;
- RNA was extracted using Trizol (invitrogen), and cDNA was further synthesized using SuperScript III cDNA synthesis kit (Invitrogen).
- the HMGB1 cDNA is amplified using the PCR (polymerase chain reaction) method, and a mammalian protein is expressed so that the GST tag and 6XHis tag sequences are added to the N-terminus of the amino acid sequence for purification. It was inserted into a plasmid vector pCAGGS for expressing proteins in cells.
- Human fetal kidney cell-derived HEK293 cultured cell line was transfected with pCAGGS-GST-His-HMGB1 using polyethyleneimine (PEI), and cells and culture supernatant were collected after 48 hours.
- the cells and culture supernatant were centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant and cells were separated and collected.
- the collected supernatant was further passed through a cellulose acetate filter having a pore size of 0.8 ⁇ m, and then passed through a nitrocellulose filter having a pore size of 0.45 ⁇ m, thereby preparing a sample from which the insoluble fraction was removed.
- This sample was inserted into 5 mL HisTrap FF (GE) equilibrated with 50 mM NaCl-containing 50 mM Tris HCl pH 8.0 (50 mL), and the adsorbed component was further washed with 10 mM imidazole-containing 50 mM NaCl 50 mM Tris HCl pH 8.0. The nonspecific adsorption component was removed. Specific adsorbed components were eluted from the column with 50 mM NaCl 50 mM Tris HCl pH 8.0 containing 100 mM imidazole.
- Each adsorbed fraction was fractionated into 500 ⁇ L silicon-coated plastic tubes, and the protein-containing fractions were collected, and then imidazole was removed using a desalting column PD10 (GE). 50 mM Tris HCl pH. 7.5, 150 mM NaCl was used to elute. HRV3C (Novagen) was added to the eluted sample and reacted at 4 ° C. for 8 hours.
- the sample was bound to a HiTrap Heparin 1 mL column (GE) equilibrated with 50 mM Tris HCl pH.7.5, 150 mM NaCl, the interior of the column was washed with 50 mM Tris HCl pH.7.5, 150 mM NaCl, and then 50 mM Tris The bound protein was eluted with HCl pH.7.5, 1000 mM NaCl. The eluted sample was diluted 50-fold with 50 mM Tris HCl pH.8.8 20 mM NaCl and adsorbed to 1 mL HiTrap Q FF (GE) equilibrated with the same buffer.
- HiTrap Heparin 1 mL column equilibrated with 50 mM Tris HCl pH.7.5, 150 mM NaCl
- Adsorbed protein was eluted by gradually increasing the concentration of NaCl using 50 mM Tris HCl pH.8.8 500 mM NaCl. Proteins bound to the nickel column, heparin column, and Q column were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue to confirm the presence of the protein. As a result, as shown in FIG. 1, high purity HMGB-1 was purified. In the following examples, HMGB-1 using this purification method is used.
- PCR polymerase chain reaction
- the cells and culture supernatant were centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant (supernatant A) and cells were separated and collected. Cells were disrupted by adding 0.1% Tween20-containing PBS and applying ultrasonic waves for 30 seconds under ice-cooling. Further, the mixture was centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant was collected (supernatant B). Supernatant A and supernatant B were mixed and added to HiTrap GST FF column (GE healthcare, 5 mL) whose buffer was replaced with 30 mL of PBS in advance.
- HiTrap GST FF column GE healthcare, 5 mL
- the column was washed with 100 mL of PBS, and the adsorbed protein was eluted with reduced glutathione-containing 20 mM phosphate buffer (pH.8).
- the buffer was replaced with PBS using a gel filtration column PD-10 (manufactured by GE).
- a disk made of silicon having an outer diameter of 10 mm, an inner diameter of 6 mm, and a thickness of 1 mm was adhered to the ulcer using double-sided adhesive tape and medical adhesive Aron Alpha A (Sankyo).
- the ulcer was covered with a silicon disk having a diameter of 10 mm and a thickness of 1 mm.
- tegaderm 3M
- HMGB-1 (40 ⁇ g) or S100A8 (250 ng) was administered from the tail vein 5 times at 24-hour intervals from the day of skin ulcer preparation.
- mice Two weeks after the preparation of the ulcer, mice were inhaled by anesthesia using isoflurane, and the degree of accumulation of GFP fluorescence in the skin ulcer preparation part prepared on the back was observed using a fluorescent stereomicroscope.
- cut the skin of the skin ulcer area in a circle fix it with 4% paraformaldehyde in PBS (phosphate buffer solution, nacalai), embed it in an OCT compound, and then use a microtome with a cooling device (Leica) for 8 microns.
- Meter slices were made. After the section was attached to a preparation, the compound was washed with PBS, and the nucleus was stained with DAPI. Further, excess DAPI was washed with PBS, and encapsulated using an encapsulating material containing a fluorescent fading inhibitor. The fluorescence of GFP was detected for each sample using a fluorescence microscope.
- Bone marrow pluripotent stem cells can be differentiated into osteoblasts, chondrocytes, adipocytes, etc., but in skin tissue, it is thought that they can differentiate into epidermal cells, hair follicle cells, dermal fibroblasts, etc. ing.
- HMGB-1 and S100A8 have the activity of mobilizing bone marrow pluripotent stem cells, and the effect of reducing skin ulcer in mice administered HMGB-1 and S100A8 directly to the skin ulcer site has been clarified.
- the results revealed that bone marrow-derived cells were mobilized at the site of skin ulcer by administering HMGB-1 or S100A8 into venous blood that is distal to and away from the skin ulcer.
- HMGB-1 40 ⁇ g
- S100A8 250 ng
- HMGB-1 showed a reduction in the ulcer area from the third day after the ulcer was created.
- S100A8 showed a reduction in the ulcer area from the 7th day after the creation of the ulcer compared to the negative control (PPS administration).
- HMGB-1 and S100A8 were administered directly to the skin ulcer area, and the effect of promoting the healing of skin ulcer was obtained.
- HMGB-1 and S100A8 were administered intravascularly and distally from the ulcer site. Succeeded in promoting the healing of skin ulcers. Since the present invention makes it possible to heal skin ulcers without directly administering to the ulcer part, it can be administered directly to the ulcer part, such as a wide range of skin ulcers, skin defects, ulcers with infected or necrotic lesions, etc. It has become possible to develop pharmaceuticals that can be used even in difficult situations.
- Non-scarring healing promoting effect of skin ulcer by intravenous administration of HMGB-1 A circular skin ulcer with a diameter of 6 mm was prepared on the back of C57BL / 6 male mice (8 weeks old).
- a disk made of silicon having an outer diameter of 10 mm, an inner diameter of 6 mm, and a thickness of 1 mm was adhered to the ulcer using double-sided adhesive tape and medical adhesive Aron Alpha A (Sankyo).
- the ulcer was covered with a silicon disk having a diameter of 10 mm and a thickness of 1 mm.
- HMGB-1 (40 ⁇ g) was administered 5 times from the tail vein at 24-hour intervals from the day of skin ulcer preparation. The ulcer part 4 weeks after the creation of the ulcer was collected and fixed with 10% buffered formaldehyde. After the sample was embedded in paraffin, a thin slice was prepared using a microtome. After deparaffinization, HE (hematoxylin and eosin) staining and MT (Masson trichrome) staining were performed.
- HMGB-1 can be administered intravenously to mobilize bone marrow-derived cells to the ulcer area and promote early closure of skin ulcers and non-scarring healing.
- HMGB-1 100 ⁇ g
- PBS 1% paraformaldehyde under inhalation anesthesia
- the brain was removed from the skull and allowed to penetrate into 10% sucrose solution for 12 hours and 20% sucrose solution for 24 hours to dehydrate the brain tissue. After dehydration, it was put into an OTC compound and frozen on dry ice to produce a block.
- the block was prepared by using a microtome for frozen sections to prepare a section having a thickness of 8 ⁇ m and extended on a silane-coated preparation. After stretching, the sample was sufficiently dried, and the compound was washed with PBS. After 2% skim milk-containing PBS was infiltrated into the sample, anti-mouse Nestin antibody and ⁇ III Tubulin antibody were diluted 500 times in PBS containing 2% skim milk and allowed to infiltrate the sample at 4 ° C. for 8 hours. After thoroughly washing 5 times with PBS for 5 minutes each, PE labeled anti-rat IgG antibody diluted 500 times with PBS containing 2% skim milk was allowed to permeate the sample at room temperature for 1 hour.
- the DAPI solution was permeated at room temperature for 10 minutes and thoroughly washed with PBS.
- the fluorescence of GFP, DAPI, and PE was observed using a confocal laser microscope.
- Bone marrow-derived cells are found in the brain of HMGB-1-treated mice, some of which are bone marrow-derived cells, Nestin-positive cells (yellow cells on the right), bone marrow-derived cells, and ⁇ III tubulin -positive cells (left) Yellow cells) were observed. In the PBS-administered group, bone marrow-derived cells were observed, but cells that simultaneously expressed Nestin and ⁇ III- ⁇ ⁇ tubulin were not observed (photos not shown).
- Bone marrow-derived cells are known to differentiate into neurons in vitro (culture system). In vivo (in vivo), bone marrow-derived cells are known to rarely express neuronal markers in the brain, but it is not clear whether they possess functions as cranial nerves. On the other hand, it has been confirmed that non-inflammatory bone marrow cells such as bone marrow mesenchymal stem cells are therapeutically effective against cerebral infarction, but the healing mechanism is not clear. From these results, bone marrow-derived cells expressing a neuronal marker were observed in the brain in mice to which HMGB-1 was intravenously administered after preparation of cerebral infarction. These GFP positive cells are expected to be derived from non-inflammatory cells such as bone marrow mesenchymal stem cells.
- HMGB1 monofilament nylon thread (emboli thread) with a thermoformed tip of 700 ⁇ m was inserted.
- the embolic thread was advanced toward the internal carotid artery, and the common carotid artery thread was loosened at the site where it was inserted approximately 8 mm from the tip of the embolic thread. It was confirmed by laser Doppler blood flow meter that the value of blood flow meter becomes 1/10 before and after blood flow interruption. The embolic thread was removed 30 minutes after blood flow interruption to restore blood flow.
- purified HMGB1 (10 ⁇ g) was diluted in 500 ⁇ L of PBS and administered from the tail vein of the prepared disease model mice. Thereafter, HMGB-1 was similarly administered 4 times every 24 hours.
- PBS was administered to control mice. Five days after the last day of treatment, perfusion fixation was performed using 2% paraformaldehyde under inhalation anesthesia with isoflurane. The brain was removed from the skull, dehydrated, placed in an OTC compound, and frozen on dry ice to make a block. The block was prepared by using a microtome for frozen sections to prepare a section having a thickness of 8 ⁇ m and extended on a silane-coated preparation. After stretching, the sample was sufficiently dried, and the compound was washed with PBS. After fixing with 4% paraformaldehyde-containing PBS for 10 minutes, it was washed with a phosphate buffer solution for 5 minutes and permeated with distilled water for 10 minutes.
- mice Compared with PBS-administered mice, HMGB-1-administered mice confirmed a marked improvement effect in cerebral infarctions.
- HMGB-1 was administered intravascularly after the creation of cerebral infarction, and the effect of reducing cerebral infarction was observed. So far, it has been known that intravenous administration of bone marrow cells of the person after cerebral infarction has an effect of improving cerebral infarction, but HMGB-1 has an activity to mobilize bone marrow-derived pluripotent stem cells, so that bone marrow cells It is expected to have the same effect as when intravenously administered. In addition, direct administration of HMGB-1 directly to the cerebral infarction site may induce brain tissue damage or inflammation. Intrinsic administration is an excellent administration method that enables treatment of cerebral infarction.
- HMGB1 monofilament nylon thread (emboli thread) with a thermoformed tip of 700 ⁇ m was inserted.
- the embolic thread was advanced toward the internal carotid artery, and the common carotid artery thread was loosened at the site where it was inserted approximately 8 mm from the tip of the embolic thread. It was confirmed by laser Doppler blood flow meter that the value of blood flow meter becomes 1/10 before and after blood flow interruption. After blocking blood flow for a certain time (45 minutes or 60 minutes) for each mouse, the embolic thread was removed to restore blood flow. Twelve hours later, purified HMGB1 (10 ⁇ g) was administered from the tail vein of a disease model mouse prepared by diluting in 500 ⁇ L of PBS. Thereafter, HMGB-1 was similarly administered 4 times every 24 hours. PBS was administered to control mice. The survival rate was observed for 7 days after the infarct preparation.
- mice with an infarction time of 30 minutes survived until 7 days after all cases (N 3 each) (not shown).
- the survival rate after 7 days was 40% in the case of PBS administration, whereas all the mice were alive in the case of HMGB-1 administration.
- the survival rate after 7 days was 50% in the case of PBS ⁇ administration, whereas all the mice were alive in the case of HMGB-1 administration.
- Cerebral infarction is a disease that affects life prognosis depending on the site, range, and time of infarction.
- cerebral infarction often involves paralysis and loss of consciousness, it may be delayed for medical institutions. Since pharmaceuticals such as t-PA preparations that are currently effective need to be administered within 3 to 4 hours after onset, this preparation is very rare in patients with global cerebral infarction.
- the present invention is an extremely simple and minimally invasive method of intravenous administration, it is possible to improve the life prognosis even if administration is started after a long time after the onset of cerebral infarction (12 hours later), It enables the development of new cerebral infarction drugs that can be administered to many cerebral infarction cases.
- tissue section after fracture healing was prepared.
- the sections were blocked with 4% skimmed milk-containing PBS, reacted with anti-mouse osteocalcin antibody diluted in 4% skimmed milk-containing PBS, washed with PBS, diluted with 4% skimmed milk-containing PBS, and room temperature. For 1 hour. After washing with PBS, nuclei were stained with DAPI and further washed with PBS. After encapsulation, fluorescence was observed using a confocal laser microscope.
- Osteocalcin is a red fluorescent moiety, and GFP-positive cells that are bone marrow-derived cells are green fluorescent moieties.
- the yellow cells in the superimposed image (Merge) are osteocalcin positive bone marrow-derived cells. That is, FIG. 8 shows that the bone marrow cells of the left GFP bone marrow chimeric mouse migrated to the fracture site of the right foot of the wild type mouse and differentiated into osteoblasts.
- mice in which the GFP protein was knocked into the genome downstream of the PDGF receptor ⁇ promoter (reference: Hamilton et al., Mol Cell Biol. 2003 Jun; 23 (11): 4013-25 )It was used. Since this mouse expresses GFP protein in cells expressing PDGF receptor ⁇ , it can detect green fluorescence when observed using a fluorescence microscope.
- Bone marrow cells of PDGFR ⁇ -GFP mice were collected, seeded on a cell culture dish, and cultured in ⁇ -MEM containing 10% FBS. The medium was changed every 3-4 days, and attached cells were collected after about 14 days. The collected cells were divided into CD11b positive cells and CD11b negative cells using anti-CD11b MACS beads. When observed with a fluorescence microscope, it was confirmed that CD11b-positive cells were GFP-negative (A1, A2 in FIG. 42) and CD11b-negative cells were GFP-positive (B1, B2 in FIG. 42). In order to investigate whether HMGB1 migrates these cells, the Boyden chamber method was performed.
- CD11b positive cells or CD11b negative cells were placed in the upper layer of the Boyden chamber, respectively, and HMGB1 was diluted in DMEM containing 10% FBS so as to be 0 ⁇ 50 ⁇ 100 ⁇ g / mL in the lower layer.
- the chamber was allowed to stand in an incubator at 37 ° C. and 5% CO2. After 4 hours, the membrane in the chamber was recovered and detected by staining the cells that migrated toward the lower layer (FIG. 43).
- a 12-week-old male PDGFR ⁇ -GFP mouse was general anesthetized with isoflurane, and a fracture model of the left lower thigh tibia was prepared.
- CD11b positive cells were GFP negative, suggesting that they did not express PDGF receptor ⁇ (A1, A2 in FIG. 42). It was suggested that CD11b-negative cells were GFP-positive and expressed PDGF receptor ⁇ (B1, B2 in FIG. 42).
- CD11b positive (PDGF receptor ⁇ negative) cells did not migrate with HMGB1, whereas CD11b negative (PDGF receptor ⁇ positive) cells migrated with HMGB1 (FIG. 43).
- PBS control D1 in FIG. 44
- 4 out of 6 mice in the HMGB1 administration were GFP positive (PDGF receptor ⁇ positive) in the bone around the fracture site. Cells were observed.
- Bone marrow cells include hematopoietic cells (red blood cells, white blood cells, macrophages, etc.) and mesenchymal cells. Of these cells, cells that adhere to the cell culture dish are macrophages (CD11b positive) and bone marrow mesenchymal stem cells ( CD11b negative).
- PDGF receptor ⁇ is a kind of bone marrow mesenchymal stem cell marker
- CD11b-negative and GFP-positive (PDGF receptor ⁇ -positive) obtained in this experiment are considered bone marrow mesenchymal stem cells.
- the results of the Boyden chamber method revealed that HMGB1 migrates bone marrow mesenchymal stem cells (PDGF receptor ⁇ -positive, CD11b-negative) without migrating macrophages (CD11b-positive cells).
- GFP positive cells PDGF receptor ⁇ positive cells
- These GFP positive cells are thought to be bone marrow mesenchymal stem cells mobilized by HMGB1.
- Bone marrow mesenchymal stem cells are known to be pluripotent stem cells that differentiate into osteoblasts, chondrocytes, adipocytes, and the like.
- the healing mechanism of fracture is thought to be carried out by migration of osteoblasts from or near the fracture site, but as shown in the experimental results of parabiosis in Example 8, bones other than the fracture site are shown. It is considered that bone marrow mesenchymal stem cells also contribute to the regeneration of fractures.
- HMGB1 bone marrow mesenchymal stem cells mobilized in the blood by intravenous administration of HMGB1 gathered at the fracture site, so HMGB1 can be used as a therapeutic agent for fractures It is done.
- HMGB1 has a migration activity specific to mesenchymal stem cells effective for tissue regeneration.
- the conventional treatment of fractures is mainly non-invasive reduction, surgery, Gibbs fixation, etc., and there are few drugs for actively promoting healing of fracture sites. Since this method is performed by intravenous administration of a drug, it can be applied to intractable fractures, fractures difficult to be performed, and the like, and provides an unprecedented therapeutic method.
- New mouse skin (female) was transplanted to the dorsal skin of a GFP bone marrow transplanted mouse obtained after engraftment of transplanted bone marrow cells (6 weeks).
- the transplanted skin piece was collected by biopsy under inhalation anesthesia, a frozen skin section (6 ⁇ m) was prepared using a microtome with a cooling device, 4% paraformaldehyde fixed (30 minutes), and the cell nucleus in the tissue was stained with DAPI.
- bone marrow-derived mesenchymal stem cell mobilization factor is released from transplanted skin tissue in the direction of ischemia / necrosis immediately after skin grafting, and mesenchymal stem cells are mobilized from bone marrow into the transplanted skin piece via peripheral blood circulation, It was expected to induce functional skin tissue regeneration.
- the cells When the area occupied by the cells grew from 70 to 100% of the culture dish bottom area, the cells were detached from the culture dish using 0.25% trypsin 1 mM EDTA (manufactured by Nacalai) and further subcultured under the same conditions. The passage work was repeated at least 5 times. Furthermore, these adherent cells were isolated and cultured, and cell surface antigens were analyzed by flow cytometry, and confirmed to be Lin negative, CD45 negative, CD44 positive, Sca-1 positive, and c-kit negative. It was confirmed that these cells can be differentiated into bone cells and adipocytes and have the properties of bone marrow mesenchymal stem cells.
- the skin extract (25 ⁇ l) is inserted into the lower tank (volume 25 ⁇ l) of the Boyden chamber, a polycarbonate membrane with 8 ⁇ m fine holes is placed on it, and in contact with this, the upper tank of the Boyden chamber (volume 50 ⁇ l)
- the bone marrow-derived mesenchymal stem cell suspension (5 ⁇ 10 4 cells / 50 ml culture medium: DMEM / 10% fetal bovine serum) was placed therein, and cultured in a CO 2 incubator at 37 ° C. for 4 to 24 hours.
- a candidate protein having bone marrow-derived mesenchymal stem cell mobilization activity is selected from the identified protein group, and a purified preparation containing the candidate protein is treated with a neutralizing antibody (100 ⁇ l of the purified preparation solution is treated with a polyclonal of the protein). After incubation for 30 minutes on ice under a 100-fold antibody dilution condition, the degree of inhibition of bone marrow-derived mesenchymal stem cell mobilization activity was examined by a migration ability assay using Boyden chambers.
- the obtained bone marrow-derived mesenchymal stem cell purified sample is mixed in Matrigel so that the volume is about 10%, and is inserted into a silicon tube having a diameter of about 1 mm and a length of 5 mm. Transplanted subcutaneously on the back. Two weeks later, the insertion tube was taken out, and the GFP fluorescence emitted from the bone marrow-derived cells migrated into the tube was quantitatively analyzed by a fluorescence measuring device. Furthermore, migratory cells were removed from the tube, seeded in DMEM / 10% fetal bovine serum medium, and cultured in a CO 2 incubator to examine the in vivo mobilization activity of bone marrow-derived mesenchymal stem cells.
- the anti-HMGB1 polyclonal antibody strongly inhibits the bone marrow-derived mesenchymal stem cell migration activity in the purified preparation (FIG. 13), and the bone marrow-derived stem cell mobilization factor present in the skin extract is HMGB1. It became clear that there was. Furthermore, in order to confirm that HMGB1 has bone marrow-derived mesenchymal stem cell mobilization activity in vivo, a silicon tube containing this purified sample was inserted subcutaneously into the back of a GFP bone marrow-transplanted mouse, and 2 weeks later, The nature of the mobilized cells was examined. As a result, compared to the control (purified sample used in SDS-PAGE lane 4 in FIG.
- the HMGB1 purified sample contained many GFP-positive bone marrow-derived cells in the tube (about three times the control control). He was mobilized (Figure 14).
- FIG. 15 shows a strong magnified image by a fluorescent stereomicroscope. Further, the GFP positive cells mobilized in the tube were taken out and cultured in DMEM / 10% fetal calf serum medium. As a result, the cells were in a circular floating cell state immediately after the start of the culture (FIG. 16), but after 24 hours, It was confirmed that GFP-positive bone marrow-derived cells adhered to the culture dish and proliferated as spindle-type fibroblast-like morphologies, and also like epithelial cell-like morphologies (FIG. 17).
- FIG. 18A shows that when these cells were cultured continuously for 2 weeks, cells exhibiting hair follicle morphology were confirmed in GFP-positive bone marrow-derived cells (FIG. 18A; bright field, weakly enlarged, FIG. 18B; GFP fluorescence, weakly enlarged, FIG. 18C bright field, strong magnification, FIG. 18D; GFP fluorescence, strong magnification).
- FIG. 18E shows that keratin 5 positive cells were confirmed in GFP positive bone marrow-derived cells.
- the present inventors are the first in the world that free skin pieces produce HMGB1, and that the produced HMGB1 has the activity of mobilizing bone marrow-derived mesenchymal stem cells in large quantities in the skin pieces.
- the bone marrow-derived mesenchymal stem cells mobilized inside the cell differentiate into mesenchymal cells such as fibroblasts, adipocytes, and smooth muscle cells, and further differentiate into cells that form epidermal hair follicles.
- mesenchymal cells such as fibroblasts, adipocytes, and smooth muscle cells
- HMGB1 This mobilization of bone marrow-derived mesenchymal stem cells and the resulting functional tissue regeneration by HMGB1 functions not only for transplanted skin regeneration, but also as a mechanism for inducing functional tissue regeneration at the time of many organ / tissue damages associated with ischemia / necrosis It is expected easily. If the development of pharmaceuticals based on HMGB1 formulation enables local recruitment of bone marrow-derived mesenchymal stem cells during regeneration of damaged tissue, functional organs that do not fall into dysfunction due to fibrotic scar healing I am convinced that functional tissue regeneration-guided medical treatment will be possible.
- HEK293 human embryonic kidney cell-derived cultured cell line
- HEK293 human embryonic kidney cell-derived cultured cell line
- 100 ⁇ L of Anti Flag antibody Gel (Sigma) per 50 mL of this supernatant was mixed and incubated at 4 ° C for 16 hours. The gel was collected by centrifugation and washed 5 times with PBS. Further, elution was carried out using 3X Flag peptide (final 100 ⁇ g / ml).
- Recombinant protein expression was confirmed by the Western blot method using a mouse anti-Flag® antibody diluted 1000-fold with S-T-PBS and a peroxidase-labeled anti-mouse IgG antibody (GE-Healthcare) diluted 2000-fold with S-T-PBS.
- the migration activity of these purified recombinant proteins in mouse bone marrow mesenchymal stem cells was evaluated using a Boyden chamber in the same manner as in Reference Example 2.
- the back skin of 8-week-old C57BL / 6 mice was excised into a circular shape with a diameter of 8 ⁇ m, and a skin ulcer model was prepared, and purified HMGB1, HMGB2, HMGB3 Each (100 ng) was mixed with an equal volume of hyaluronic acid solution with a concentration of 1 g / 100 mL PBS, and 100 ⁇ L was administered to the ulcer surface.
- the ulcer surface was covered with an adhesive transparent wound covering / protective material Tegaderm® (3M Healthcare) so as not to dry, and the wound area was measured over time to determine the healing effect.
- human bone marrow mesenchymal stem cells were evaluated using a Boyden chamber in the same manner as in [Reference Example 2] in order to examine whether the human skin extract and human purified HMGB1 had the activity of migrating.
- Human skin having an area of 1 cm 2 was soaked in 1 ml of PBS, incubated for 16 hours at 4 ° C., and then centrifuged at 44 OG for 10 minutes at 4 ° C. Only the supernatant was collected and used as a human skin extract.
- human bone marrow mesenchymal stem cells (Cambrex) were used as cells to be placed in the upper part of the Boyden chamber. (This cell was analyzed by cell surface antigen by flow cytometry.
- CD105 positive, CD166 positive, CD29 positive, CD44 were used.
- human HMGB1 was diluted 10-fold with 100 ng / well (R & D) and PBS at the bottom of the chamber, and PBS was used as a control.
- HMGB2 and HMGB3 bands were detected in addition to the HMGB1 band. Therefore, it was confirmed that the newborn mouse skin extract contained HMGB2 and HMGB3, which are family proteins, in addition to HMGB1 (FIG. 19).
- Expression vectors of HMGB1, HMGB2, and HMGB3 were prepared by adding a Flag tag to the N-terminus of each protein (FIG. 20). After introducing an expression vector into HEK293 cells and purifying the expressed protein using Flag tag, the protein was confirmed using Western blot method (Fig. 21). When the migration activity of mouse bone marrow mesenchymal stem cells using these purified proteins was measured, the activity of any protein was confirmed (FIG. 22).
- HMGB2 and HMGB3 are known as proteins having high homology with HMGB1. These proteins are also expected to have the same properties as HMGB1. Therefore, it was confirmed that HMGB2 and HMGB3, which are family of HMGB1, were also produced from the free skin piece extract. Furthermore, recombinant proteins of HMGB1, HMGB2, and HMGB3 were prepared, bone marrow mesenchymal stem cell migration activity in in vitro was confirmed, and skin ulcer treatment effect in in vivo was also confirmed.
- the HMGB family (HMGB1, HMGB2, HMGB3) and the recombinant HMGB family in newborn mouse free skin have bone marrow mesenchymal stem cell-inducing activity and activity to locally induce stem cells that can differentiate into bone marrow-derived epithelial system. It was revealed that these induced bone marrow-derived cell groups differentiated into various cells such as epidermal keratinocytes, hair follicles and fibroblasts in the damaged tissue, and promoted healing of the damaged tissue.
- bone marrow mesenchymal stem cells are pluripotent stem cells, the HMGB family can be administered systemically or locally to treat other tissue damage conditions such as brain damage, myocardial infarction, fractures, etc. I am confident that a therapeutic effect can be expected.
- human and mouse HMGB1 have 98% (213/215) homology in the amino acid sequence constituting each, and HMGB2 has 96% (202/210) homology in the amino acid sequence constituting each, It is known that HMGB3 has 97% (195/200) homology in the amino acid sequence constituting each. Therefore, human HMGB may have the same activity as mouse HMGB. From the results of this reference example, human skin extract and HMGB1 are the same as mouse skin extract and HMGB1. It was revealed that it has activity to induce stem cell.
- the Boyden chamber was used to examine the migration activity against bone marrow-derived mesenchymal stem cells in the same manner as in Reference Example 2. .
- concentration of HMGB1 contained in the same sample was measured using HMGB1 ELISA kit (Sinotest).
- brain, heart and skin tissue extracts were bound to a heparin affinity column in the same manner as in [Reference Example 2], and the bone marrow mesenchymal stem cell inducing activity of the bound fraction protein was confirmed using a Boyden chamber.
- mice brain extract contained HMGB1 equivalent to the newborn mouse skin extract. Furthermore, the inducing activity of bone marrow mesenchymal stem cells was observed in the mouse brain as well as the skin.
- the mouse intestinal extract and mouse heart extract contained almost no HMGB1, but bone marrow mesenchymal stem cell-inducing activity was observed.
- the heparin column-binding fraction of mouse brain and mouse heart had the activity of inducing bone marrow mesenchymal stem cells in the same manner as the heparin column-binding fraction of mouse skin (FIG. 25). Table 1 shows the results of measuring the HMGB1 concentration and the bone marrow mesenchymal stem cell inducing activity of each mouse tissue extract.
- a novel method for functionally and simply extracting a plurality of bone marrow mesenchymal stem cell inducers containing HMGB1 from various organs has been developed. Furthermore, in order to purify the bone marrow mesenchymal stem cell inducer from the tissue extract, a method for binding to a heparin column was developed. In addition, these components having bone marrow mesenchymal stem cell inducing activity can be purified from the brain and heart using a heparin column in the same manner as for skin.
- each of the adsorbed fractions was evaluated for the migration activity of the mouse bone marrow-derived cell line using the Boyden chamber method described above, and fractions having the migration ability were collected.
- the solution having this activity was used in the following experiments as a skin extract heparin purified fraction.
- mice were irradiated with 10 Gy X-rays once to prepare myelosuppressed mice.
- mice transplanted with bone marrow cells of GFP mice were subjected to inhalation anesthesia with isoflurane and pentobarbital (45 mg / kg) was injected intraperitoneally.
- the mouse was fixed to a stereotaxic apparatus, and a midline incision was made in the head with a scalpel.
- a burr was drilled from the bregma to the right outside 2.5 mm and the front 1 mm using a drill (FIG. 27A).
- An outer cylinder of a 20G Surfflow needle was inserted and fixed with the tip at a depth of 3 mm from this part.
- a negative pressure was applied using a syringe, and a part of the brain tissue was sucked (FIG. 27B).
- Bone marrow-derived mesenchymal stem cells are known to differentiate into nerve cells, and it was clarified from this result that nerve cell regeneration at the brain injury site can be induced by the purified fraction of skin extract heparin column. This can also be applied to nerve regeneration at a brain tissue injury site in cerebral ischemic disease or cerebral contusion.
- cDNA was amplified using the PCR (polymerase chain reaction) method, and the Flag tag sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Lys; SEQ ID NO: The protein added with 30) was inserted into a plasmid vector pCAGGS for expressing the protein in mammalian cells (FIG. 20).
- HEK293 human embryonic kidney cell-derived cultured cell line
- HEK293 human embryonic kidney cell-derived cultured cell line
- Cells and culture supernatants each expressing HMGB1 protein were incubated at 4 ° C. for 16 hours, and then centrifuged at 4400 g for 5 minutes to recover the supernatant.
- 100 ⁇ L of Anti Flag antibody Gel (Sigma) per 50 mL of the supernatant was mixed and incubated at 4 ° C. for 16 hours. The gel was collected by centrifugation and washed 5 times with PBS. Further, elution was performed using 3X Flag peptide (final 100 ⁇ g / ml). The concentration of the eluted protein was confirmed using HMGB1 ELISA kit (Sinotest), lyophilized and adjusted to 200 ⁇ g / mL using PBS.
- the cells were centrifuged at 440 g for 5 minutes at 25 ° C., and the supernatant was removed. 400 ⁇ L of PBS containing 1% paraformaldehyde was added to prepare a sample for flow cytometry analysis.
- Centrifugation was performed at 400 G (18 ° C.) for 40 minutes, and the intermediate layer containing mononuclear cells was collected in a new centrifuge tube, 45 mL of PBS was added, and centrifugation was performed at 800 G (18 ° C.) for 5 minutes to remove the supernatant. Further, 45 mL of PBS was added once more and centrifuged at 800 G (18 ° C.) for 5 minutes to remove the supernatant.
- the resulting mononuclear cells were reacted with Phycoerythrobilin (PE) -labeled anti-mouse PDGFR ⁇ antibody and Fluorescein isothiocyanate (FITC) -labeled anti-mouse CD44 antibody, and then subjected to flow cytometry (Facscan; Becton, Dickinson and Company). The frequency of PDGFR ⁇ -positive / CD44-positive cells in the fraction was evaluated.
- PE Phycoerythrobilin
- FITC Fluorescein isothiocyanate
- PDGFR ⁇ and CD44 are known as surface markers for bone marrow mesenchymal stem cells that represent bone marrow-derived pluripotent stem cells.
- Bone marrow mesenchymal stem cells are pluripotent stem cells that can differentiate into bone cells, chondrocytes, and adipocytes, and can also differentiate into nerve cells, epithelial cells, and the like.
- the skin pieces used in this experiment are ischemic, the tissue gradually becomes necrotic, and intracellular proteins such as nuclei are released from the proteins on the surface of the cells to the surroundings.
- HMGB1 is a protein contained in the skin extract.
- cells mobilized in peripheral blood can be collected in the same way as normal blood collection.
- bone marrow-derived tissue stem cells that are simpler and safer can be used. Collection method became possible.
- bone marrow cells gather at the damaged site during skin injury and contribute to the functional regeneration of the skin by differentiating into various organs that make up the skin.
- the skin graft contains a substance that collects bone marrow cells that can differentiate into various organs.
- stem cell systems in the bone marrow there are two stem cell systems in the bone marrow: hematopoietic stem cells and mesenchymal stem cells. It is difficult to predict that bone marrow-derived epithelial cells and mesenchymal cells mobilized in the transplanted skin in large quantities are supplied from bone marrow-derived hematopoietic stem cells. It is strongly suggested that bone marrow-derived mesenchymal stem cells may contribute to the targeted regeneration.
- the bone marrow-derived mesenchymal stem cell mobilization factor is released from the transplanted skin tissue in the direction of ischemia / necrosis immediately after transplanting, and the mesenchymal stem cells are mobilized from the bone marrow into the transplanted skin piece via peripheral blood circulation. It was expected to induce functional skin tissue regeneration.
- the cells When the area occupied by the cells grew from 70 to 100% of the culture dish bottom area, the cells were detached from the culture dish using 0.25% trypsin 1 mM EDTA (manufactured by Nacalai) and further subcultured under the same conditions. The passage work was repeated at least 5 times. Furthermore, these adherent cells were isolated and cultured, and cell surface antigens were analyzed by flow cytometry, and confirmed to be Lin negative, CD45 negative, CD44 positive, Sca-1 positive, and c-kit negative. It was confirmed that these cells can be differentiated into bone cells and adipocytes and have the properties of bone marrow mesenchymal stem cells.
- the gel was collected, and the protein in the gel was transferred to a PVDF membrane (Millipore) measuring 7 cm in length and 9 cm in width using a blotting apparatus (ATTO) in advance. Transcription was performed at 120 mA for 75 minutes. After completion of the transfer, the PVDF membrane was collected and shaken in 4% skim milk-containing PBS (nacalai) for 30 minutes at room temperature. Thereafter, the recovered PVDF membrane was immersed in a solution obtained by diluting 5 ⁇ l of anti-S100A8 antibody (R & D) or anti-S100A9 (R & D) in 10 mL of PBS containing 4% skim milk, and shaken at room temperature for 60 minutes.
- the membrane was washed with 30 mL of 0.1% Tween 20-containing PBS for 5 minutes at room temperature. Washing was repeated 5 times. After washing, the membrane was placed in a solution obtained by diluting 5 ⁇ l of HRP-labeled anti-goat IgG antibody (GE healthcare) in 10 mL of 4% skim milk-containing PBS and shaken at room temperature for 45 minutes. After removing the antibody solution, the membrane was washed with 30 mL of 0.1% Tween 20-containing PBS for 5 minutes at room temperature. Washing was repeated 5 times. The membrane was exposed to light with an ECL detection kit (GE healthcare) to expose the film. The film was developed with a developing device, and S100A8 and S100A9 protein signals were obtained (FIG. 35).
- HRP-labeled anti-goat IgG antibody GE healthcare
- Heparin affinity column chromatography was performed in order to purify the factor having bone marrow-derived mesenchymal stem cell mobilization activity in the skin extract.
- the following operations were performed using an FPLC apparatus (GE healthcare).
- 2-day-old mouse skin extract was diluted 10-fold with 9-fold 20 mM phosphate buffer pH 7.5 at 4 ° C (Dilution A).
- 20 mM phosphate buffer pH 7.5 300 ml
- Diluent A was bound to the column.
- the column was then washed with 20 mM phosphate buffer pH 7.5, (300 ml).
- (A solution) 20 mM phosphate buffer pH 7.5, 10 mM NaCl and (B solution) 20 mM phosphate buffer pH 7.5, 500 mM NaCl were prepared.
- the solution A was sent at 100% / B solution 0%, the ratio of the B solution was gradually increased, and finally the solution A was sent at 0% / 100% B solution.
- the total liquid feeding amount was 150 mL.
- the eluate was fractionated by 3 mL into a silicon-coated tube. 5 ⁇ l of each fractionated sample and 5 ⁇ l of SDS-PAGE sample buffer (Bio-Rad) were mixed, heated in a heat block at 98 ° C.
- S100A8 and S100A9 cDNAs were amplified by PCR (polymerase chain reaction) method, and the GST tag sequence (SEQ ID NO: 31 (amino acid sequence), SEQ ID NO: 32 (DNA) It was inserted into a plasmid vector pCAGGS for expressing the protein in mammalian cells so as to express the protein to which the sequence)) was added (FIG. 39).
- Human fetal kidney cell-derived HEK293 cultured cell line was transfected with pCAGGS-GST-S100A8 or pCAGGS-GST-S100A9 using a lipofection reagent (Invitrogen), and the cells and culture supernatant were collected 48 hours later.
- the cells and the culture supernatant were centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant (supernatant A) and cells were separated and collected.
- Cells were disrupted by adding 0.1% Tween20-containing PBS and applying ultrasonic waves for 30 seconds under ice-cooling. Furthermore, the supernatant was collected by centrifugation at 4400 g for 5 minutes at 4 ° C.
- Supernatant B Supernatant A and supernatant B were mixed and added to HiTrap GST FF column (GE healthcare, 5 mL) whose buffer was replaced with 30 mL of PBS in advance. After the addition, the column was washed with 100 mL of PBS, and the adsorbed protein was eluted with 20 mM phosphate buffer (pH.8) containing reduced glutathione. Bone marrow mesenchymal stem cell migration activity was studied using recombinant S100A8 and S100A9 Boyden chambers.
- a sample prepared by adjusting purified S100A8 and S100A9 proteins to a concentration of 0.1 ng / ⁇ L and dissolved in DMEM, or a sample of 2-day-old mouse skin extract diluted with 4 volumes of DMEM was inserted.
- proteins were extracted from cells transfected with a control vector into which S100A and S100A9 cDNA had not been inserted, and fractions eluted from HiTrap GST FF column were used in the same manner.
- HLB solution (Immuno-Biological Laboratories) was added and incubated at room temperature for 5 minutes. This hemolysis operation was repeated twice. 10 mL of PBS was added, centrifuged at 440 g for 5 minutes at 25 ° C., and the supernatant was removed to recover the cells.
- the antibody was used in the combination of (I) PDGFR ⁇ / CD45 / CD44 (II) PDGF ⁇ / CD45 / CD44.
- II PDGFR ⁇ / CD45 / CD44
- PDGF ⁇ / CD45 / CD44 the ratio of PDGFR ⁇ (or ⁇ ) and CD44 expressing cells to CD45 weakly positive-negative cells was examined (FIGS. 41A and 41B).
- Bone marrow mesenchymal stem cells are known to be pluripotent stem cells that differentiate into bone tissue, adipose tissue, cartilage tissue, fibroblasts, etc. It has also been pointed out that pluripotent stem cells that differentiate into tissues such as cells and epidermal cells also exist.
- S100A8 and S100A9 mobilize bone marrow-derived tissue stem cells to the skin graft, and the function of the damaged tissue. It is thought that it induces sexual repair.
- S100A8 and S100A9 can be mobilized by intravenous injection of bone marrow mesenchymal stem cells in peripheral blood, so they can be administered to peripheral tissues that are difficult to administer locally (brain, heart, spinal cord, etc.) via the peripheral circulation .
- bone marrow-derived tissue stem cells including mesenchymal stem cells can be mobilized locally during regeneration of damaged tissue, not only tissue damage healing of skin tissue but also brain, muscle, We are convinced that effects such as shortening the healing period and functional regeneration of damaged tissues are expected in the healing process of various tissue damages such as bone.
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Abstract
Description
一方、生体は臓器の損傷時の為に、損傷部位の修復再生機構を備えているが、損傷部位が大きい場合には非機能的な瘢痕組織によって損傷部位を塞ぐことが知られている。このような瘢痕組織による損傷治癒は、脳梗塞や脊髄損傷では、神経再生の阻害因子となり、心筋梗塞では心破裂の要因となり、広範囲熱傷や手術創ではケロイドとなるなど、生命予後、美容面においてQOLを著しく損なう原因となっている。生体が持つ損傷組織の修復再生機構を賦活化することが可能になれば、瘢痕治癒することなく機能的な組織による損傷組織(臓器)の再生を誘導することが期待できる。
骨髄内には、白血球や赤血球などに分化する造血系幹細胞の他、骨、軟骨、脂肪などに分化可能な間葉系の幹細胞が存在することが知られているが、近年になって上皮系や神経系の細胞に分化することが可能な多能性幹細胞が存在することが明らかになってきている。
また、脳梗塞モデルマウス脳における骨髄由来細胞の存在を確認したところ、脳梗塞作製後HMGB-1を静脈投与したマウスにおいて、神経細胞マーカーを発現する骨髄由来細胞を脳内に認めた。次に、脳梗塞巣の縮小効果を確認したところ、コントロールマウスと比較して、HMGB-1静脈投与マウスにおいて、脳梗塞巣において顕著な改善効果が確認された。また、脳梗塞後の生存率改善を確認したところ、HMGB-1静脈投与例ではマウスの生存率が上昇した。
さらに、マウスを用いて、骨折治癒過程における、骨折部以外の部位の骨髄多能性幹細胞の関与を確認したところ、損傷部位遠方の骨髄由来細胞が骨折部分に移動して損傷組織の修復を行っていることが明らかになった。
さらに、骨折モデルマウスを用いて、HMGB1静脈投与による損傷部位への骨髄間葉系幹細胞の動員活性を評価したところ、HMGB1の静脈内投与によって血中内に動員された骨髄間葉系幹細胞が骨折部分に集まってくることが明らかになった。
〔1〕以下の(a)から(q)のいずれかに記載の物質を含有する組織再生促進剤であって、再生が必要な組織とは異なる組織に投与される薬剤;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
〔2〕非経口投与される、〔1〕記載の薬剤。
〔3〕注射投与である、〔2〕記載の薬剤。
〔4〕血管内、筋肉内、皮下、皮内又は腹腔内に投与される、〔1〕記載の薬剤。
〔5〕細胞又は組織の抽出液が細胞又は組織を溶媒に浸す工程を含む方法で製造される、〔1〕~〔4〕のいずれかに記載の薬剤。
〔6〕細胞又は組織の抽出液のヘパリン結合画分が以下の工程を含む方法で製造される、〔1〕~〔4〕のいずれかに記載の薬剤;
(a)細胞又は組織を溶媒に浸す工程、
(b)工程(a)で得られる抽出液を固定化ヘパリンに接触させる工程、および
(c)固定化ヘパリンからヘパリン結合画分を溶出する工程。
〔7〕神経組織、骨組織、又は皮膚組織の再生促進に用いられる、〔1〕~〔6〕のいずれかに記載の薬剤。
〔8〕以下の(a)から(q)のいずれかに記載の物質を含有する組成物を含む、組織再生促進キットであって、該組成物が、再生が必要な組織とは異なる組織に投与される前記キット;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
〔9〕非経口投与される、〔8〕記載のキット。
〔10〕注射投与である、〔9〕記載のキット。
〔11〕血管内、筋肉内、皮下、皮内又は腹腔内に投与される、〔8〕記載のキット。
〔12〕神経組織、骨組織、又は皮膚組織の再生促進に用いられる、〔8〕~〔11〕のいずれかに記載のキット。
〔13〕以下の(a)から(q)のいずれかに記載の物質を含有する組成物を、再生が必要な組織とは異なる組織にその有効量を投与する工程を含む、組織の再生を促進する方法;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
〔14〕非経口投与である、〔13〕記載の方法。
〔15〕注射投与である、〔14〕記載の方法。
〔16〕血管内、筋肉内、皮下、皮内又は腹腔内に投与する、〔13〕記載の方法。
〔17〕神経組織、骨組織、又は皮膚組織の再生を促進する、〔13〕~〔16〕のいずれかに記載の方法。
〔18〕組織再生促進剤の製造における以下の(a)から(q)のいずれかに記載の物質を含有する組成物の使用であって、該組織再生促進剤が、再生が必要な組織とは異なる組織に投与される、前記使用;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
〔19〕該組織再生促進剤が非経口投与される、〔18〕記載の使用。
〔20〕注射投与である、〔19〕記載の使用。
〔21〕該組織再生促進剤が血管内、筋肉内、皮下、皮内又は腹腔内に投与される、〔18〕記載の使用。
〔22〕該組織再生促進剤が、神経組織、骨組織、又は皮膚組織の再生促進剤である、〔18〕~〔21〕のいずれかに記載の使用。
〔23〕組織の再生を促進する方法に使用するための、以下の(a)から(q)のいずれかに記載の物質を含有する組成物であって、再生が必要な組織とは異なる組織に投与される、前記組成物;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
〔24〕非経口投与される、〔23〕記載の組成物。
〔25〕注射投与である、〔24〕記載の組成物。
〔26〕血管内、筋肉内、皮下、皮内又は腹腔内に投与される、〔23〕記載の組成物。
〔27〕組織の再生を促進する方法が、神経組織、骨組織、又は皮膚組織の再生を促進する方法である、〔23〕~〔26〕のいずれかに記載の組成物。
HMGB1、HMGB2、HMGB3、S100A8、S100A9は骨髄多能性幹細胞を動員する活性を有している。骨髄多能性幹細胞は間葉系の他、上皮系や神経系の細胞にも分化可能である。広範囲の組織損傷の際に、骨髄多能性幹細胞を血流を介し損傷部位に動員する事が可能になれば、骨髄多能性幹細胞による生理的な損傷組織の修復再生を促進することが期待できる。
本発明により、骨髄多能性幹細胞動員因子である、HMGB1、HMGB2、HMGB3、S100A8、S100A9を静脈内投与などの方法で損傷部位から遠位部に投与することで、末梢血中内に骨髄多能性幹細胞を動員し、損傷組織修復を促進する方法が提供された。例えば脳梗塞の様な体内深部臓器の疾患を治療する場合、損傷部位(脳)への治療薬の直接投与は困難である。一方、本発明では、一般診療において広く施行されている静脈投与による治療を可能にしたため、治療薬を任意の回数、濃度で安全かつ簡便に投与することが可能となり、このことは従来の治療方法と比較して優れた効果である。
また、最近開発された骨髄細胞を使用した脳梗塞の治療法としては、患者本人の骨髄から細胞を採取し血液循環中に再投与する方法が有効であることが知られているが、骨髄細胞採取の際には体内深部の骨髄に太い針を刺して吸引する必要があるため大きな侵襲を伴うことが避けられない。これに対し、本発明では、薬剤を静脈内投与することで骨髄細胞を直接血液循環中に動員する事が可能であるため、脳梗塞の患者に頻回に投与しても大きな侵襲を伴うことがない。
骨髄由来の多能性幹細胞は間葉系、上皮系、神経系などの多様な細胞に分化する潜在能力を有するが、損傷部位に移動した後は周囲のニッチ環境に応じて分化し組織修復を誘導すると考えられる。再生医療や細胞治療では希少な骨髄多能性幹細胞を生体外で培養し増殖させた後に治療に利用するが、従来の医薬品と異なり、培養過程で起きうる細胞の劣化(癌化や細菌、ウイルスなどのコンタミネーション)の危険があるため十分な安全管理が必要である。本発明では、HMGB1、HMGB2、HMGB3、S100A8、S100A9を投与することで、骨髄多能性幹細胞を末梢循環血内に動員するが、細胞を体外に取り出し人工的操作を加えることはしないため安全性の高い治療法である。
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
該組織再生促進剤は、再生が必要な組織とは異なる組織に投与されることで、骨髄細胞を骨髄から再生が必要な組織に末梢循環を介して動員(誘導または局所誘導とも表現される)することを特徴とする。ここで、「末梢循環」とは、「血液循環」、「末梢循環血流」とも称される。
非瘢痕性組織再生促進剤であって、再生が必要な組織とは異なる組織に投与される薬剤、
非瘢痕性組織再生促進剤であって、再生が必要な組織とは異なる組織に投与され、骨髄から骨髄細胞を末梢血中に動員し、末梢循環系を介して、再生が必要な組織に骨髄由来細胞を動員することにより、組織の再生を促進することを特徴とする薬剤
とも表現することもできる。
本発明において、再生が必要な組織とは異なる組織への投与とは、再生が必要な部位以外の部位(再生が必要な部位とは異なる部位)に投与することを意味する。したがって、「再生が必要な組織とは異なる組織」は、再生が必要な組織とは異なる部位、再生が必要な部位とは異なる部位、再生が必要な組織から離れた部位、再生が必要な部位から離れた部位、再生が必要な部位から遠位にある部位、再生が必要な組織から遠位にある組織、遠位部、遠位組織と表現することもできる。
特に、本発明の薬剤は、体外から直接薬剤を投与することが困難な組織(脳、心臓など)を再生するために、有効に利用される。
また、再生が必要な組織とは異なる組織としては、血液組織、筋肉組織、皮下組織、皮内組織、腹腔等が例示できる。
本発明の薬剤には、好ましくは、神経組織、骨組織、皮膚組織等の再生促進剤が含まれるが、これらに限定されない。神経組織の再生促進剤としては、中枢神経組織の再生促進剤が挙げられるが、これに限定されない。また、神経組織の再生促進剤は、例えば、脳梗塞、脳出血、脳挫傷等の治療に使用できるが、これらに制限されない。また骨組織の再生促進剤は、例えば、骨折の治療に使用できるが、これに限定されない。また皮膚組織の再生促進剤は、例えば、皮膚潰瘍、手術創の縫合不全、熱傷、切傷、挫傷、皮膚びらん、擦過傷等の治療に使用できるが、これらに制限されない。
本明細書において、「骨髄細胞」とは、骨髄内に存在する細胞を意味し、一方、「骨髄由来細胞」とは、骨髄外に動員された「骨髄細胞」を意味する。
配列番号:1、3、5、7、9、11、13、15、17、19、21、23、25または27に記載のアミノ酸配列を含むタンパク質と機能的に同等なタンパク質としては、骨髄由来細胞を再生が必要な組織に動員する活性を有するタンパク質、又は骨髄由来細胞を遊走する活性を有するタンパク質が挙げられる。
改変されるアミノ酸の数は、典型的には50アミノ酸以内であり、好ましくは30アミノ酸以内であり、さらに好ましくは5アミノ酸以内(例えば、1アミノ酸)であると考えられる。
また、非保存的置換によりタンパク質の活性などをより上昇(例えば恒常的活性化型タンパク質などを含む)させることも考えられる。
ハイブリダイゼーションを利用することによって、たとえば配列番号:1、3、5、7、9、11、13、15、17、19、21、23、25または27に記載のアミノ酸配列を含むタンパク質以外のHMGB1、HMGB2、HMGB3、S100A8またはS100A9タンパク質のホモログをコードするDNAの単離が可能である。
酵母では、Pichia属酵母が糖鎖を備えたタンパク質の発現に有効なことが公知である。糖鎖の付加という点では、昆虫細胞をホストとするバキュロウイルスベクターを利用した発現系も有用である(Bio/Technology, 6:47-55, 1988)。更に、哺乳動物の細胞を利用して、CMV、RSV、あるいはSV40等のプロモーターを利用したベクターのトランスフェクションが行われており、これらのホスト/ベクター系は、いずれもHMGB1、HMGB2、HMGB3、S100A8またはS100A9タンパク質の発現系として利用することができる。また、レトロウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター等のウイルスベクターを利用して遺伝子を導入することもできる。
溶媒に浸される細胞又は組織としては、特に制限はないが、組織由来細胞、組織由来細胞から樹立された株化細胞(例えば、HeLa、HEK293が例示できるが、これらに制限されない)、単離された細胞、単離されていない細胞(例えば単離された組織中に存在する細胞)、HMGB1、HMGB2、HMGB3、S100A8またはS100A9タンパク質をコードするDNAが導入された細胞などが例示できる。上記組織としては、どのような組織でもよく、例えば、生体皮膚組織、体内生検(手術)組織(脳、肺、心臓、肝臓、胃、小腸、大腸、膵臓、腎臓、膀胱、脾臓、子宮、精巣や血液など)が例示できるが、これらに制限されるものではない。
末梢血抽出液の調整方法は、注射器などを用いて採血した後、冷凍庫や液体窒素、ドライアイスなどで細胞を凍結し、その後0℃以上の温度下で再融解する。さらに、細胞の不溶成分を取り除くために、例えば、4℃~25℃(例えば4℃)、重力加速度10G~100000G(例えば440G)で遠心し、上清を分取することにより、溶媒から細胞の不溶成分を取り除くことができるが、これに制限されない。該上清を細胞や組織の抽出液として利用できる。細胞の不溶成分を除去するためには、遠心操作の代わりに、0.45μmの微少の孔をもつニトロセルロースフィルターなどを通過させることで、不溶成分を取り除くことができる。また、採血した末梢血を3時間から48時間4℃の状態に置くことで、細胞の壊死を誘発し、末梢血中の細胞から細胞内成分を分泌させることができる。この後重力加速度10G~100000G(例えば440G)で遠心し、上清を分取することにより、溶媒から細胞の不溶成分を取り除くことができるが、これに制限されない。該上清を細胞や組織の抽出液として利用できる。細胞の不溶成分を除去するためには、遠心操作の代わりに、0.45μmの微少の孔をもつニトロセルロースフィルターなどを通過させることで、不溶成分を取り除くことができる。
(a)細胞又は組織を溶媒に浸す工程、
(b)工程(a)で得られる抽出液を固定化ヘパリンに接触させる工程、および
(c)固定化ヘパリンからヘパリン結合画分(ヘパリン精製画分、ヘパリンカラム精製画分とも表現しうる)を溶出する工程
固定化ヘパリンとは、ヘパリンを不溶性担体に共有結合させたものである。上記不溶性担体としては、Sepharose beads(Sepharose 4B,Sepharose 6B等:GE Healthcare)が例示されるが、これに制限されるものではない。本発明においては、市販の固定化ヘパリン(Hitrap Hepalin HP column: GE Healthcare)を用いてもよい。
細胞や組織の抽出液と固定化ヘパリンの接触条件としては、pH7~8程度(好ましくはpH7.5)、塩濃度は0~200mM、好ましくは100~200mM程度が例示されるが、これらに制限されない。抽出液と固定化ヘパリンとが接触している時間は特に限定されないが、ヘパリン結合画分を固定化ヘパリンに十分吸着させるという観点では5分以上保持されることが好ましい。また、温度としては、4~8℃、好ましくは4℃が挙げられるが、これらに制限されない。さらに、固定化ヘパリンに吸着したヘパリン結合画分の溶出条件としては、pH7~8程度、塩濃度200~1000mM(好ましくは1000mM程度)が例示されるが、これらに制限されるものではない。
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
該組織再生促進用キットは、再生が必要な組織とは異なる組織に投与されることで、骨髄細胞を骨髄から再生が必要な組織に末梢循環を介して動員することを特徴とする。
また、本発明のキットには、上記再生が必要な組織を治療するためのキットが含まれる。好ましくは、本発明のキットには、非経口投与されるキット、さらに好ましくは注射投与されるキットが含まれる。また、本発明のキットには、好ましくは、血管内、筋肉内、皮下、皮内又は腹腔内に投与されるキットが含まれる。
また、好ましくは、本発明のキットには、神経組織、骨組織、又は皮膚組織の再生促進に用いられるキットが含まれる。
組織再生促進用キットとしては、(1)フィブリノゲンに溶解した上記物質、および(2)トロンビンを含む組織再生促進用キット、または、(1)上記物質、(2)フィブリノゲン、および(3)トロンビンを含む組織再生促進用キットが例示できる。本発明においては、市販のフィブリノゲンやトロンビンを使用することができる。例えば、フィブリノゲンHT-Wf(ベネシスー三菱ウェルファーマー)、ベリプラスト(ZLBベーリング)、ティシール(バクスター)、ボルヒール(化血研)、タココンブ(ZLBベーリング)が挙げられるが、これらに制限されるものではない。
(1)以下の(a)から(q)のいずれかに記載の物質を含有する組成物を、再生が必要な組織とは異なる組織にその有効量を投与する工程を含む、組織の再生を促進する方法;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
(2)組織再生促進剤の製造における以下の(a)から(q)のいずれかに記載の物質を含有する組成物の使用であって、該組織再生促進剤が、再生が必要な組織とは異なる組織に投与される、前記使用;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
(3)組織の再生を促進する方法に使用するための、以下の(a)から(q)のいずれかに記載の物質を含有する組成物であって、再生が必要な組織とは異なる組織に投与される、前記組成物;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。
新生マウス皮膚からTrizol (invitrogen) を用いてRNAを抽出し、更にSuperScript III cDNA synthesis kit(Invitrogen)を用いてcDNAを合成した。このcDNAをテンプレートとしてHMGB1のcDNAをPCR(ポリメラーゼ連鎖反応)法を用いて増幅し、精製のためにアミノ酸配列のN末端にGST tagおよび6XHis tagの配列を付加したタンパクを発現するように、哺乳類細胞でタンパクを発現させるプラスミドベクターpCAGGSに挿入した。
ヒト胎児腎細胞由来HEK293培養細胞株にポリエチレンイミン(PEI)を用いてpCAGGS-GST-His-HMGB1をトランスフェクションし、48時間後細胞及び培養上清を回収した。細胞及び培養上清は4℃で4400g・5分間遠心し、上清と細胞を分離しそれぞれ回収した。回収した上清はさらに直径0.8μm孔をもつセルロースアセテートフィルターを通過させた後、0.45μmの孔を持つニトロセルロースフィルターを通過させ、それにより不溶画分を除去したサンプルを調整した。本サンプルを、50mM NaCl含50mM Tris HCl pH8.0 (50mL)で平衡化した5 mL HisTrap FF(GE)に挿入し、吸着成分をさらに、10mM イミダゾール含有50mM NaCl 50mM Tris HCl pH8.0で洗浄して非特異的吸着成分を除去した。特異的吸着成分を、カラムから、100mM イミダゾール含有50mM NaCl 50mM Tris HCl pH8.0で溶出した。吸着画分はそれぞれ、500μLずつシリコンコートしたプラスチックチューブに分画し、タンパク含有フラクションをまとめた後、脱塩カラムPD10(GE)を用いてイミダゾールを除去し、50mM Tris HCl pH. 7.5, 150mM NaCl を用いて溶出した。溶出したサンプルにHRV3C(Novagen)を添加し、4℃、8時間反応させた。切断後、サンプルを50mM Tris HCl pH.7.5,150 mM NaClで平衡化したHiTrap Heparin 1 mL カラム(GE)に結合させ、50mM Tris HCl pH.7.5,150 mM NaClでカラム内部を洗浄後、50mM Tris HCl pH.7.5,1000 mM NaClで結合タンパク質を溶出した。溶出したサンプルは50mM Tris HCl pH.8.8 20mM NaClにて50倍希釈し同じバッファーで平衡化した1mL HiTrap Q FF (GE)に吸着させた。50mM Tris HCl pH.8.8 500mM NaClを使用しNaClの濃度を徐々に上昇させることで吸着タンパク質を溶出した。ニッケルカラム、ヘパリンカラム、Qカラムに結合したタンパク質はSDS-PAGEしクマシーブリリアントブルー染色を行いタンパク質の存在を確認した。
その結果、図1に示すように、高純度のHMGB-1が精製された。以降の実施例では本精製方法を使用したHMGB-1を使用する。
ヒト胎児腎細胞由来HEK293培養細胞株にリポフェクション試薬(Invitrogen)を用いてpCAGGS-GST-S100A8をトランスフェクションし、48時間後細胞及び培養上清を回収した。細胞及び培養上清は4℃で4400g・5分間遠心し、上清(上清A)と細胞を分離しそれぞれ回収した。細胞は0.1%Tween20含PBSを加え氷冷下で30秒間超音波をあてることで細胞膜を破壊した。さらに、4℃で4400g・5分間遠心し、上清を回収した(上清B)。上清Aおよび上清Bを混合し、あらかじめ30mLのPBSでバッファーを置換したHiTrap GST FF column (GE healthcare,5mL)に添加した。添加後PBS100mLでカラムを洗浄し、還元型グルタチオン含20mMリン酸バッファー(pH.8)で吸着したタンパク質を溶出した。グルタチオンを除くために、ゲル濾過カラムPD-10(GE社製)にてバッファーをPBSに置換した。
C57BL/6雄マウス(6週齢)に致死量放射線(10Gy)を照射し、その直後に尾静脈よりGFP(green fluorescent protein)トランスジェニックマウス(Okabe M. et al, FEBS Lett. 407, 313-319, 1997)由来骨髄細胞(5x106個/0.1ml 生理的リン酸緩衝溶液pH7.4)を移植した。8週間後、背部に直径6mmの円形の皮膚潰瘍を作製した。マウスの皮膚が収縮することを防ぐため、シリコン製の外径10mm内径6mm厚さ1mmの円盤を両面粘着テープおよび医療用接着剤アロンアルファA(三共)を用いて潰瘍部に接着した。潰瘍部分の乾燥と細菌感染を防ぐため直径10mm厚さ1mmのシリコン製の円盤で潰瘍部を覆った。潰瘍部を保護するため、テガダーム(3M)で被覆した。
皮膚潰瘍作製当日から24時間間隔で5回尾静脈からHMGB-1(40μg)もしくはS100A8(250ng)を投与した。潰瘍作製から2週間後、マウスにイソフルランを用いて吸入麻酔した後、背部に作製した皮膚潰瘍作製部分のGFP蛍光の集積程度を蛍光実体顕微鏡使用して観察した。さらに、皮膚潰瘍作製部分の皮膚を円形に切除し4%パラホルムアルデヒド含PBS(リン酸緩衝液,nacalai)で固定し、OCTコンパウンドに包埋後冷却装置付ミクロトーム(Leica)を使用して8マイクロメートルの薄切切片を作製した。切片をプレパラートに貼り付けた後、PBSにてコンパウンドを洗浄し、核をDAPIによって染色した。さらにPBSにて余剰のDAPIを洗浄し、蛍光退色防止剤入り封入材を用いて封入した。それぞれのサンプルについて蛍光顕微鏡を用いてGFPの蛍光を検出した。
C57BL/6雄マウス(8週齢)に背部に直径6mmの円形の皮膚潰瘍を作製した。マウスの皮膚が収縮することを防ぐため、シリコン製の外径10mm内径6mm厚さ1mmの円盤を両面粘着テープおよび医療用接着剤アロンアルファA(三共)を用いて潰瘍部に接着した。潰瘍部分の乾燥と細菌感染を防ぐため直径10mm厚さ1mmのシリコン製の円盤で潰瘍部を覆った。潰瘍部を保護するため、テガダーム(3M)で被覆した。
皮膚潰瘍作製当日から24時間間隔で尾静脈からHMGB-1(40μg)もしくはS100A8(250ng)を計五回投与した。潰瘍作成後3日目、5日目、10日目の潰瘍部分の面積を計測した。
C57BL/6雄マウス(8週齢)に背部に直径6mmの円形の皮膚潰瘍を作製した。マウスの皮膚が収縮することを防ぐため、シリコン製の外径10mm内径6mm厚さ1mmの円盤を両面粘着テープおよび医療用接着剤アロンアルファA(三共)を用いて潰瘍部に接着した。潰瘍部分の乾燥と細菌感染を防ぐため直径10mm厚さ1mmのシリコン製の円盤で潰瘍部を覆った。潰瘍部を保護するため、テガダーム(3M)で被覆した。
皮膚潰瘍作製当日から24時間間隔で尾静脈からHMGB-1(40μg)を計五回投与した。潰瘍作成後4週後の潰瘍部分を採取し10%緩衝ホルムアルデヒドで固定した。サンプルをパラフィン包埋した後、ミクロトームを使用して薄切切片を作製した。脱パラフィン後HE(ヘマトキシリン・エオジン)染色、およびMT(マッソン・トリクローム)染色を行った。
GFP骨髄移植マウスに脳梗塞を作成した(中大動脈寒栓糸モデル)。具体的には、上述の実施例の方法で作成したGFP骨髄移植マウスにイソフルランを用いて吸入麻酔を行った後、頭部皮膚を切開し、頭蓋骨に直接レーザードップラー血流計プローブを接着し脳血流を確認した。さらに胸骨から下顎まで正中皮膚を切開し、右総頚動脈を剥離し4号絹糸を用いて緩く結紮した。右外頚動脈の遠位部に6号絹糸をかけて結紮した。総頚動脈の糸に緊張を加えながら右外頚動脈近位部に穴を開けて、先端700μmを加熱成形した6号モノフィラメントナイロン糸(塞栓糸)を挿入した。内頚動脈に向けて塞栓糸を進め、塞栓糸の先端から約8 mmまで挿入した部位で総頚動脈糸をゆるめた。レーザードップラー血流計によって血流遮断前後で血流計の数値が10分の1になることを確認した。
血流遮断30分後に塞栓糸を抜去し血流を回復させた。12時間後、精製HMGB-1(100μg)を500μLのPBSに希釈しで作製した疾患モデルマウスの尾静脈から投与した。以後4回24時間おきに同様にHMGB-1を投与した。コントロールマウスにはPBSを投与した。
治療最終日から2週間後、イソフルランによる吸入麻酔下で2%パラホルムアルデヒドを使用して灌流固定を行った。脳を頭蓋骨から取り出し10%スクロース液に12時間、20%スクロース液に24時間浸透させて脳組織から脱水を行った。脱水後OTC コンパウンド内に投入し、ドライアイス上で凍結させブロックを作製した。ブロックは凍結切片用のミクロトームを使用して厚さ8μmの切片を作製し、シランコーティングのプレパラート上に伸展した。伸展後十分に乾燥させ、PBSを用いてコンパウンドを洗浄した。
2%スキムミルク含PBSをサンプルに浸透させた後、2%スキムミルク含PBSに抗マウスNestin 抗体およびβIII Tubulin抗体を500倍に希釈しサンプルに4℃で8時間浸透させた。PBSにて5分間ずつ5回充分に洗浄した後、2%スキムミルク含PBS で500倍に希釈したPE 標識抗ラットIgG抗体をサンプルに室温で1時間浸透させた。同様にPBSにて充分に洗浄した後DAPI溶液を室温で10分間浸透させ、PBSで充分に洗浄した。蛍光消退防止剤含有封入剤で封入後、共焦点レーザー顕微鏡を用いて、GFP・DAPI・PEの蛍光を観察した。
本結果から、脳梗塞作製後HMGB-1を静脈投与したマウスにおいて、神経細胞マーカーを発現する骨髄由来細胞を脳内に認めた。これらのGFP陽性細胞の由来は、骨髄間葉系幹細胞などの非炎症系細胞に由来すると予想される。
8週齢C57/Bl6 メスマウスにイソフルランを用いて吸入麻酔を行った後、頭部皮膚を切開し、頭蓋骨に直接レーザードップラー血流計プローブを接着し脳血流を確認した。さらに胸骨から下顎まで正中皮膚を切開し、右総頚動脈を剥離し4号絹糸を用いて緩く結紮した。右外頚動脈の遠位部に6号絹糸をかけて結紮した。総頚動脈の糸に緊張を加えながら右外頚動脈近位部に穴を開けて、先端700μmを加熱成形した6号モノフィラメントナイロン糸(塞栓糸)を挿入した。内頚動脈に向けて塞栓糸を進め、塞栓糸の先端から約8 mmまで挿入した部位で総頚動脈糸をゆるめた。レーザードップラー血流計によって血流遮断前後で血流計の数値が10分の1になることを確認した。
血流遮断30分後に塞栓糸を抜去し血流を回復させた。12時間後、精製HMGB1(10μg)を500μLのPBSに希釈し、作製した疾患モデルマウスの尾静脈から投与した。以後4回24時間おきに同様にHMGB-1を投与した。コントロールマウスにはPBSを投与した。
治療最終日から5日後、イソフルランによる吸入麻酔下で2%パラホルムアルデヒドを使用して灌流固定を行った。脳を頭蓋骨から取り出し、脱水後OTC コンパウンド内に投入し、ドライアイス上で凍結させブロックを作製した。ブロックは凍結切片用のミクロトームを使用して厚さ8μmの切片を作製し、シランコーティングのプレパラート上に伸展した。伸展後十分に乾燥させ、PBSを用いてコンパウンドを洗浄した。4%パラホルムアルデヒド含PBSにて10分間固定した後、リン酸緩衝液で5分間洗浄し、蒸留水にて10分間浸透させた。さらに0.5%クレシルバイオレット溶液にて13分間染色した。蒸留水で1分間洗浄した後、50%エタノール、75%エタノール、95%エタノール、100%エタノール、にそれぞれ10秒浸透させた後キシレンに2分間浸透させることを2回繰り返した。さらにエンテラン液を用いて封入した。
8週齢C57/Bl6 雄マウスにイソフルランを用いて吸入麻酔を行った後、頭部皮膚を切開し、頭蓋骨に直接レーザードップラー血流計プローブを接着し脳血流を確認した。さらに胸骨から下顎まで正中皮膚を切開し、右総頚動脈を剥離し4号絹糸を用いて緩く結紮した。右外頚動脈の遠位部に6号絹糸をかけて結紮した。総頚動脈の糸に緊張を加えながら右外頚動脈近位部に穴を開けて、先端700μmを加熱成形した6号モノフィラメントナイロン糸(塞栓糸)を挿入した。内頚動脈に向けて塞栓糸を進め、塞栓糸の先端から約8 mmまで挿入した部位で総頚動脈糸をゆるめた。レーザードップラー血流計によって血流遮断前後で血流計の数値が10分の1になることを確認した。
血流遮断を一定時間(45分間もしくは60分間)それぞれのマウスに行った後で塞栓糸を抜去し血流を回復させた。12時間後、精製HMGB1(10μg)を500μLのPBSに希釈しで作製した疾患モデルマウスの尾静脈から投与した。以後4回24時間おきに同様にHMGB-1を投与した。コントロールマウスにはPBSを投与した。梗塞作製後7日間の生存率の観察を行った。
C57BL/6雄マウス(6週齢)に致死量放射線(10Gy)を照射し、その直後に尾静脈よりGFP(green fluorescent protein)トランスジェニックマウス由来骨髄細胞(5x106個/0.1ml 生理的リン酸緩衝溶液pH7.4)を移植した(GFP骨髄キメラマウス)。8週間後、GFP骨髄キメラマウス(図8 左のマウス)と野生型マウス(図8 右のマウス)を皮膚で結合したパラビオーシスを作製した。さらに、野生型マウス(図8 右のマウス)の右下肢に骨折を作製し、骨折治癒後の組織切片を作製した。切片に対し、4%スキムミルク含PBSでブロッキング後、4%スキムミルク含PBSに希釈した抗マウスオステオカルシン抗体を反応させ、PBSで洗浄後、4%スキムミルク含PBSに希釈したPE標識抗ラットIgG抗体と室温で1時間反応させた。PBSで洗浄後、DAPIを用いて核を染色しさらにPBSで洗浄した。封入後共焦点レーザー顕微鏡を利用して蛍光を観察した。
方法:上記の目的に対して以下の方法により研究を行った。
1)実験ではPDGFレセプターαのプロモーターの下流にGFPタンパクをゲノムにノックインしたマウス(PDGFRα-GFPマウス)(参考文献:Hamilton et al., Mol Cell Biol. 2003 Jun;23(11):4013-25)を使用した。このマウスはPDGFレセプターαが発現している細胞においてGFPタンパクが発現するため、蛍光顕微鏡を用いて観察すると緑色の蛍光を検出できる。
2)PDGFRα-GFPマウスの骨髄細胞を採取し、細胞培養用皿に播種し10% FBS含有α-MEMで培養を行った。3-4日ごとに培地を交換し約14日後に付着している細胞を回収した。回収した細胞を抗CD11b MACSビーズを使用してCD11b陽性細胞とCD11b陰性細胞にわけた。蛍光顕微鏡で観察したところCD11b陽性細胞はGFP陰性(図42のA1,A2)であり、CD11b陰性細胞はGFP陽性(図42のB1,B2)であることを確認した。HMGB1がこれらの細胞をマイグレーションさせるかを調べるためにボイデンチャンバー法を行った。ボイデンチャンバーの上層にはそれぞれCD11b陽性細胞またはCD11b陰性細胞を入れ、下層にはHMGB1を0・50・100μg/mLになるように10%FBS含有DMEMに希釈して入れた。チャンバーは37℃、5%CO2のインキュベーター内で静置した。4時間後チャンバーのメンブレンを回収し下層に向かってマイグレーションした細胞を染色することで検出した(図43)。
3)12週齢雄PDGFRα-GFPマウスをイソフルランにて全身麻酔し、左下腿頸骨の骨折モデルを作成した。骨折作成直後、24時間後、48時間後の計三回10μgのHMGB1を500μLのPBSに希釈し尾静脈から投与した。陰性コントロールは500μLのPBSを尾静脈から投与した(N=6)。
4)骨折作成72時間後左下腿脛骨を取り出し4%パラホルムアルデヒド含PBSを用いて24時間静置し組織を固定した。PBSで洗浄した後、蛍光実体顕微鏡を用いてGFPの蛍光を検出した(図44)。
CD11b陽性の細胞はGFP陰性でありPDGFレセプターαを発現していないことが示唆された(図42のA1,A2)。CD11b陰性の細胞はGFP陽性でありPDGFレセプターαを発現していることが示唆された(図42のB1,B2)。CD11b陽性(PDGFレセプターα陰性)細胞はHMGB1によってマイグレーションしなかったが、CD11b陰性(PDGFレセプターα陽性)細胞はHMGB1によってマイグレーションした(図43)。
陰性コントロールマウスであるPBS投与(図44のD1)群に比較すると、HMGB1投与(図44のD2)群のうち6匹中4匹において、骨折部位周囲の骨にGFP陽性(PDGFレセプターα陽性)細胞が認められた。
目的:生体移植皮膚組織の機能的再生時における骨髄由来細胞寄与の評価
方法:上記の目的に対して以下の方法により研究を行った。
1)GFP骨髄移植マウスに対する生体皮膚移植系を利用して、移植皮膚片の機能的再生時の骨髄由来細胞寄与程度を検討した。具体的には、C57BL/6雄マウス(6~8週齢)に致死量放射線(10Gy)を照射し、その直後に尾静脈よりGFP(green fluorescent protein)トランスジェニックマウス由来骨髄細胞(5x106個/0.1ml 生理的リン酸緩衝溶液pH7.4)を移植した。
2)移植骨髄細胞の生着を待って(6週間)得られたGFP骨髄移植マウスの背部皮膚に、新生マウス皮膚(雌)を移植した。
3)移植皮膚片の生着と十分な皮膚組織再生を待って(4週間)、移植皮膚片領域におけるGFP蛍光の集積程度を、蛍光実体顕微鏡を利用して観察した。
4)吸入麻酔下に移植皮膚片を生検により採取し、冷却装置付ミクロトームを用いて皮膚凍結切片(6μm)を作製、4%パラホルムアルデヒド固定(30分間)、組織内細胞核をDAPIで染色、表皮細胞特異的ケラチン5の抗体を用いて免疫染色を施行した後、組織を封入して共焦点レーザー顕微鏡によりGFP陽性骨髄由来細胞の存在を検討した。また一部の標本はHE染色によりその組織構築を検討した。
結果:GFP骨髄移植マウスへの生体皮膚移植系において、再生した皮膚領域に一致した強いGFP蛍光の集積が観察された(図9)。また、移植皮膚片のHE標本を用いた組織学的観察では、多数の毛包を含む皮膚組織の機能的再生が確認された(図9)。共焦点レーザー顕微鏡による観察では、ケラチン5を発現している表皮角化細胞、真皮線維芽細胞、さらには平滑筋細胞や脂肪細胞の多くがGFP蛍光を示し、これらの細胞が骨髄由来であることが示された(図10)。即ち、移植皮膚の機能的再生時に必要な上皮系および間葉系細胞の多くが、骨髄由来幹細胞により供給されていることが初めて明らかとなった。
考察:これらの研究結果は、これまで臨床現場で日常的に行われている皮膚移植における皮膚再生時に、骨髄由来細胞が大きく寄与していることを初めて明確に示したものであり、極めて画期的な発見である。
目的:皮膚組織抽出液内に存在する骨髄間葉系幹細胞誘導因子の同定
方法:疎血状態にある切除皮膚からの放出が予想される骨髄間葉系幹細胞動員因子の同定を目的として、以下の方法により研究を進めた。
1)マウス骨髄由来間葉系幹細胞を得るために、C57BL/6マウスの骨髄細胞を大腿骨もしくは下腿の骨から採取し、10%胎仔ウシ血清含D-MEM(Nacalai 社製)を細胞培養培地として細胞培養皿に撒き、37℃、炭酸ガス濃度5%の条件下で培養した。細胞が占める面積が培養皿底面積に対して70から100%に増殖した時点で、0.25%トリプシン1mMEDTA(Nacalai社製)を用いて細胞を培養皿からはがし、さらに同じ条件で継代培養した。継代作業は少なくとも5回以上繰り返した。さらにこれらの付着細胞を単離培養しフローサイトメトリーによる細胞表面抗原の分析を行いLin陰性、CD45陰性、CD44陽性、Sca-1陽性、c-kit陰性であることを確認した。これらの細胞は骨細胞、脂肪細胞に分化可能で骨髄間葉系幹細胞の性質を有することを確認した。
2)新生マウス400匹から得た遊離皮膚片を生理的リン酸緩衝液pH7.4(PBS)400ml内に浸し、4℃で24時間インキュベーションした後、組織を取り除くために、4℃の条件下で10分間、440Gで遠心し上清を回収して皮膚抽出液を作製した。
3)得られた皮膚抽出液の中に骨髄間葉系幹細胞誘導活性が存在することを確認するため、ボイデン・チャンバーを用い、本発明者らが既に株化しているC57BL6マウス骨髄由来間葉系幹細胞に対する遊走活性を検討した。具体的にはボイデン・チャンバーの下槽(容量25μl)に皮膚抽出液(25μl)を挿入し、8μmの微細穴を持つポリカーボネートメンブレンを乗せ、さらにこれに接してボイデン・チャンバー上槽(容量50μl)を載せて、その中に骨髄由来間葉系幹細胞浮遊液(5x104個/50ml培養液:DMEM/10%ウシ胎仔血清)を入れ、CO2インキュベーター内で37℃、4~24時間培養した。培養後、チャンバーの上槽をはずし、シリコン薄膜を取り出して、その微細穴を通過してチャンバー下槽に遊走した骨髄由来間葉系幹細胞の数を染色により定量的に検討した。
4)皮膚抽出液内の骨髄由来間葉幹細胞動員活性をもつ因子を精製するために、ヘパリンアフィニティーカラム・クロマトグラフィー、および陰イオン交換カラム(Qカラム)クロマトグラフィーを施行した。皮膚抽出液を4℃の9倍容20 mM リン酸バッファー pH 7.5を用い 10倍に希釈した(希釈液A)。あらかじめ、20 mM リン酸バッファー pH 7.5(30ml)をHiTrap Hepalin HP column(カラム容量: 5ml、GE Healthcare)の中に流しカラムを平衡化した。さらに、希釈液Aをカラムに結合させた。その後20 mM リン酸バッファー pH 7.5, 100mM NaCl(30ml)でカラムを洗浄した。吸着したタンパクを溶出するため20 mM リン酸バッファー pH 7.5, 1000mM NaClをカラム内に流入し、溶出液をチューブに分画した。吸着画分をそれぞれ、2)で説明したボイデン・チャンバーを利用した遊走活性評価法により遊走能を有する画分を集めた。これを9倍容50 mM Tris HCl pH 8.0で希釈した(希釈液B)。あらかじめ、50 mM Tris HCl pH 8.0(30ml)をHiTrap mono Q column(カラム容量: 1ml、GE Healthcare)の中に流しカラムを平衡化した。さらに、希釈液Bをカラムに結合させた。吸着したタンパクを溶出するためTris HCl pH 8.0, 1000mM NaClをカラム内に流入し、溶出液をチューブに分画した。以上の精製過程はすべて4~16℃で行うことが可能であるが、4~8℃で行うことが好ましく、4℃で行うことが更に好ましい。この溶出液を2)で説明したボイデン・チャンバーを利用した遊走活性評価方法により評価した。
6)5)のSDS-PAGE電気泳動および銀染色により単一バンドとしてゲル内分画された皮膚抽出液由来蛋白群の中で、骨髄由来間葉系幹細胞動員活性の最も強いクロマトグラフィー精製標品から得られた蛋白バンドをすべて切り出して、質量分析およびデータベース解析により当該蛋白の同定を進めた。
7)同定した蛋白群の中から骨髄由来間葉系幹細胞動員活性を持つ候補蛋白を選定し、その候補蛋白を含む精製標品を中和抗体で処理(精製標品溶液100μlを当該蛋白のポリクロナール抗体100倍希釈条件にて氷上で30分間インキュベート)した後、骨髄由来間葉系幹細胞動員活性の阻害程度を、ボイデン・チャンバーを用いた遊走能アッセイにて検討した。
8)得られた骨髄由来間葉系幹細胞精製標品をマトリゲルの中に約10%ボリュームとなるように混入し、直径約1mm、5mm長のシリコンチューブ内に挿入し、これをGFP骨髄移植マウス背部皮下に移植した。2週間後に挿入チューブを取り出して、チューブ内に遊走した骨髄由来細胞から出るGFP蛍光を蛍光測定装置により定量的に解析した。さらに、チューブ内から遊走細胞を取り出し、DMEM/10%ウシ胎仔血清培地に播種してCO2インキュベーター内で培養することにより、骨髄由来間葉系幹細胞の生体内動員活性を検討した。2週間継続して培養した細胞は2%パラホルムアルデヒドで25℃の条件下で、10分間固定した後、5分間ずつ4回PBSを用いてパラホルムアルデヒドを洗浄し、2%スキムミルク液で処理をし、0.5% tween20含有2%スキムミルクで1000倍に希釈した抗マウスケラチン5抗体を4℃の条件下で16時間反応させた。5分間ずつ4回PBSを用いて抗体を洗浄し、2%スキムミルクで1000倍に希釈したAlexa546標識抗ウサギIgG抗体を25℃の条件下で1時間反応させた。
さらに、HMGB1が生体内で骨髄由来間葉系幹細胞動員活性を持つことを確認する目的で、この精製標品を入れたシリコンチューブをGFP骨髄移植マウス背部皮下に挿入し、2週間後にチューブ内に動員された細胞の性質を検討した。その結果、対照コントロール(図12におけるSDS-PAGEレーン4に用いた精製標品)に比較して、HMGB1精製標品はGFP陽性の骨髄由来細胞をチューブ内に多数(対照コントロールの約3倍)動員していた(図14)。図15に蛍光実体顕微鏡による強拡大像を示す。さらに、チューブ内に動員されたGFP陽性細胞を取り出し、DMEM/10%ウシ胎仔血清培地内で培養した結果、培養開始直後は円形の浮遊細胞状態であったが(図16)、24時間後にはGFP陽性骨髄由来細胞は培養シャーレに付着し、紡錘系の線維芽細胞様形態、さらには類円形の上皮細胞様形態の細胞として増殖することが確認された(図17)。さらにこれらの細胞を2週間継続して培養するとGFP陽性骨髄由来細胞の中に毛包の形態を呈する細胞を確認した(図18A;明視野、弱拡大、図18B;GFP蛍光、弱拡大、図18C明視野、強拡大、図18D;GFP蛍光、強拡大)。また、上皮ケラチノサイトのマーカーであるケラチン5に対する免疫組織化学を行ったところ、GFP陽性骨髄由来細胞の中にケラチン5陽性細胞を確認した(図18E;明視野、図18F;ケラチン5陽性細胞の蛍光)。
目的:皮膚抽出液中HMGB1ファミリーの同定と骨髄間葉系幹細胞誘導活性の検討
方法:新生マウス皮膚抽出液中に含まれるHMGB蛋白ファミリーの有無をWestern blot 法を用いて確認した。サンプルとして〔参考例2〕で得られた皮膚抽出液を10μl をSDS-PAGE法を用いて電気泳動し、ゲル中で分離した蛋白をブロッティング装置(ATTO)を用いPVDF膜にトランスファーした。3%スキムミルク0.1% Tween 20 含PBS(S-T-PBS)にて、室温で1時間インキュベートした後、S-T-PBSで1000倍に希釈したラビット抗マウスHMGB1 抗体、ラビット抗マウスHMGB2抗体、ラビット抗マウスHMGB3抗体をそれぞれ4℃で16時間反応させた。反応後、同PVDF膜をS-T-PBSにて5分間5回洗浄後、S-T-PBSで2000倍希釈したペルオキシダーゼ標識ヤギ抗ラビットIgG抗体(GE Healthcare)にて同PVDF膜を25℃で1時間インキュベートした。さらにS-T-PBSにて5分間5回洗浄後ECL Western Blotting Detection System (GE Healthcare)を同PVDF膜と反応させ、ECL film を感光させた後現像してHMGB1、HMGB2、HMGB3タンパクの存在を検出した。
目的:骨髄間葉系幹細胞誘導因子組織抽出液の作製方法の確立
方法:6週齢C57BL6一匹分の脳、心臓、腸、腎臓、肝臓及び新生マウス皮膚一匹分を生理的リン酸緩衝液pH7.4(PBS)1ml内に浸し、4℃で24時間インキュベーションした後、組織を取り除くために、4℃の条件下で10分間、440Gで遠心し上清を回収して組織抽出液とした。得られた抽出液の中に骨髄間葉系幹細胞誘導活性が存在することを確認するため、ボイデン・チャンバーを用い、〔参考例2〕と同様に骨髄由来間葉系幹細胞に対する遊走活性を検討した。また、同じサンプル中に含まれるHMGB1の濃度をHMGB1 ELISA kit(シノテスト)を用いて計測した。更に脳、心臓および皮膚の組織抽出液を〔参考例2〕と同様に、ヘパリンアフィニティーカラムに結合させ、結合画分のタンパクの骨髄間葉系幹細胞誘導活性をボイデン・チャンバーを用いて確認した。
目的:培養細胞から間葉系幹細胞遊走活性物質を抽出する方法を確立する。
方法:ヒト胎児腎由来培養細胞株HEK293及びヒト子宮頸癌細胞株HeLaはそれぞれ10%胎仔牛血清含D-MEM(nacalai 社製)で培養した。それぞれの細胞をPBSで洗浄後、細胞107個を4℃の5mlのPBS(nacalai社製)に16時間浸した。重力加速度440Gで4℃で5分間遠心し上清を回収した。ボイデン・チャンバーの上層にヒト骨髄間葉系幹細胞をいれ、下層にDMEMで5倍希釈した細胞抽出液をいれ、ヒト骨髄間葉系幹細胞遊走活性を確認した。
結果:HEK293抽出液もHeLa抽出液も同様に骨髄間葉系幹細胞を遊走する活性を示した(図26)。
考察:培養細胞をPBSに浸すという簡便な方法で骨髄間葉系幹細胞を遊走する活性物質を抽出することに成功した。
目的:マウス脳欠損モデルを作成し、局所損傷部位に皮膚抽出液ヘパリンカラム精製画分を徐放化して投与することで、自己の骨髄系に含まれる幹細胞を局所損傷部位に遊走させ、神経系細胞の再生を誘導できないか検討する。
方法:
(1) 皮膚抽出液ヘパリンカラム精製画分の作製
切除した新生マウス皮膚からPBS(一匹/ml)中に4℃で16時間インキュベーションし抽出した皮膚抽出液を4℃の9倍容20 mM リン酸バッファー pH 7.5を用い 10倍に希釈した。あらかじめ、20 mM リン酸バッファー pH 7.5(30ml)をHiTrap Hepalin HP column(カラム容量: 5ml、GE Healthcare)の中に流しカラムを平衡化した。さらに、希釈液をカラムに結合させた。その後20 mM リン酸バッファー pH 7.5, 100mM NaCl(30ml)でカラムを洗浄した。吸着したタンパクを溶出するため20 mM リン酸バッファー pH 7.5, 1000mM NaClをカラム内に流入し、溶出液をチューブに分画した。吸着画分をそれぞれ、マウス骨髄由来細胞株の遊走活性を上記に示したボイデン・チャンバー法を用いて評価し遊走能を有する画分を集めた。この活性を有する溶液を皮膚抽出液ヘパリン精製画分として以下の実験に使用した。
マウスに10GyのX線単回照射を行い、骨髄抑制マウスを作成した。
GFPマウスの両側大腿骨および下腿骨より骨髄細胞を採取した。これを照射後24時間経過した骨髄抑制マウスの尾静脈より投与した。なお、投与はイソフルランによる吸入麻酔下に施行した。
GFPマウスの骨髄細胞を移植した骨髄抑制マウスにイソフルランにて吸入麻酔を行い、ペントバルビタール(45mg/kg)を腹腔内に注入した。マウスを脳定位固定装置に固定し、メスにて頭部に正中切開を加えた。ブレグマから右外側2.5mm、前方1mmにドリルを用いて穿頭を施した(図27A)。この部位から深さ3mmの位置を先端にして、20Gサーフロー針の外筒を挿入して固定した。ここでシリンジを用いて、陰圧をかけ、脳組織を一部吸引した(図27B)。
前述の位置に、ハミルトンシリンジと26Gシリンジを用いて、フィブリン糊製剤のフィブリノゲン(ボルヒール(化血研))に溶解した皮膚抽出液ヘパリンカラム精製画分を5μl注入し、次にフィブリン糊製剤のトロンビン(ボルヒール(化血研))を5μl注入した(図27C)。この操作によって、皮膚抽出液ヘパリンカラム精製画分の徐放剤としての効果を狙った。
コントロール群と治療群のマウスとを用いて評価した。適切な経過設定を決め(経時的に)、マウスを4%パラホルムアルデヒドにて灌流固定後、脳の切り出しを行った。さらに、4%パラホルムアルデヒドを外固定した。15%と30%の勾配をつけたショ糖にて脱水後、凍結切片を作成した。
DAPI(4',6-Diamidino-2-phenylindole, dihydrochloride)solusionにて核染色を行い、退光防止剤を用いて封入した。共焦点レーザー顕微鏡にて損傷部位(脳組織欠損部)でのGFP陽性細胞の集積を評価した。
結果:投与後、2週間および6週間後のGFP陽性細胞集積を定性的に示す。2週間後(コントロール;図27D, 皮膚抽出液ヘパリンカラム精製画分;図27E)および6週間後(コントロール;図27F 皮膚抽出液ヘパリンカラム精製画分;図27G)ともに、コントロール群に比して治療群の損傷部位にGFP陽性細胞の集積が高い傾向にあった。
考察:皮膚抽出液ヘパリンカラム精製画分投与により、骨髄由来細胞が脳組織欠損部位に集積し神経細胞の形態を示した。骨髄由来間葉系幹細胞は神経細胞にも分化することが知れており、本結果から、皮膚抽出液ヘパリンカラム精製画分によって脳損傷部位における神経系細胞再生の誘導できることが明らかになった。また、これは、脳虚血性疾患や脳挫傷における脳組織障害部位での神経再生にも応用可能である。
目的:皮膚組織抽出液内に存在する骨髄由来組織幹細胞誘導因子による骨髄組織幹細胞の末梢血への動員
方法:上記の目的に対して以下の方法により研究を行った。
1)骨髄由来組織幹細胞誘導剤の調製:新生マウス(2日齢)25匹から得た遊離皮膚片を生理的リン酸緩衝液pH 7.4(PBS)25 mlに浸し、4℃で24時間インキュベーションした後、組織を取り除くために、4℃の条件下で10分間、440 Gで遠心し上清を回収して皮膚抽出液(SE)を作製した。
また、C57/Bl6新生マウス皮膚からTrizol (invitrogen) を用いてRNA を抽出し更にSuperScript III cDNA synthesis kit(Invitrogen) を用いてcDNA を合成した。このcDNA をテンプレートとしてHMGB1のcDNAをPCR (ポリメラーゼ連鎖反応)法を用いて増幅し、アミノ酸配列のN末端にFlag tagの配列(Asp-Tyr-Lys-Asp-Asp-Asp-Lys;配列番号:30)を付加したタンパク質を発現するように、哺乳類細胞でタンパク質を発現させるプラスミドベクターpCAGGSに挿入した(図20)。これらのプラスミドベクターをHEK293 (ヒト胎児腎細胞由来培養細胞株)に遺伝子導入し48時間培養しタンパク質を発現させた。HMGB1タンパク質をそれぞれ発現させた細胞及び培養上清は4℃で16時間インキュベートした後、4400 g・5分間遠心し上清を回収した。この上清50 mL あたり100μLのAnti Flag 抗体Gel(Sigma)を混合し4℃で16時間インキュベートした。遠心しGelを回収した後PBSを用いて、5回洗浄した。更に 3X Flag peptide(final 100μg/ml)を用いて溶出した。溶出したタンパク質はHMGB1 ELISA kit (シノテスト)を用いて濃度を確認し、凍結乾燥後PBSを用いて200μg/mLに調整した。
8週齢雄マウス(C57/Bl6)の尾静脈からマウスHMGB1を250μL(1μg/μL)もしくは陰性コントロール群としてPBS250μLを30G1/2の注射針を装着したシリンジを用い投与した(図29)。投与12時間後、イソフルランによる吸入麻酔下、マウスの心臓からヘパリンコートした1 mLのシリンジを用いて末梢血1 mLを採血し、3 mLのPBSと混合した後、3 mLのFicol(GE healthcare)の上に静かに重層した。遠心機を用い、25℃で400 g、40分間遠心した。中間層の白濁した層の細胞を単核球画分として回収した。回収した細胞に1 mLの溶血剤であるHLB solution(免疫生物研究所)を加え室温で5分インキュベートした。この溶血操作を2回繰り返した。10 mLのPBSを加え、25℃で440 g、5分間遠心し上清を除去して細胞を回収した。この細胞1,000,000個に抗マウスPE標識PDGFRα抗体(e-Bioscience)、PerCy5標識抗マウスCD44抗体(BD biosciences)それぞれをPBSで100倍希釈し20分間室温でインキュベートした。その後この細胞を25℃、440 g、5分間遠心し上清を除去した。1%パラホルムアルデヒド含PBSを400μL加え、フローサイトメトリー解析のサンプルとした。
目的:組み換えHMGB1蛋白の静脈内投与によって、間葉系幹細胞が末梢血中へ動員されるかを確認した。
方法:C57BL6マウス(8~10週齢、オス)尾静脈より組み換えHMGB1蛋白/生理食塩水(100 μg/ml)を400μl (40μg HMGB1)あるいは生理食塩水400μl投与した。12時間後にマウス末梢血を採取してPBSを加え全量を4mLに希釈した。遠心管にFicoll-Paque Plus(GE)液を3mL 挿入後その上に希釈血液を重層した。400G (18℃)で40分間遠心し、単核球を含む中間層を新しい遠心管に回収し、45 mL のPBSを加え800G(18℃)5分で遠心し上清を除去した。さらにもう一度45 mL のPBSを加え800G(18℃)5分で遠心し上清を除去した。得られた単核球をPhycoerythrobilin(PE)標識抗マウスPDGFRα抗体およびFluorescein isothiocyanate(FITC)標識抗マウスCD44抗体で反応させた後、フローサイトメトリー(Facscan ; Becton, Dickinson and Company)により単核球分画内のPDGFRα陽性/CD44陽性細胞の存在頻度を評価した。
目的:生体移植皮膚組織の機能的再生時における骨髄由来細胞寄与の評価
方法:上記の目的に対して以下の方法により研究を行った。
1)GFP骨髄移植マウスに対する生体皮膚移植系を利用して、移植皮膚片の機能的再生時の骨髄由来細胞寄与程度を検討した。具体的には、C57BL/6雄マウス(6~8週齢)に致死量放射線(10Gy)を照射し、その直後に尾静脈よりGFP(green fluorescent protein)トランスジェニックマウス由来骨髄細胞(5x106個/0.1ml 生理的リン酸緩衝溶液pH7.4)を移植した。
2)移植骨髄細胞の生着を待って(6週間)、得られたGFP骨髄移植マウスの背部皮膚に、新生マウス皮膚(雌)を移植した。
3)移植皮膚片の生着と十分な皮膚組織再生を待って(4週間)、移植皮膚片領域におけるGFP蛍光の集積程度を、蛍光実体顕微鏡を利用して観察した。
4)吸入麻酔下に移植皮膚片を生検により採取し、冷却装置付ミクロトームを用いて皮膚凍結切片(6μm)を作製、4%パラホルムアルデヒド固定(30分間)、組織内細胞核をDAPIで染色、蛍光退色防止剤入り封入材を用いて組織を封入した後共焦点レーザー顕微鏡によりGFP陽性骨髄由来細胞の存在を検討した。
骨髄内には造血系幹細胞と間葉系幹細胞の二つの幹細胞システムが存在することが報告されている。今回の研究で示された、移植皮膚内に大量動員された骨髄由来上皮系細胞および間葉系細胞が骨髄由来造血系幹細胞から供給されていると予想するのは困難であり、移植組織の機能的再生には骨髄由来間葉系幹細胞が寄与している可能性が強く示唆される。即ち、植皮直後に疎血・壊死方向にある移植皮膚組織から骨髄由来間葉系幹細胞動員因子が放出され、骨髄から間葉系幹細胞を、末梢血液循環を介して移植皮膚片内に動員し、機能的皮膚組織再生を誘導していることが予想された。
目的:皮膚組織抽出液内に存在する骨髄由来組織幹細胞誘導因子の同定
方法:疎血状態にある切除皮膚からの放出が予想される骨髄間葉系幹細胞動員因子の同定を目的として以下の方法のように研究を進めた。
1)マウス骨髄由来間葉系幹細胞を得るために、C57BL/6マウスの骨髄細胞を大腿骨もしくは下腿の骨から採取し、10%胎仔ウシ血清含D-MEM(Nacalai 社製)を細胞培養培地として細胞培養皿に撒き、37℃、炭酸ガス濃度5%の条件下で培養した。細胞が占める面積が培養皿底面積に対して70から100%に増殖した時点で、0.25%トリプシン1mMEDTA(Nacalai社製)を用いて細胞を培養皿からはがし、さらに同じ条件で継代培養した。継代作業は少なくとも5回以上繰り返した。さらにこれらの付着細胞を単離培養しフローサイトメトリーによる細胞表面抗原の分析を行いLin陰性、CD45陰性、CD44陽性、Sca-1陽性、c-kit陰性であることを確認した。これらの細胞は骨細胞、脂肪細胞に分化可能で骨髄間葉系幹細胞の性質を有することを確認した。
2)新生マウス(2日齢)5匹から得た遊離皮膚片を生理的リン酸緩衝液pH7.4(PBS)5ml内に浸し、4℃で24時間インキュベーションした後、組織を取り除くために、4℃の条件下で10分間、440Gで遠心し上清を回収して皮膚抽出液を作製した。また同様に、6週齢マウス1匹から得た遊離皮膚片を生理的リン酸緩衝液pH7.4(PBS)5ml内に浸し、4℃で24時間インキュベーションした後、組織を取り除くために、4℃の条件下で10分間、440Gで遠心し上清を回収して皮膚抽出液を作製した。
3)得られた皮膚抽出液の中に骨髄間葉系幹細胞誘導活性が存在することを確認するため、ボイデン・チャンバーを用い、本発明者らが既に株化しているC57BL6マウス骨髄由来間葉系幹細胞に対する遊走活性を検討した。具体的にはボイデン・チャンバーの下槽(容量25μl)に2日齢もしくは6週齢のマウス皮膚抽出液(5μl)とDMEM(20μl)の混合液を挿入し、8μmの微細穴を持つポリカーボネートメンブレンを乗せ、さらにこれに接してボイデン・チャンバー上槽(容量50μl)を載せて、その中に骨髄由来間葉系幹細胞浮遊液(5x104個/50ml培養液:DMEM/10%ウシ胎仔血清)を入れ、CO2インキュベーター内で37℃、4~24時間培養した。培養後、チャンバーの上槽をはずし、シリコン薄膜を取り出して、その微細穴を通過してチャンバー下槽に遊走した骨髄由来間葉系幹細胞の数を染色により定量的に検討した(図34)。
分画したサンプルをそれぞれ、上記と同様にボイデン・チャンバーを利用した遊走活性を評価した(図37)。
分画したサンプルをそれぞれ、上記と同様にWestern blot法を用いてS100A8とS100A9のタンパク質の存在を検出した(図38)。
Claims (7)
- 以下の(a)から(q)のいずれかに記載の物質を含有する組織再生促進剤であって、再生が必要な組織とは異なる組織に投与される薬剤;
(a)HMGB1タンパク質
(b)HMGB1タンパク質を分泌する細胞
(c)HMGB1タンパク質をコードするDNAが挿入されたベクター
(d)HMGB2タンパク質
(e)HMGB2タンパク質を分泌する細胞
(f)HMGB2タンパク質をコードするDNAが挿入されたベクター
(g)HMGB3タンパク質
(h)HMGB3タンパク質を分泌する細胞
(i)HMGB3タンパク質をコードするDNAが挿入されたベクター
(j)S100A8タンパク質
(k)S100A8タンパク質を分泌する細胞
(l)S100A8タンパク質をコードするDNAが挿入されたベクター
(m)S100A9タンパク質
(n)S100A9タンパク質を分泌する細胞
(o)S100A9タンパク質をコードするDNAが挿入されたベクター
(p)細胞又は組織の抽出液
(q)細胞又は組織の抽出液のヘパリン結合画分。 - 非経口投与される、請求項1記載の薬剤。
- 注射投与である、請求項2記載の薬剤。
- 血管内、筋肉内、皮下、皮内又は腹腔内に投与される、請求項1記載の薬剤。
- 細胞又は組織の抽出液が細胞又は組織を溶媒に浸す工程を含む方法で製造される、請求項1~4のいずれかに記載の薬剤。
- 細胞又は組織の抽出液のヘパリン結合画分が以下の工程を含む方法で製造される、請求項1~4のいずれかに記載の薬剤;
(a)細胞又は組織を溶媒に浸す工程、
(b)工程(a)で得られる抽出液を固定化ヘパリンに接触させる工程、および
(c)固定化ヘパリンからヘパリン結合画分を溶出する工程。 - 神経組織、骨組織、又は皮膚組織の再生促進に用いられる、請求項1~6のいずれかに記載の薬剤。
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JP2016034964A (ja) | 2016-03-17 |
CN102711777A (zh) | 2012-10-03 |
AU2010312537A2 (en) | 2012-08-23 |
CA2778759A1 (en) | 2011-05-05 |
JP2017137350A (ja) | 2017-08-10 |
US20120251510A1 (en) | 2012-10-04 |
CN102711777B (zh) | 2015-04-15 |
US20220047642A1 (en) | 2022-02-17 |
EP2494977A1 (en) | 2012-09-05 |
BR112012009973A2 (pt) | 2017-07-25 |
CN104825491A (zh) | 2015-08-12 |
JPWO2011052668A1 (ja) | 2013-03-21 |
KR20120135190A (ko) | 2012-12-12 |
HK1212626A1 (zh) | 2016-06-17 |
RU2599448C2 (ru) | 2016-10-10 |
RU2012121811A (ru) | 2013-12-10 |
JP5865703B2 (ja) | 2016-02-17 |
RU2016135937A3 (ja) | 2018-12-11 |
EP2494977A4 (en) | 2013-06-05 |
US11191786B2 (en) | 2021-12-07 |
JP6199936B2 (ja) | 2017-09-20 |
RU2016135937A (ru) | 2018-12-11 |
AU2015213373A1 (en) | 2015-09-03 |
EP2494977B1 (en) | 2018-06-13 |
AU2010312537A1 (en) | 2012-05-17 |
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