WO2009133940A1 - 損傷組織の機能的再生促進医薬 - Google Patents
損傷組織の機能的再生促進医薬 Download PDFInfo
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Definitions
- the present invention relates to a pharmaceutical for promoting functional regeneration of damaged tissue.
- Stem cells that can be mobilized to the damaged site include tissue stem cells that are present in the damaged part or in the vicinity thereof, and bone marrow-derived stem cells that are present in peripheral blood.
- tissue stem cells that are present in the damaged part or in the vicinity thereof
- bone marrow-derived stem cells that are present in peripheral blood.
- Bone marrow-derived cells here are distinguished from hematopoietic stem cells capable of differentiating into blood cells (leukocytes, erythrocytes), and are present in stem cells or bone marrow represented by cells that have been called bone marrow mesenchymal stem cells.
- a tissue progenitor cell population A tissue progenitor cell population.
- Bone marrow mesenchymal stem cells are undifferentiated stem cells capable of differentiating into osteoblasts, adipocytes, and chondrocytes, and further differentiate into other mesenchymal cells such as fibroblasts, muscle cells, stromal cells, and tendon cells. It is possible to do. In recent years, it has been proved that bone marrow mesenchymal stem cells are differentiated into nerve cells, epithelial cells (skin keratinocytes, etc.) and vascular endothelial cells (Non-patent Document 1).
- 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 S100 protein family consists of approximately 20 types of proteins, and is mainly present in the cytoplasm, but part of it is secreted extracellularly and is known to play various functions during tissue damage, skin cancer, and inflammation. ing. It also plays an important role in the differentiation of epidermal cells in the skin.
- S100A8 and S100A9 are known to be strongly induced after about 1 week of skin injury (Non-patent Document 2).
- S100A8, S100A9, and S100A8 / A9 heterodimer have an activity of inducing neutrophil migration to the inflammatory site (Non-patent Document 4).
- central nerve cells in the brain and spinal cord have not been regenerated once damaged.
- a neural stem cell niche in the normal nervous system has also been identified. Therefore, recovery of damaged central nervous system cells, which was no longer possible, can be expected.
- the main causes of brain tissue (cell) damage are cerebral contusion caused by trauma and cerebral ischemic disease.
- Other causes include those resulting from brain surgery such as brain tumor removal surgery. In particular, it is difficult to remove all gliomas that arise from brain parenchymal cells, and in order to avoid motor and language dysfunction, it is necessary to stay at palliative removal.
- malignant glioma has a poor prognosis, and immunization and gene therapy, which has been actively studied in recent years, including chemotherapy and radiotherapy, have not yet been sufficiently effective. Therefore, it is ideal if there is a treatment that removes as many tumor cells as possible and recovers the resulting brain function damage.
- An object of the present invention is to isolate and identify a factor that mobilizes a cell that differentiates into damaged tissue to a damaged site, and to provide an invention using the factor.
- the mobilization factor of cells that differentiate into damaged tissues is clarified, cells that differentiate into damaged tissues existing in peripheral blood or cells that differentiate into damaged tissues in local tissues can be administered by administering this factor into the body. It becomes possible to mobilize a large number of damaged sites and develop new regenerative medicine that promotes functional tissue regeneration.
- the present inventors examined the possibility that bone marrow-derived cells were mobilized from an extra-skin tissue to the transplanted skin piece during the process of engrafting the transplanted skin piece in the living tissue, and 1) bone marrow.
- Derived cells are mobilized in large quantities in the transplanted skin
- mobilized bone marrow-derived cells are any of dermal fibroblasts, adipocytes, muscle cells, vascular endothelial cells, epidermal keratinocytes in the transplanted skin piece
- the mesenchymal stem cells derived from bone marrow are included in the mobilized bone marrow-derived cells
- the bone marrow-derived mesenchymal stem cells are mobilized from peripheral blood to the skin graft.
- An agent for inducing bone marrow cells comprising the component according to any one of (a) to (f) below: (A) S100A8 protein (b) Cell that secretes S100A8 protein (c) Vector inserted with DNA encoding S100A8 protein (d) S100A9 protein (e) Cell that secretes S100A9 protein (f) Encodes S100A9 protein A vector into which DNA has been inserted.
- a method for inducing bone marrow cells in a damaged tissue comprising a step of administering the substance described in any of (a) to (f) below to a subject with damaged tissue; (A) S100A8 protein (b) Cell that secretes S100A8 protein (c) Vector inserted with DNA encoding S100A8 protein (d) S100A9 protein (e) Cell that secretes S100A9 protein (f) Encodes S100A9 protein A vector into which DNA has been inserted.
- a method of promoting regeneration of damaged tissue comprising a step of administering the substance described in any of (a) to (f) below to a subject with damaged tissue; (A) S100A8 protein (b) Cell that secretes S100A8 protein (c) Vector inserted with DNA encoding S100A8 protein (d) S100A9 protein (e) Cell that secretes S100A9 protein (f) Encodes S100A9 protein A vector into which DNA has been inserted.
- FIG. 1 It is a figure which shows the GFP bone marrow transplantation 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 figure which shows the regeneration induction factor extraction method from an excised skin piece.
- A is a diagram in which GST-S100A8 and GST-S100A9 were administered from the tail vein of mice and CD44, PDGFR ⁇ , and PDGFR ⁇ FACS were performed on CD45-negative cell fractions in peripheral blood 12 hours later.
- 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 figure which shows the skin ulcer therapeutic effect of S100A8 in a normal mouse. It is a figure which shows the skin ulcer treatment effect of S100A8 in a diabetic mouse.
- the present invention provides an agent for inducing bone marrow cells, which contains at least one component described in the following (a) to (f).
- (A) S100A8 protein (b) Cell that secretes S100A8 protein (c) Vector inserted with DNA encoding S100A8 protein (d) S100A9 protein (e) Cell that secretes S100A9 protein (f) Encodes S100A9 protein A vector into which DNA has been inserted.
- the inducer it becomes possible to induce bone marrow-derived cells locally (sites where the above-mentioned components are administered / added or in the vicinity thereof, or damaged sites) to promote functional regeneration of the tissue.
- the inducer can be used as a reagent necessary for regenerative medicine, basic research for regenerative medicine development and clinical research.
- bone marrow-derived cells can be mobilized to necessary living tissues in experimental animals, and the degree of tissue repair and tissue function reconstruction can be examined.
- tissue regeneration induction studies by mobilization of bone marrow-derived cells can be performed in vitro.
- regeneration of damaged tissue can be accelerated
- the inducer is not only used as a functional tissue regeneration inducer / promoter, but also used as a preventive medicine for preventing a decrease in tissue / organ function due to a decrease in tissue stem cells, or an aging change. It can be expected to be used as an anti-aging drug that delays the progression of cancer.
- the inducer of the present invention includes a drug used for mobilizing bone marrow cells from bone marrow to peripheral blood, containing the components described in any of (a) to (f) below.
- A S100A8 protein
- b Cell that secretes S100A8 protein
- c Vector inserted with DNA encoding S100A8 protein
- d S100A9 protein
- e Cell that secretes S100A9 protein
- f Encodes S100A9 protein A vector into which DNA has been inserted.
- the drug is administered to blood vessels or muscles.
- Agents used to mobilize bone marrow cells from bone marrow to peripheral blood are characterized by mobilizing bone marrow cells from bone marrow to peripheral blood when administered to blood vessels or muscles.
- Bone marrow cells mobilized in the peripheral blood are induced into the damaged tissue from the peripheral blood. Therefore, by using the drug, it becomes possible to induce bone marrow cells at the damaged site and promote functional regeneration of the tissue. Therefore, the drug is used for basic research for development of regenerative medicine and regeneration-guided medicine. And can be used as a reagent necessary for clinical research. Moreover, regeneration of damaged tissue can be promoted by using the above-mentioned drug.
- the above drugs induce bone marrow-derived cells at the site of injury by being administered to blood vessels or muscles. Therefore, it is difficult to administer the drug directly from the outside. It becomes possible to promote regeneration.
- the present invention also provides a tissue regeneration promoter or a tissue regeneration promotion kit containing at least one of the components described in the following (a) to (f).
- (A) S100A8 protein (b) Cell that secretes S100A8 protein (c) Vector inserted with DNA encoding S100A8 protein (d) S100A9 protein (e) Cell that secretes S100A9 protein (f) Encodes S100A9 protein A vector into which DNA has been inserted.
- the tissue regeneration-promoting agent or tissue regeneration-promoting kit is administered to a damaged tissue site or the vicinity thereof, blood vessels, or muscles, so that bone marrow-derived cells circulating in the blood are removed from peripheral blood from the damaged tissue. It is characterized in that it is induced (locally induced).
- the tissue regeneration promotion kit includes (1) the above component dissolved in fibrinogen, and (2) a tissue regeneration promotion kit containing thrombin, or (1) the above component, (2) fibrinogen, and (3) thrombin.
- a tissue regeneration promotion kit containing 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 tissue to be regenerated is not particularly limited, and may be any tissue as long as it is damaged, for example, living skin tissue, biopsy (surgical) tissue (brain, lung, heart, liver, Examples include stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testis and blood.
- 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 the damaged tissue differentiate into various cells and contribute to functional regeneration, functional maintenance, and functional enhancement of the damaged tissue.
- the damaged tissue include tissues damaged by various pathological conditions causing ischemia, ischemia / hypoxia, trauma, burn, inflammation, autoimmunity, gene abnormality, etc., but are not limited to these causes. It is not something. Injured tissues also include necrotic tissues.
- 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 in vivo tissues such as gastrointestinal tissue, liver / bile / pancreatic tissue, urinary / genital organs, and the like.
- tissue regeneration promoter not only skin diseases such as refractory skin ulcers, skin wounds, blistering, and alopecia, but also tissues such as cerebral infarction, myocardial infarction, fracture, lung infarction, gastric ulcer, enteritis, etc. Injuries allow treatments that induce functional tissue regeneration.
- tissue regeneration promoter examples include humans and non-human animals, and examples include humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, and the like. It is not limited.
- the drug of the present invention can be administered to diabetic patients. It is known that intractable skin ulcers, which are skin complications in diabetes, are more difficult to heal than normal human skin ulcers, but the drug of the present invention is also effectively used for such diabetic patients. Is done.
- the bone marrow cells of the present invention are hematopoietic stem cells and cells other than leukocytes, erythrocytes, and platelets derived therefrom, and so far called bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, or bone marrow pluripotent stem cells.
- Stem cell typified by living cells or tissue progenitor cell populations present in the bone marrow.
- the bone marrow cells of the present invention are cells that can be isolated by bone marrow collection (bone marrow cell collection) or peripheral blood collection.
- the hematopoietic stem cells are non-adherent cells, and the bone marrow cells of the present invention are obtained as adherent cells by bone marrow collection (bone marrow cell collection) and mononuclear cell fractionation in blood obtained by peripheral blood collection.
- the bone marrow cells of the present invention include mesenchymal stem cells, such as osteoblasts (which can be identified by the recognition of calcium deposition when differentiation is induced), chondrocytes (positive for Alcian blue staining, positive for safranin-O staining, etc.) Identifiable), adipocytes (identified with positive Zudan III staining), mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, tendon cells, and also neurons, epithelial cells (eg epidermis angle) Cells and intestinal epithelial cells express the cytokeratin family), and preferably have the ability to differentiate into vascular endothelial cells, but the differentiated cells are not limited to the above cells, such as liver, kidney, pancreas, etc.
- mesenchymal stem cells such as osteoblasts (which can be identified by the recognition of calcium deposition when differentiation is induced), chondrocytes (positive for Alcian blue staining, positive
- bone marrow-derived mesenchymal stem cells are cells existing in the bone marrow and directly from the bone marrow or other tissues (blood, skin, fat , Other tissues) indirectly, and can be cultured and propagated as adherent cells (made of plastic or glass) to culture dishes, such as bone, cartilage, fat and other mesenchymal tissues (mesenchymal stem cells), Alternatively, it is a cell having the ability to differentiate into skeletal muscle, myocardium, nerve tissue, epithelial tissue (pluripotent stem cells), bone marrow blood collection, peripheral blood collection, and mesenchymal tissue such as fat, skin It can be obtained by collecting from epithelial tissues such as brain and nerve tissues such as brain.
- bone marrow-derived mesenchymal stem cells or bone marrow-derived pluripotent stem cells or bone marrow pluripotent stem cells are administered once to cells that have been attached to a culture dish to the damaged part of the living body, for example, epithelial systems such as keratinocytes that constitute the skin. It also has the feature of having the ability to differentiate into tissues of the nervous system that constitute the tissue and brain.
- Bone marrow mesenchymal stem cells or bone marrow stromal pluripotent stem cells or bone marrow pluripotent stem cells of the present invention can be identified as osteoblasts (which can be identified by the presence of calcium deposition when differentiation is induced), chondrocytes (Alcian blue) In addition to staining positive, safranin-O staining positive, etc.), adipocytes (identified by Zudan III staining positive, etc.), for example, fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, tendon cells, etc.
- Mesenchymal cells neurons, pigment cells, epidermal cells, hair follicle cells (expressing cytokeratin family, hair keratin family, etc.), epithelial cells (eg epidermal keratinocytes, intestinal epithelial cells, cytokeratin family, etc.) Expressed), endothelial cells, and preferably have the ability to differentiate into parenchymal organ cells such as liver, kidney, pancreas, etc., but the differentiated cells are limited to the above cells is not.
- human bone marrow mesenchymal stem cells or bone marrow stromal pluripotent stem cells or bone marrow pluripotent stem cells are collected from bone marrow (bone marrow cells), peripheral blood, and fat, and cultured directly or after separation of mononuclear cells. Examples of cells that can be obtained as adherent cells are not limited thereto. Examples of markers of human bone marrow mesenchymal stem cells or bone marrow stromal pluripotent stem cells or bone marrow pluripotent stem cells can include all or part of Lin negative, CD45 negative, CD44 positive, but are limited to these is not.
- mouse bone marrow mesenchymal stem cells examples include, but are not limited to, cells that can be obtained by the methods described in the Examples.
- markers for mouse bone marrow mesenchymal stem cells or bone marrow stromal pluripotent stem cells or bone marrow pluripotent stem cells CD44 positive, PDGFR ⁇ positive, PDGFR ⁇ positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, Although all or a part of can be illustrated, it is not limited to these.
- 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 components other than at least one of the components described in (a) to (f) above can induce bone marrow-derived cells and promote tissue regeneration.
- the tissue regeneration-promoting agent of the present invention includes related molecules (groups) that enhance the functional tissue regeneration-inducing function of S100A8 or S100A9 in addition to at least one of the components described in (a) to (f) above. ), Molecules (group) that suppress actions other than the expected effects of S100A8 or S100A9, factors that control the growth and differentiation of bone marrow-derived cells, and other factors that enhance or maintain the function of the cells Is possible.
- Examples of the animal species that is the source of the S100A8 or S100A9 protein in the inducer or tissue regeneration promoter of the present invention include humans and non-human animals, such as humans, mice, rats, monkeys, pigs, dogs, rabbits, and hamsters. Guinea pigs and the like can be exemplified, but the animal species is preferably the same as the animal species to which the above components are administered.
- S100A8 protein in the inducer or tissue regeneration promoter of 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 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: 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.
- S100A9 protein in the inducer or tissue regeneration promoter of the present invention include, but are not limited to, a protein containing the amino acid sequence set forth in SEQ ID NO: 7, 9, or 11.
- 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: 7, 9 or 11.
- 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: 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 An isolated protein encoded by DNA that hybridizes under conditions, which is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 7, 9, or 11. An isolated protein that is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 is represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11.
- a protein functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9 or 11 is prepared by a method known by those skilled in the art (Experimental Medicine Separate Volume, Genetic Engineering Handbook, pp246-251, It can be isolated at Yodosha (published in 1991).
- Examples of the protein functionally equivalent to the protein containing the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 include a protein having an activity of inducing bone marrow-derived cells.
- a protein functionally equivalent to a protein comprising the amino acid sequence described in 5, 7, 9 or 11 includes a naturally occurring protein.
- 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.
- an artificially prepared mutant protein is also included in the present invention as long as the protein is functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11.
- substitution of base pairs by nitrite treatment of DNA is known (Hirose, S. et al., Proc. Natl. Acad. Sci. USA., 79: 7258-7260, 1982).
- base pair substitution can be randomly introduced into a specific segment by treating the segment into which a mutation is to be introduced with nitrous acid.
- there is a gapped duplex method or the like as a technique for bringing a target mutation to an arbitrary place Kramer W.
- 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.
- 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 the 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 to which a conservative substitution is added in the above amino acid substitution, and is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9 or 11 Protein. 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 permanently activated protein).
- non-conservative substitution for example, including a permanently activated protein.
- a method using hybridization can be mentioned. That is, a DNA encoding the S100A8 or S100A9 protein according to the present invention as shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12 or a fragment thereof is used as a probe, and a DNA that can hybridize with this DNA is simply used. Release. If hybridization is performed under stringent conditions, DNA with high homology is selected as the base sequence, and the protein isolated as a result may include proteins functionally equivalent to the S100A8 or S100A9 protein. Increases nature.
- 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, or temperature, but those skilled in the art will readily set these conditions to obtain the required stringency.
- hybridization for example, DNA encoding a homologue of S100A8 or S100A9 protein other than the protein containing the amino acid sequence described in SEQ ID NO: 1, 3, 5, 7, 9, or 11 can be isolated. .
- a protein functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 is usually described in SEQ ID NO: 1, 3, 5, 7, 9, or 11.
- 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 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)]
- NCBI National Center for Biotechnology Information
- 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).
- 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.
- various modifications such as physiological modifications such as sugar chains, labels such as fluorescence and radioactive substances, or fusion with other proteins are added.
- any of the S100A8 or S100A9 protein according to the present invention or functional Is a protein equivalent to
- the S100A8 or S100A9 protein can be obtained not only as a biomaterial but also as a gene recombinant by incorporating a gene encoding it into an appropriate expression system.
- the DNA encoding the S100A8 or S100A9 protein described above may be incorporated into an appropriate expression system for expression.
- 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 pGEX incorporating a gene encoding S100A8 or S100A9 protein is heat shocked.
- GST glutathione S-transferase
- IPTG isopropylthio- ⁇ -D-galactoside
- a host / vector system for obtaining “recombinant” of S100A8 or S100A9 protein.
- a fusion protein expression vector using a histidine tag, an HA tag, a FLAG tag or the like is commercially available.
- yeast it is known that yeasts of the genus Pichia are effective for the expression of proteins with sugar chains.
- expression systems using baculovirus vectors hosted by insect cells are also useful (Bio / Technology, 6: 47-55, 1988).
- mammalian cells are used to transfect vectors using promoters such as CMV, RSV, or SV40.
- host / vector systems are all S100A8 or S100A9 protein expression systems.
- 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 the S100A8 or S100A9 protein in the inducer or tissue regeneration promoter of 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 S100A8 or S100A9 protein may be DNA encoding S100A8 or S100A9 protein or DNA encoding S100A8 or S100A9 protein (ATG CAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG CTG A vector prepared by inserting a DNA combined with TGG GTT CCA GGT TCC ACT GGT GAC; SEQ ID NO: 15) into a known expression vector or gene therapy vector is used as a fibroblast (for example, normal skin fibroblast) It can also be produced by introducing it into mammalian cells such as cell lines derived therefrom and insect cells, and other cells.
- fibroblast for example, normal skin fibroblast
- Examples of the DNA encoding the secretion signal include, but are not limited to, DNAs having the above-described sequences.
- the animal species from which these cells are derived is not particularly limited, but the cells of the subject animal species in which tissue regeneration is performed, the cells of the subject themselves, or cells derived from the relatives of the subject in which tissue regeneration is performed are used. It is preferable.
- the DNA encoding the S100A8 or S100A9 protein in the inducer or tissue regeneration promoter of the present invention may be cDNA, genomic DNA, or natural DNA. Alternatively, it may be an artificially synthesized DNA.
- DNA encoding the S100A8 or S100A9 protein is usually contained in the inducer or tissue regeneration promoter of the present invention in a state of being inserted into a vector (for example, a gene therapy vector).
- Examples of the vector for gene therapy in the present invention include 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 limited to.
- the gene therapy vector may contain a promoter DNA sequence that effectively induces gene expression, a factor that controls gene expression, and a molecule necessary to maintain the stability of DNA.
- a peptide that is a partial peptide of the S100A8 or S100A9 protein and has an activity of inducing bone marrow-derived cells, a cell that secretes the partial peptide, or the It can also contain a vector into which a DNA encoding a partial peptide has been inserted.
- the administration method of the inducer or tissue regeneration promoter of the present invention examples include oral administration and parenteral administration. Specific examples of such administration methods include injection administration, nasal administration, pulmonary administration, and transdermal administration. Is mentioned.
- the inducer or tissue regeneration promoter of the present invention can be administered systemically (blood or muscle) or locally by intravascular injection (intraarterial injection, intravenous injection, etc.), intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. (For example, subcutaneous, intradermal, skin surface, eyeball or eyelid conjunctiva, nasal mucosa, oral and gastrointestinal mucosa, vaginal / intrauterine mucosa, or damaged site).
- 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.
- administering cells that secrete S100A8 or S100A9 protein or gene therapy vectors into which DNA encoding S100A8 or S100A9 protein is inserted it is administered so that the amount of S100A8 or S100A9 protein is within the above range in the damaged tissue. can do.
- the inducer and tissue regeneration promoter of the present invention are not limited to these doses.
- the inducer and tissue regeneration promoter of the present invention can be formulated according to conventional methods (for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing Company, Easton, USA), and pharmaceutically acceptable carriers and additives. May be included.
- 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.
- the use of a vector into which a DNA encoding a partial peptide is inserted can also be expressed as (1) to (5) below.
- An S100A8 or S100A9 protein a cell that secretes the protein, a vector into which a DNA encoding the protein is inserted, a partial peptide of the protein, a cell that secretes the partial peptide, or a DNA encoding the partial peptide
- a method of promoting regeneration of the damaged tissue comprising the step of administering the inserted vector to a subject with damaged tissue;
- S100A8 or S100A9 protein a cell that secretes the protein, a vector into which a DNA encoding the protein is inserted, a partial peptide of the protein
- Examples of the target in which the tissue is damaged include humans and non-human animals, and examples thereof include, but are not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, and guinea pigs. It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
- Example 1 Evaluation method of bone marrow-derived cell contribution during functional regeneration of living transplanted skin tissue: For the above objective, the following method was used for study. 1) Using a living skin transplantation system for GFP bone marrow transplanted mice, the degree of bone marrow-derived cell contribution during functional regeneration of transplanted skin pieces was examined.
- GFP green fluorescent protein
- C57BL / 6 male mice (6-8 weeks old) were irradiated with lethal dose radiation (10 Gy), and immediately after that, GFP (green fluorescent protein) transgenic bone marrow cells (5x10 6 cells) from the tail vein /0.1 ml physiological phosphate buffer solution pH 7.4) was implanted (FIG. 1).
- GFP green fluorescent protein
- the skin of new mouse (female) was transplanted to the back skin of the obtained GFP bone marrow transplanted mouse.
- the degree of accumulation of GFP fluorescence in the transplanted skin piece region was observed using a fluorescence stereomicroscope.
- 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. After the tissue was encapsulated using an encapsulant containing a fluorescent fading inhibitor, the presence of GFP-positive bone marrow-derived cells was examined using a confocal laser microscope.
- 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.
- Example 2 Identification method of bone marrow-derived tissue stem cell inducer present in skin tissue extract: To identify bone marrow mesenchymal stem cell mobilization factor that is expected to be released from resected skin in an anemic state I proceeded with the research. 1) In order to obtain mouse bone marrow-derived mesenchymal stem cells, bone marrow cells of C57BL / 6 mice were collected from the femur or lower leg bone, and 10% fetal bovine serum-containing D-MEM (manufactured by Nacalai) was used as the cell culture medium. As a cell culture dish and cultured under conditions of 37 ° C. and carbon dioxide concentration of 5%.
- 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 recovered, 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 recovered 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 4% skim milk-containing PBS, and shaken at room temperature for 60 minutes.
- a solution obtained by diluting 5 ⁇ l of anti-S100A8 antibody (R & D) or anti-S100A9 (R & D) in 10 mL of 4% skim milk-containing PBS, 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 in which 5 ⁇ l of HRP-labeled anti-goat IgG antibody (GE healthcare) was diluted 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. 5).
- 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).
- the 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
- 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% / solution B 0%, the ratio of solution B was gradually increased, and finally solution A was sent at 0% / solution B 100%.
- 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 using the PCR (polymerase chain reaction) method, and the GST tag sequence (SEQ ID NO: 13 (amino acid sequence), SEQ ID NO: 14 (DNA) was added to the N-terminus of the amino acid sequence. 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. 9).
- 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 after 48 hours, cells and culture supernatant were collected.
- 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 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 reduced glutathione-containing 20 mM phosphate buffer (pH.8). Bone marrow mesenchymal stem cell migration activity was studied using recombinant S100A8 and S100A9 Boyden chambers.
- 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. 11A and 11B).
- 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.
- Example 3 Objective Method for confirming the therapeutic effect of S100A8 on skin ulcer in normal mice and diabetic mice: Recombinant S100A8 protein was administered to a mouse skin ulcer model, and the therapeutic effect on ulcer was examined.
- the test mice used were C57 / Bl6 mice transplanted with bone marrow cells expressing GFP or BKS.Cg-m + / + Leprdb / J (db mice), which is a diabetes model mouse.
- a skin ulcer with a diameter of 6 mm was created in the mouse skin. In skin ulcers made in mice, the skin around the skin defect rapidly contracts.
- a silicon rubber disc with an outer diameter of 10 mm, an inner diameter of 6 mm, and a thickness of 0.5 mm was used for skin surgery in order to create a model to treat the defective skin by covering it with regenerated skin instead of contraction.
- the skin around the ulcer was fixed with an adhesive (Aron Alpha A) and nylon thread.
- recombinant S100A8 protein was directly administered to the ulcer surface for 1.5 days at 1.5 ⁇ g / day.
- the surface was protected with a film dressing agent Tegaderm (3M) to prevent drying of the ulcer surface. The ulcer area was measured over time to confirm the therapeutic effect.
- a bone marrow-derived cell inducer or tissue regeneration promoter containing S100A8 or S100A9 is provided.
- Development of S100A8 or S100A9 as a bone marrow-derived cell mobilization drug enables new regenerative medicine that promotes functional regeneration of intractable damaged tissue.
- S100A8 or S100A9 falls into hypoxia due to a decrease in blood flow, is released from tissue cells in a necrotic direction, and has an action of mobilizing bone marrow-derived cells or various cells derived therefrom in the local area.
- the mobilized bone marrow-derived cells are differentiated into the cell lineage required for each tissue, thereby promoting the recovery of the function lost due to anaemia and necrosis, so-called functional tissue regeneration.
- S100A8 or S100A9 For example, by administering S100A8 or S100A9 to an intractable skin ulcer caused by skin blood flow disorder, mobilize bone marrow-derived cells on the ulcer surface, and if the mobilized cells differentiate into vascular endothelial cells, local Blood flow is improved. At the same time, differentiation into fibroblasts, nerve cells, hair follicle cells, and even epidermis cells induces and promotes functional skin regeneration with the necessary skin appendages, not fibrotic scar healing. S100A8 and S100A9 can also be expected to be effective as functional tissue regeneration inducers in the ischemic necrosis of other organs such as myocardial infarction and cerebral infarction.
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Abstract
Description
脳組織(細胞)損傷の原因としては、外傷による脳挫傷、脳虚血性疾患が主要なものである。他の原因として、脳腫瘍摘出手術をはじめとした脳外科的手術に起因するものがあげられる。とくに、脳実質細胞から発生するような、神経膠腫の全摘出は困難であり、運動や言語機能障害を避けるためには、姑息的摘出にとどまらざるを得えない。また、悪性神経膠腫は生命予後も悪く、化学療法や放射線療法をはじめ、近年研究が盛んに行われている免疫・遺伝子治療も十分な効果を示すまでには至っていない。よって、可及的に多くの腫瘍細胞の摘出を行い、その結果生じる脳機能損傷を回復させるような治療があれば理想的である。
〔1〕以下の(a)から(f)のいずれかに記載の成分を含有する、骨髄細胞の誘導剤;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔2〕以下の(a)から(f)のいずれかに記載の成分を含有する、組織再生促進剤;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔3〕以下の(a)から(f)のいずれかに記載の物質を、組織が損傷した対象に投与する工程を含む、該損傷した組織に骨髄細胞を誘導する方法;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔4〕以下の(a)から(f)のいずれかに記載の物質を、組織が損傷した対象に投与する工程を含む、該損傷した組織の再生を促進する方法;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔5〕骨髄細胞の誘導剤の製造における以下の(a)から(f)のいずれかに記載の物質の使用;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔6〕組織再生促進剤の製造における以下の(a)から(f)のいずれかに記載の物質の使用;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔7〕損傷した組織に骨髄細胞を誘導する方法に使用するための、以下の(a)から(f)のいずれかに記載の物質;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
〔8〕損傷した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(f)のいずれかに記載の物質;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
該誘導剤を使用することにより、局所(上記成分の投与・添加部位やその近傍、または損傷部位)に骨髄由来細胞を誘導して組織の機能的再生を促進することが可能になるため、該誘導剤は、再生医療、再生誘導医療開発のための基礎研究および臨床研究に必要な試薬として使用することができる。例えば、実験動物における必要な生体組織に骨髄由来細胞を動員し、組織修復、組織機能再建の程度を検討することが可能になる。また試験管内で骨髄由来細胞の動員による組織再生誘導研究を行うことができる。
また、上記誘導剤を使用することにより、損傷組織の再生を促進することができる。また、上記誘導剤には、機能的組織再生誘導・促進剤としての用途のみならず、組織幹細胞の減少による組織・臓器機能の低下を予防する、いわゆる予防医薬としての用途、あるいは加齢性変化の進行を遅延させる、抗加齢医薬としての用途が期待できる。
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
上記薬剤は、血管または筋肉に投与される。
骨髄細胞を骨髄から末梢血に動員するために用いる薬剤は、血管または筋肉に投与されることで、骨髄細胞を骨髄から末梢血に動員することを特徴とする。末梢血中に動員された骨髄細胞は、末梢血中から損傷組織に誘導される。
したがって、該薬剤を使用することにより、損傷部位に骨髄細胞を誘導して組織の機能的再生を促進することが可能になるため、該薬剤は、再生医療、再生誘導医療開発のための基礎研究および臨床研究に必要な試薬として使用することができる。
また、上記薬剤を使用することにより、損傷組織の再生を促進することができる。特に、上記薬剤は血管または筋肉に投与されることにより損傷部位に骨髄由来細胞を誘導するため、外部から直接薬剤を投与することが難しい脳の組織損傷や心臓の組織損傷において、組織の機能的再生を促進することが可能になる。
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
該組織再生促進剤または組織再生促進用キットは、損傷組織部位若しくはその近傍、血管、または筋肉に投与されることで、血液中に循環している骨髄由来細胞を、末梢血中から該損傷組織に誘導(局所誘導する)することを特徴とする。
また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成分、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)上記成分、(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。
本発明において、骨髄由来間葉系幹細胞あるいは骨髄間質多能性細胞あるいは骨髄多能性幹細胞とは、骨髄内に存在する細胞であって、骨髄から直接あるいはその他の組織(血液や皮膚、脂肪、その他の組織)から間接的に採取され、(プラスチックあるいはガラス製)培養皿への付着細胞として培養・増殖可能であり、骨、軟骨、脂肪などの間葉系組織(間葉系幹細胞)、あるいは骨格筋、心筋、さらには神経組織、上皮組織(多能性幹細胞)への分化能を有するという特徴を持つ細胞であり、骨髄血採血、末梢血採血、さらには脂肪など間葉組織、皮膚などの上皮組織、脳などの神経組織からの採取によって取得することができる。また骨髄由来間葉系幹細胞あるいは骨髄由来多能性幹細胞あるいは骨髄多能性幹細胞は一度培養皿へ付着させた細胞を生体の損傷部に投与することにより、例えば皮膚を構成するケラチノサイトなどの上皮系組織、脳を構成する神経系の組織への分化能も有するという特徴も持つ。
また、ヒト骨髄間葉系幹細胞あるいは骨髄間質多能性幹細胞あるいは骨髄多能性幹細胞は骨髄採取(骨髄細胞採取)、末梢血採血、脂肪採取し、直接あるいは単核球分画を分離後に培養して付着細胞として取得することができる細胞が例示できるが、これに制限されるものではない。ヒト骨髄間葉系幹細胞あるいは骨髄間質多能性幹細胞あるいは骨髄多能性幹細胞のマーカーとしては、Lin陰性、CD45陰性、CD44陽性、の全部または一部が例示できるが、これらに制限されるものではない。
また、マウス骨髄間葉系幹細胞あるいは骨髄間質多能性幹細胞あるいは骨髄多能性幹細胞は、例えば、実施例に記載の方法によって取得できる細胞が例示できるが、これに制限されるものではない。マウス骨髄間葉系幹細胞あるいは骨髄間質多能性幹細胞あるいは骨髄多能性幹細胞のマーカーとしては、CD44陽性、PDGFRα陽性、PDGFRβ陽性、CD45陰性、Lin陰性、Sca-1陽性、c-kit陰性、の全部または一部が例示できるが、これらに制限されるものではない。
組織前駆細胞は、血液系以外の特定組織細胞への一方向性分化能を持つ未分化細胞と定義され、上述した間葉系組織、上皮系組織、神経組織、実質臓器、血管内皮への分化能を有する未分化細胞を含む。
本発明の誘導剤や組織再生促進剤におけるS100A8またはS100A9タンパク質の起源となる動物種としては、ヒト又は非ヒト動物が挙げられ、例えば、ヒト、マウス、ラット、サル、ブタ、イヌ、ウサギ、ハムスター、モルモットなどが例示できるが、上記成分が投与される動物種と同じ動物種であることが好ましい。
本発明の誘導剤や組織再生促進剤におけるS100A9タンパク質としては、配列番号:7、9または11に記載のアミノ酸配列を含むタンパク質が例示できるが、これらに限定されるものではない。本発明のS100A9タンパク質には、配列番号:7、9または11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も含まれる。そのようなタンパク質としては、例えば、1)配列番号:7、9または11に記載のアミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、挿入、および/または付加したアミノ酸配列からなり、配列番号:7、9または11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単離されたタンパク質、および、2)配列番号:8、10または12に記載の塩基配列を含むDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、配列番号:7、9または11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単離されたタンパク質が挙げられる。
配列番号:1、3、5、7、9または11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単離されたタンパク質は、配列番号:1、3、5、7、9または11に記載のアミノ酸配列を含むタンパク質のホモログあるいはパラログでありうる。配列番号:1、3、5、7、9または11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は、当業者によって公知の方法(実験医学別冊・遺伝子工学ハンドブック, pp246-251、羊土社、1991年発行)で単離することができる。
配列番号:1、3、5、7、9または11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、骨髄由来細胞の誘導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または11に記載のアミノ酸配列を含むタンパク質以外の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。
(1)S100A8またはS100A9タンパク質、該タンパク質を分泌する細胞、該タンパク質をコードするDNAが挿入されたベクター、該タンパク質の部分ペプチド、該部分ペプチドを分泌する細胞、または該部分ペプチドをコードするDNAが挿入されたベクターを、組織が損傷した対象に投与する工程を含む、該損傷した組織の再生を促進する方法、
(2)S100A8またはS100A9タンパク質、該タンパク質を分泌する細胞、該タンパク質をコードするDNAが挿入されたベクター、該タンパク質の部分ペプチド、該部分ペプチドを分泌する細胞、または該部分ペプチドをコードするDNAが挿入されたベクターを、組織が損傷した対象に投与する工程を含む、該損傷した組織に骨髄細胞を誘導する方法、
(3)骨髄細胞の誘導剤または組織再生促進剤の製造における、S100A8またはS100A9タンパク質、該タンパク質を分泌する細胞、該タンパク質をコードするDNAが挿入されたベクター、該タンパク質の部分ペプチド、該部分ペプチドを分泌する細胞、または該部分ペプチドをコードするDNAが挿入されたベクターの使用、
(4)損傷した組織に骨髄細胞を誘導する方法に使用するための、S100A8またはS100A9タンパク質、該タンパク質を分泌する細胞、該タンパク質をコードするDNAが挿入されたベクター、該タンパク質の部分ペプチド、該部分ペプチドを分泌する細胞、または該部分ペプチドをコードするDNAが挿入されたベクター、
(5)損傷した組織の再生を促進する方法に使用するための、S100A8またはS100A9タンパク質、該タンパク質を分泌する細胞、該タンパク質をコードするDNAが挿入されたベクター、該タンパク質の部分ペプチド、該部分ペプチドを分泌する細胞、または該部分ペプチドをコードするDNAが挿入されたベクター。
上記組織が損傷した対象としては、ヒト又は非ヒト動物が挙げられ、例えば、ヒト、マウス、ラット、サル、ブタ、イヌ、ウサギ、ハムスター、モルモットなどが例示できるが、これらに制限されない。
なお本明細書において引用されたすべての先行技術文献は、参照として本明細書に組み入れられる。
〔実施例1〕
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(図1)。
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4)。
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7)。
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8)。
目的:正常マウスおよび糖尿病マウスにおけるS100A8の皮膚潰瘍治療効果の確認
方法:マウス皮膚潰瘍モデルにリコンビナントS100A8タンパク質を投与し、潰瘍治療効果を検討した。被験マウスはGFPを発現する骨髄細胞を移植したC57/Bl6 マウスもしくは糖尿病モデルマウスであるBKS.Cg-m+/+Leprdb/J(dbマウス)を使用した。マウス皮膚に直径6mmの皮膚潰瘍を作成した。マウスに作製した皮膚潰瘍は皮膚欠損部分の周囲の皮膚が速やかに収縮する。本実験では、欠損皮膚を収縮ではなく再生皮膚で覆うことにより治療するモデルを作製するために、皮膚欠損部に外径10mm 、内径6mm 、厚さ0.5mm のシリコンゴム製の円盤を皮膚手術用接着剤(アロンアルファA)およびナイロン糸で潰瘍周囲の皮膚を固定した。さらにリコンビナントS100A8タンパク質を1.5μg /日、7日間連日潰瘍面に直接投与した。また潰瘍面の乾燥を防ぐため表面をフィルムドレッシング剤 Tegaderm(3M)で保護した。治療効果を確認するため経時的に潰瘍面積を測定した。
Claims (2)
- 以下の(a)から(f)のいずれかに記載の成分を含有する、骨髄細胞の誘導剤;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。 - 以下の(a)から(f)のいずれかに記載の成分を含有する、組織再生促進剤;
(a)S100A8タンパク質
(b)S100A8タンパク質を分泌する細胞
(c)S100A8タンパク質をコードするDNAが挿入されたベクター
(d)S100A9タンパク質
(e)S100A9タンパク質を分泌する細胞
(f)S100A9タンパク質をコードするDNAが挿入されたベクター。
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Also Published As
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JP5814549B2 (ja) | 2015-11-17 |
JPWO2009133940A1 (ja) | 2011-09-01 |
EP2301560A4 (en) | 2012-05-09 |
JP2016104790A (ja) | 2016-06-09 |
US20110097309A1 (en) | 2011-04-28 |
JP5960735B2 (ja) | 2016-08-02 |
RU2010148785A (ru) | 2012-06-10 |
JP2014139177A (ja) | 2014-07-31 |
AU2009240885A1 (en) | 2009-11-05 |
CN102076351A (zh) | 2011-05-25 |
CA2722906A1 (en) | 2009-11-05 |
KR20110027665A (ko) | 2011-03-16 |
EP2301560A1 (en) | 2011-03-30 |
AU2009240885A2 (en) | 2011-01-27 |
BRPI0911436A2 (pt) | 2016-07-12 |
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