WO2012172887A1 - Agent thérapeutique pour maladies cardiaques et feuillet cellulaire pour traiter les maladies cardiaques - Google Patents

Agent thérapeutique pour maladies cardiaques et feuillet cellulaire pour traiter les maladies cardiaques Download PDF

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WO2012172887A1
WO2012172887A1 PCT/JP2012/061898 JP2012061898W WO2012172887A1 WO 2012172887 A1 WO2012172887 A1 WO 2012172887A1 JP 2012061898 W JP2012061898 W JP 2012061898W WO 2012172887 A1 WO2012172887 A1 WO 2012172887A1
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peptide
amino acid
heart disease
acid sequence
cell sheet
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PCT/JP2012/061898
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English (en)
Japanese (ja)
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吉之輔 濱田
成昭 松浦
芳樹 澤
直正 河口
繁 宮川
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a heart disease therapeutic drug and a cell sheet for heart disease treatment.
  • Cardiovascular disease is one of the top causes of death in the world, and the number of patients is expected to increase in the future.
  • Heart failure a condition in which the heart's pumping function is compromised and cannot pump enough blood to the body, is caused by various heart conditions. And the incidence and prevalence are increasing year by year with aging.
  • Myocardial infarction which is a type of ischemic heart disease, is a disease in which the blood flow in the coronary artery that nourishes the myocardium is reduced or interrupted for a certain period of time and the myocardium in the perfusion region falls into necrosis.
  • necrotic part is eventually replaced with scar tissue, but the scar part has no contractile force and gradually deteriorates cardiac function.
  • research on new therapeutic methods using cell transplantation, gene therapy, cytokines, and angiogenic factors has been promoted for such pathological conditions.
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • the present inventors have clarified that a peptide consisting of 7 amino acids (SVVYGLR) present in osteopontin (OPN), which is a kind of extracellular matrix, has an angiogenic action. It has been found that it is as high as VEGF, which plays a central role in angiogenic factors (see Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2). Further, the present inventors have found that the peptide has a mesenchymal cell proliferation promoting action (see Patent Document 2). However, it is not known that the peptide has an ability to improve cardiac function.
  • An object of the present invention is to find a peptide having an ability to improve cardiac function, provide a therapeutic agent for heart disease containing this as an active ingredient, and a cell sheet for treating cardiac disease that secretes the peptide.
  • the present invention includes the following inventions in order to solve the above problems.
  • a peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having a cardiac function improving action, or a pharmaceutically acceptable salt thereof is effective.
  • the therapeutic agent for heart disease according to any one of [1] to [6], wherein the heart disease is ischemic heart disease or cardiomyopathy.
  • the heart disease is ischemic heart disease or cardiomyopathy.
  • For the treatment of heart disease characterized by secreting a peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having an effect of improving cardiac function Cell sheet.
  • the peptide is a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and the amino acid sequence of a secretory signal peptide.
  • the cell sheet according to any one of [8] to [11], wherein the cells are myoblasts, smooth muscle cells, mesenchymal cells, or adipocytes.
  • the present invention it is possible to provide a therapeutic agent for heart disease containing a peptide having an ability to improve cardiac function as an active ingredient and a cell sheet for treating heart disease that secretes the peptide. Since the active ingredient is a peptide, the therapeutic agent for heart disease of the present invention has the advantages that side effects hardly occur from the viewpoint of antigenicity and safety is high.
  • the peptide that is an active ingredient of the therapeutic agent for heart disease of the present invention can be allowed to act on the target site for a long period of time.
  • FIG. 1 It is a figure which shows the result of having evaluated the myocardial fibrosis rate of the myocardial infarction peripheral part of the 3rd, 6th, and 9th week after an operation
  • (A) is a Sirius red dyeing
  • (B) is a myocardial fibrosis rate. It is a figure which shows the analysis result. It is a figure which shows the result of having measured the myocardial cell lateral diameter of the peripheral part of myocardial infarction of 3, 6, and 9 weeks after an operation, (A) is a PAS dyeing
  • FIG. 1 It is a figure which shows the result of having measured the capillary density of the myocardial infarction peripheral part of the 3rd, 6th, and 9th week after an operation
  • (A) is an immunohistochemical dyeing
  • (B ) Is a diagram showing the measurement results of capillary density. It is the chart which tested the synthesized peptide with the high performance liquid chromatograph mass spectrometer. It is the photograph of the isolated myoblast, The left is a phase-contrast microscope image, The right is a fluorescence microscope image.
  • EF left ventricular ejection fraction
  • % FS left ventricular diameter shortening rate
  • EDV left ventricular end-diastolic volume 8 weeks after a cell sheet transplant.
  • HW / BW heart weight ratio
  • FIG. 1 It is a figure which shows the result of having evaluated the left ventricle wall thickness of the infarcted part of the left ventricular cavity at 8 weeks after cell sheet transplantation, (A) is a Masson trichrome stained image of the heart, and (B) is the left ventricular cavity. The figure which shows the evaluation result of the diameter of this, (C) is a figure which shows the evaluation result of the left ventricle wall thickness of an infarction part.
  • FIG. 1 It is a figure which shows the result of having evaluated the myocardial fibrosis rate of the myocardial infarction periphery 8 weeks after cell sheet transplantation,
  • (A) is a Sirius red dyeing
  • (B) is an analysis of myocardial fibrosis rate It is a figure which shows a result. It is a figure which shows the result of having measured the myocardial cell horizontal diameter of the myocardial infarction periphery 8 weeks after a cell sheet transplant, (A) is a PAS dyeing
  • (A) is an immunohistochemical staining image using an antibody against Von WillbrandbFactor
  • (B) It is a figure which shows the measurement result of capillary blood vessel density. It is a figure which shows the result of having examined the distribution of the smooth muscle actin (SMA) positive cell in the ventricle 8 weeks after cell sheet transplantation.
  • SMA smooth muscle actin
  • the therapeutic agent for heart disease according to the present invention contains a peptide having a cardiac function improving action or a pharmaceutically acceptable salt thereof as an active ingredient.
  • a peptide having a cardiac function improving action or a pharmaceutically acceptable salt thereof include peptides having an amino acid sequence represented by the following formula (I), (II), (III) or (IV).
  • X 1 is not particularly limited, but for example, serine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable.
  • X 2 is not particularly limited, and for example, valine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable.
  • X 3 is not particularly limited, for example, valine, alanine, arginine, lysine, histidine, tryptophan, phenylalanine is preferred.
  • X 5 is not particularly limited, and for example, glycine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable.
  • X 6 is not particularly limited, and for example, leucine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable.
  • X 7 is not particularly limited, and for example, arginine, alanine, lysine, histidine, tryptophan, and phenylalanine are preferable.
  • the peptide as an active ingredient of the therapeutic agent for heart disease of the present invention may be any peptide having any one of the amino acid sequences represented by the above formulas (I) to (IV), and has an amino acid sequence other than these. May be.
  • the size of the peptide serving as the active ingredient of the therapeutic agent for heart disease of the present invention is not particularly limited, but the total number of amino acid residues is about 50 or less from the viewpoint of side effects such as ease of handling, production efficiency, and antigenicity. Is preferred. More preferably, it is about 30 residues or less, More preferably, it is about 20 residues or less, Most preferably, it is about 10 residues or less. The lower limit is 6 residues, preferably 7 residues or more.
  • the peptide serving as the active ingredient of the therapeutic agent for heart disease of the present invention has the amino acid sequence represented by SEQ ID NO: 1-6 or the amino acid sequence represented by SEQ ID NO: 7-9 below, and the total number of amino acid residues. Less than 20 peptides are preferred.
  • Ser-Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 7) Ser-Val-Val-Phe-Gly-Leu-Arg (SEQ ID NO: 8) Ser-Val-Val-Trp-Gly-Leu-Arg (SEQ ID NO: 9)
  • a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and having a total number of amino acid residues of 20 or less is more preferable, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 is further preferable.
  • the peptide of the present invention can be produced by a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase synthesis method according to a known general peptide synthesis protocol. Moreover, it can manufacture using the transformant which introduce
  • the fact that the obtained peptide has a cardiac function improving action can be confirmed, for example, by evaluating using a heart disease model rat as shown in the Examples. Specifically, it can be confirmed by administering a peptide to a heart disease model rat, recording an echocardiogram after a certain period, and evaluating cardiac function.
  • the cardiac function to be evaluated is not particularly limited. For example, the left ventricular diameter shortening rate (% fractional shortening;% FS), left ventricular ejection fraction (EF), left ventricular end-diastolic volume (LV end-diastolic volume; EDV), left ventricular end systolic volume (LV ESV), and the like.
  • the evaluation time after administration is not particularly limited, but is preferably after about 3 weeks, more preferably after about 4 weeks, and even more preferably after about 5 weeks. If the cardiac function of the individual administered with the peptide is improved compared to the cardiac function of the individual not administered with the peptide, it can be determined that the peptide has a cardiac function improving action.
  • cardiomyocytes are isolated from newborn rats or adult rats, peptides are added to the isolated cardiomyocytes, and changes in cell morphology, protein expression level such as hypertrophy markers, cardiomyocyte viability, etc. are determined in vitro. This can be confirmed by evaluating with
  • the amino acid constituting the peptide of the present invention may be one in which the side chain is modified with an arbitrary substituent.
  • a substituent is not specifically limited, For example, a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl group, a cycloalkyl group, an alkoxy group, an amino group etc. are mentioned.
  • the benzene ring of tryptophan or phenylalanine is modified with a substituent, and more preferably, the benzene ring of tryptophan or phenylalanine in the amino acid sequences represented by SEQ ID NOs: 1 to 9 is modified with a substituent. It is that.
  • the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ), or an ester (—COOR).
  • R in the ester is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, ⁇ - C 6-12 aryl group such as naphthyl, for example, benzyl, C 7 - 14 aralkyl such as ⁇ - naphthyl -C 1-2 alkyl group such as a phenyl -C 1-2 alkyl or ⁇ - naphthylmethyl such phenethyl
  • a pivaloyloxy a C 1-6 alkyl group such as
  • the amino group of the N-terminal methionine residue is protected with a protecting group (for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl).
  • a protecting group for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl.
  • N-terminal side cleaved in vivo glutamyl group produced by pyroglutamine oxidation, substituent on amino acid side chain in molecule (for example, —OH, —SH, amino group, imidazole group, indole group) And a guanidino group) are protected with an appropriate protecting group (for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl).
  • an appropriate protecting group for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl.
  • the peptide of the present invention may form a pharmaceutically acceptable salt.
  • the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, Salts with acids such as citric acid, oleic acid and palmitic acid; salts with alkali metals or alkaline earth metals such as sodium, potassium and calcium; or salts with aluminum hydroxides or carbonates; triethylamine, benzylamine , Salts with diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.
  • the heart disease to be treated is not particularly limited as long as it is a heart disease that exhibits a therapeutic effect by improving cardiac function.
  • ischemic heart disease myocardial infarction, angina pectoris, etc.
  • cardiomyopathy hypertrophic cardiomyopathy, secondary myocardial hypertrophy, dilated cardiomyopathy, restricted cardiomyopathy, etc.
  • myocarditis myocarditis, heart failure, Endocarditis (such as bacterial endocarditis), valvular heart disease (such as mitral regurgitation), pericarditis (such as acute pericarditis, chronic constrictive pericarditis), congenital heart disease (atrium) Septal defect, ventricular septal defect, etc.), cardiac asthma, pulmonary heart and the like.
  • ischemic heart disease cardiomyopathy and heart failure
  • cardiomyopathy and heart failure more preferred are cardiomyopathy and heart failure.
  • the therapeutic agent for heart disease of the present invention can be formulated by using the above-mentioned peptide having a cardiac function-improving action as an active ingredient and appropriately blending a pharmaceutically acceptable carrier or additive.
  • oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ
  • Carriers or additives that can be blended are not particularly limited.
  • various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption
  • Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
  • Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry.
  • a liquid carrier such as fats and oils can be further contained in the above type of material.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like.
  • aqueous liquid for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • alcohols eg, ethanol
  • polyalcohols eg, propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80 TM , HCO-50
  • oily liquid for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • Buffers eg, phosphate buffer, sodium acetate buffer
  • soothing agents eg, benzalkonium chloride, procaine, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • storage You may mix
  • the therapeutic agent for heart disease of the present invention may be in a form in which a peptide having an effect of improving cardiac function as an active ingredient is bound to a carrier.
  • the carrier is not particularly limited, and examples thereof include resins used for artificial organs, biopolymers such as proteins, and the like.
  • the therapeutic agent for heart disease of the present invention may be in a form containing cells that secrete peptides having an effect of improving cardiac function of active ingredients.
  • it is a form including a cell sheet that secretes a peptide having an effect of improving cardiac function of an active ingredient. The cells and the cell sheet will be described later.
  • the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) can do.
  • the dose varies depending on the administration subject, target disease, administration route, etc., for example, when the therapeutic agent for heart disease of the present invention is orally administered for the purpose of treating cardiomyopathy, generally in an adult (with a body weight of 60 kg).
  • the single dose of the active ingredient varies depending on the administration subject, the target disease, etc.
  • the therapeutic agent for heart disease of the present invention is usually in the form of injection.
  • the active ingredient is administered by intravenous injection per day. Is preferred.
  • the present invention further includes the following inventions.
  • A a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) and having a cardiac function-improving action for a mammal, or a pharmaceutically
  • a method of treating heart disease comprising administering an effective amount of an acceptable salt.
  • B a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) for producing a therapeutic agent for heart disease, and having a cardiac function improving action, or Use of a pharmaceutically acceptable salt thereof.
  • C a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) and having a cardiac function-improving action, for use in the treatment of heart disease, or Its pharmaceutically acceptable salt.
  • the therapeutic agent for heart disease of the present invention maintains improvement in cardiac function even in the 9th week after single administration around the infarcted myocardium in myocardial infarction model rats (see Example 1). .
  • angiogenesis therapy for inducing new blood vessels by introducing VEGF, HGF and bFGF (basic fibroblast growth factor), which are known angiogenesis-promoting factors for ischemic heart disease, has been constructed (Rissanen TT , Et al., Adv Genet. 117-167, 2004, Miyagawa S, et al., Transplantation. 81: 902-907, 2006, Rissanen TT, et al., FASEB.
  • the therapeutic agent for heart diseases of the present invention is a therapeutic agent for cardiac diseases superior to known angiogenesis-promoting factors such as VEGF, HGF, bFGF, etc. in that the effect of improving cardiac function is maintained for a long time.
  • the cell sheet for treating heart disease of the present invention is characterized by secreting a peptide having a cardiac function improving action, which is an active ingredient of the above-mentioned therapeutic agent for heart disease of the present invention.
  • Peptides having a cardiac function improving action are as described above, and include peptides having an amino acid sequence represented by the above formula (I), (II), (III) or (IV).
  • Preferred examples of the amino acid sequence represented by (IV) include the sequences of SEQ ID NOs: 1 to 6.
  • the peptide having a cardiac function improving action secreted from the cell sheet for treating heart disease of the present invention preferably contains the amino acid sequence of a secretory signal peptide.
  • the secretory signal peptide means a peptide region that plays a role of allowing a protein or peptide bound to the secretory signal peptide to permeate through the cell membrane. Therefore, by including the amino acid sequence of the secretory signal peptide, it is possible to secrete a peptide having an effect of improving cardiac function out of the cell.
  • the amino acid sequences of such secretory signal peptides and the nucleic acid sequences encoding them are well known and reported in the art (eg, von Heijine G (1988) Biochim. Biohys.
  • the amino acid sequence of the secretory signal peptide and the nucleic acid sequence encoding it can be obtained from a known database (DDBJ / GenBank / EMBL, etc.).
  • the secretory signal peptide suitable for the present invention is a secretory signal peptide that can function in mammalian cells, particularly human cells. For example, an Ig ⁇ chain leader sequence, a honey bee melittin signal sequence, an ⁇ factor secretion signal, and the like.
  • the amino acid arrangement of the secretory signal peptide is preferably arranged on the N-terminal side of the peptide having a cardiac function improving action.
  • the peptide secreted from the cell sheet for treating heart disease of the present invention is preferably a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and the amino acid sequence of the secretory signal peptide.
  • the total number of amino acid residues of the secreted peptide is preferably about 100 or less. More preferably, it is about 70 residues or less, More preferably, it is about 50 residues or less, Most preferably, it is about 40 residues or less.
  • the lower limit is not particularly limited, but the lower limit is the number obtained by adding the number of amino acid residues of the secretory signal peptide to the 6 residues that are the lower limit of the peptide having a cardiac function improving action.
  • the cell sheet for treating heart disease of the present invention can be prepared by culturing cells that secrete peptides having a cardiac function improving action.
  • a cell that secretes a peptide having a cardiac function improving action introduces a recombinant expression vector into which a polynucleotide encoding a peptide having a cardiac function improving action (including the amino acid sequence of a secretory signal peptide) is inserted into an appropriate cell.
  • a method for producing a cell secreting a peptide having a cardiac function improving action and a cell sheet for treating heart disease of the present invention will be described.
  • peptide secreting cells are not particularly limited, but cells that have already been used for transplanting cell sheets into the heart are preferred.
  • Myoblasts Ghostine S, Carrion C, Souza LC, Richard P, Bruneval P, Vilquin JT, Pouzet B, Schwartz K, Menasche P, Hagege AA : Long-term efficacy of myoblast alplantfunction card functionplant Circulation.
  • smooth muscle cells Yoo KY, LiRK, Weisel RD, Mickle DAG, Li GM, Kim EJ, Tomita S, Yau TM: Autologous smooth muscle cell transplantation improved heart function in dilated cardiomyopathy.
  • Ann Thorac Surg 70: 859-865, 2000., etc. mesenchymal cells (Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers JM, Pittenger BJ, Pittenger BJ, stem cell implantation in a swine myocardial infarct model: engraftment and functional effects.
  • adipocytes (Valina C, Pinkernell K, Song YH, Bai X, Seaut J, TH, Alt E. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. Eur Heart J. 28: 2667-77, 2007.
  • Recombinant expression vectors can be prepared using known gene recombination techniques.
  • an appropriate expression vector containing a promoter that can function in a host cell may be selected.
  • retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors animal cell expression plasmids (eg, pA-11, pxT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. ) And the like.
  • a target recombinant expression vector can be prepared.
  • the polynucleotide may be DNA or RNA, or may be a DNA / RNA chimera.
  • the polynucleotide may be double-stranded or single-stranded. Preferred is double-stranded DNA, particularly cDNA.
  • the nucleotide sequence of a polynucleotide encoding a peptide having a cardiac function improving action can be appropriately designed based on the amino acid sequence of the peptide.
  • the polynucleotide can be prepared by chemical synthesis.
  • the method for introducing the recombinant expression vector into the cell is not particularly limited, and a known method can be appropriately selected and used.
  • the cells may be infected with the virus.
  • Peptide-secreting cells are selected from the cells into which the recombinant expression vector has been introduced.
  • the peptide-secreting cell is preferably a cell that stably expresses the target peptide.
  • the cell sheet for treating heart disease of the present invention can be produced by peeling the cells while maintaining the sheet state.
  • the culture is confluent using a culture dish coated with poly-N-isopropylacrylamide, which is a temperature-responsive polymer, and is peeled after low-temperature treatment (Medical History, 195, 203-204 (2000). )reference).
  • a cell sheet having a three-dimensional structure composed of cells and extracellular matrix can be produced by a method using a culture dish coated with the temperature-responsive polymer.
  • the amount of the peptide having a cardiac function improving action secreted by the cell sheet for treating heart disease of the present invention is not particularly limited.
  • the cell sheet for treating heart disease of the present invention can be laminated in a plurality of layers so that the secretion amount of the peptide having an effect of improving cardiac function becomes the target secretion amount.
  • the transplanted cell sheet is thought to gradually fall out of the cell transplant site in about 2 weeks.
  • the SVVYGLR peptide improves the cardiac function in vivo in an amount of 20 to 100 ng. Therefore, a cell sheet capable of secreting 20-100 ng or more of a peptide within 2 weeks after transplantation is preferred.
  • the cell sheet for treating heart disease of the present invention has been demonstrated to overcome the drawbacks of conventional cell sheets and maintain the improvement effect of cardiac function even 8 weeks after cell sheet transplantation (Example 3). And 4).
  • the cell sheet for treating heart disease of the present invention has both a cardiac function improving effect and an angiogenic action, it can be expected that the cardiac function can be improved more continuously. Therefore, it is considered to be extremely useful for treating not only ischemic heart diseases such as myocardial infarction but also cardiomyopathy.
  • the peptide is continuously secreted from the sheet transplantation site, and the effect can be maintained.
  • the injection method is known to induce arrhythmia, but there has been no such report on cell sheet transplantation. Therefore, if the cell sheet for treatment of heart disease of the present invention is used, a peptide having a function of improving cardiac function can be used in combination with the treatment of heart disease using a known cell sheet, and a synergistic treatment effect can be expected.
  • Example 1 Evaluation of peptide using rat myocardial infarction model
  • Experimental method (1-1) Synthesis of peptide A peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 was synthesized by Fmoc method using a multi-item solid phase method automatic peptide synthesizer (PSSM-8; Shimadzu Corporation). did. More specifically, the graft copolymerization range Tentagel (particle size 80 ⁇ m) of polyethylene glycol and polystyrene was used as a support and was synthesized by a high-efficiency solid phase method.
  • PSSM-8 multi-item solid phase method automatic peptide synthesizer
  • the obtained synthetic peptide was tested with a high performance liquid chromatograph mass spectrometer (LCMS; Shimadzu Corporation), and confirmed to be a single component consistent with the theoretical mass value (see FIG. 6).
  • LCMS liquid chromatograph mass spectrometer
  • WiDa the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is referred to as “WiDa”.
  • % Fractional shortening % Fractional shortening
  • EF left ventricular ejection fraction
  • FAC left ventricular area change rate
  • LV% FS [(LVDd ⁇ LVDs) / LVDd] ⁇ 100
  • LVEF (%) [(LVDd 3 -LVDs 3) / LVDd 3] ⁇ 100
  • LVFAC (%) [(LVEDA ⁇ LVEDS) / LVEDA] ⁇ 100
  • the section was washed with PBS-T and reacted with a biotinylated labeled anti-Rabbit IgG antibody (Anti-Rabbit Ig, biotinyated species-specific whole antibody, DAKO) as a secondary antibody.
  • Color was developed with DAB (Diamino benzidine) using the LSAB (Labeled Streptavidin Biotinyated Antibody) method with HRP-labeled streptavidin (Streptavidin-Horseradish peroxidase conjugate, GE Healthcare). After staining, 15 fields were randomly selected with an optical microscope ( ⁇ 400), and the number of capillaries having Von Willbrand Factor-positive vascular endothelial cells was counted to obtain the capillary density.
  • the long-term effect of WiDa on ischemic myocardium was evaluated at 6 and 9 weeks after surgery.
  • the left ventricular diameter shortening rate (% FS, FIG. 1 dg) and the ejection fraction (EF, FIG. 1 eh) were evaluated, both the 6-week and 9-week models were significantly higher in the WiDa group than in the Control group.
  • the left ventricular area change rate (FAC, FIG. 1fi) was evaluated, the WiDa group was significantly improved with respect to the Control group in the 9-week model.
  • FIG. 2a-g the left is the HE-stained image and the right is the Sirius red-stained image.
  • the scale bar represents 1000 ⁇ m.
  • FIG. 2a (Sham group) only showed results for 3 weeks.
  • the wall thickness from the left ventricular anterior wall to the posterior wall became thinner and many collagen fibers were observed.
  • the left ventricular wall thickness of the WiDa group was kept thicker than that of the Control group.
  • the left ventricular cavity in the HE-stained images of FIGS. 2b to g, the left ventricular cavity was gradually expanded in the Control group in both the 3-week, 6-week and 9-week models, whereas in the WiDa group, the Control Dilation was suppressed compared to the group.
  • FIG. 3 (B) is a graph showing the analysis result of the myocardial fibrosis rate.
  • the 3-week model no significant difference was observed between the Control group and the WiDa group in the myocardial fibrosis rate (FIG. 3 (B) a).
  • FIGS 4A and 4B show the PAS-stained image and the measurement results of the myocardial cell lateral diameter.
  • A is a PAS-stained image of the infarct boundary, and the scale bar represents 100 ⁇ m.
  • (B) is a graph showing the measurement results of the myocardial cell lateral diameter, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary.
  • the myocardial cell lateral diameter was significantly higher in the Control group and the WiDa group than in the sham group (p ⁇ 0.0001).
  • FIGS. 5 (A) and (B) are an immunohistochemically stained image of the infarct boundary, and the scale bar represents 100 ⁇ m.
  • (B) is a graph showing the measurement result of capillary blood vessel density, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary.
  • HGF has an action for improving angiogenesis in addition to an angiogenesis-promoting action
  • Ueda H, et ai., CardiovascvasRes 2001; 51: 41-50. Ueda H, et ai., CardiovascvasRes 2001; 51: 41-50.
  • the left ventricular function was 4% after treatment, the left ventricular diameter shortening rate (% FS) 20%, ejection fraction (EF) 42% Although an improvement effect was observed, a downward trend was observed thereafter (Miyagawa S. et ai., Circulation 2002; 105: 2556-2561.).
  • the left ventricular function of the 3-week model is the left ventricular diameter shortening rate (% FS) of 25, even though only one administration of WiDa (100 ng / ml) was performed immediately after the creation of the myocardial infarction model.
  • WiDa is a low molecular weight peptide composed of 7 amino acid residues, and its half-life is considered to be short.
  • a single administration of WiDa to the ischemic peripheral myocardium dramatically improved left ventricular function and the effect persisted for a long time. From this, it was found that WiDa has an effect of remarkably improving the cardiac function lost due to ischemia.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • Rissanen TT et ai., Adv Genet 2004; 52: 117-167.
  • Rissanen TT et ai., The FASEB Journal 2003; 17: 100-102.
  • Hao X et ai., BiochemBiophys Res Commun 2004; 322: 292-6.
  • these factors are concerned about side effects such as edema and inflammatory reaction.
  • WiDa induced more structurally and functionally stabilized trophic blood vessels from ischemic myocardial tissue from the angiogenic action of previous studies. Therefore, it can be said that WiDa is a new factor capable of overcoming side effects such as early regression of new blood vessels and edema. From the above research results, it was considered that WiDa exhibited not only the conventionally known angiogenic action but also some function that markedly improved cardiac function.
  • Example 2 Production of WiDa-secreting myoblast sheet
  • Experimental method (1-1) Myoblast isolation Anterior tibial muscle was collected from 3-week-old Lewis rats, washed with cold HBSS (Hanks balanced salt solution), and trypsin (Invitrogen life Technologies, Carlsbad, CA, USA) was added, and tendons, fibrous tissue, adipose tissue, etc. were carefully removed to prevent mixing of fibroblasts. Mince with trypsin and treated with 0.2% type II collagenase (Worthingto Biochemical corporation, Lakewood, NJ, USA) for 45 minutes.
  • the cells were seeded on a collagen type I (Nitta Gelatin Inc, Osaka, Japan) coat dish (10 ⁇ g / ml) in order to adhere fibroblasts. After incubating at 37 ° C. with 5% CO 2 for 4 hours, the supernatant was seeded again on a collagen type I coat dish (10 ⁇ g / ml). After another 24 hours, the supernatant was matrigel (Becton Dickinson Bioscience, Flanklin Lakes, NJ, USA) Coated dishes (0.5 mg / ml) were reseeded, and the cells adhered here were used as myoblasts.
  • a collagen type I Nita Gelatin Inc, Osaka, Japan
  • FBS fetal bovine serum, Biowest, Minami, FL, USA
  • Antibiotic-Antimytic Invitrogen life Technologies
  • Orgadron Neuronal growth factor (Ga) -containing DMEM (Dulbecco modified Eagle's medium) (Nihonseiyaku , Toky, Japan).
  • DMEM Dulbecco modified Eagle's medium
  • the medium of each dish was replaced with 10% FBS DMEM, and the DNA complex was added to the culture supernatant to introduce the gene. After 12 hours, the medium was replaced with 6 ml of 10% FBS DMEM, and the virus-containing supernatant was collected 48 hours and 72 hours after gene introduction.
  • the collected virus-containing supernatant was passed through a 0.45 ⁇ m filter (Millipore, Billerica, MA, USA) to remove floating cells and the like, and Lenti-XTM Concentrator (Clontech Laboratories, Inc., Mountain View, CA, USA). Concentrated.
  • a virus-containing supernatant (mock) into which a pCS-CG empty vector used as a negative control for expression studies was introduced was also collected.
  • RT-PCR The expression of WiDa mRNA was examined by RT-PCR.
  • the medium was exchanged with 20% FBS DMEM 48 hours after infection, and 1 ml of Sepasol-RNA1 Super G (nacalai tesque, Kyoto, Japan) was added 120 hours after infection. After 5 minutes, the cells were detached with a scraper and transferred to a 1.5 ml tube, and then 200 ⁇ l of chloroform was added and mixed vigorously. It was centrifuged (12000 rpm, 15 minutes, 4 ° C.) and 400 ⁇ l of the supernatant was transferred to a new 1.5 ⁇ l tube.
  • DEPC diethypyrocarbonate
  • AMV buffer 5 ⁇ l, 10 mM dNTP 2 ⁇ l, 50 ⁇ M Oligo dT 1 ⁇ l, RNasion 1 ⁇ l, AMV reverse transcriptase (Sigma) 1 ⁇ l was added, and a reverse transcription reaction was performed under reaction conditions of 42 ° C. for 60 minutes, 65 ° C. for 10 minutes, and 4 ° C.
  • CDNA obtained by reverse transcription was used as a template.
  • the primers used are shown below.
  • GAPDH (Glycelaldehyde-3-phospate dehydrogenase) was used as an internal control.
  • a high HA polyclonal antibody was used as the primary antibody, an HRP-labeled anti-rabbit IgG antibody was used as the secondary antibody, and light was emitted with Super signal West Femto, followed by development and analysis. The image of the film was taken into Image J, and the density of Dot of each well was quantified and examined and evaluated.
  • WiDa mRNA was detected only from WiDa / pCS-CG-infected cells.
  • FIG. (A) is the result of dot blotting in which a known concentration of WiDa-HA peptide is serially diluted
  • (B) is the result of dot blotting of WiDa / pCS-CG-infected cell culture supernatant
  • (C) is (B ) Is a table showing the darkness of the numerical value. From the results shown in FIG. 9, it was confirmed that WiDa was produced and secreted in WiDa / pCS-CG-infected cell culture. Further, from the comparison of the difference in the lightness and the lightness of the serially diluted dots in FIG. 9A, it was calculated that about 3.125 to 6.25 ng / ml WiDa was secreted in 72 hours.
  • Example 3 Evaluation of WiDa secreting myoblast sheet using rat heart failure model I
  • (1) Experimental method (1-1) Preparation of rat heart failure model F344 / NJcl-rnu / rnu rats (8 weeks old, female) were inhaled and anesthetized with isoflurane. Thereafter, endotracheal intubation was performed and intraoperative management was performed under artificial ventilation. A thoracotomy was performed from the heartbeat area, and a visual field was secured using a retractor.
  • the left anterior descending coronary artery was ligated with 7-0 non-absorbable thread (nylon thread) at the height of the left atrial appendage to create a myocardial infarction in the left ventricular anterior wall.
  • Echocardiography was taken 2 weeks after ligation, and left ventricular ejection fraction (EF) of 45-35% was used as a myocardial infarction model rat in the experiment.
  • EF left ventricular ejection fraction
  • a myoblast sheet resuming 2 weeks after ligation and secreting WiDa (hereinafter referred to as “WiDa-rSkM”), a wild type myoblast sheet (hereinafter referred to as “WT-rSkM”) Transplanted to the infarct site.
  • WiDa-rSkM a myoblast sheet resuming 2 weeks after ligation and secreting WiDa
  • WT-rSkM a wild type myoblast sheet Transplanted to the infarct site.
  • a cell sheet that was not transplanted was designated as Control.
  • Each cell sheet was composed of three layers. This model starts treatment at the second week after ligation of the left anterior descending coronary artery. The motility of the left ventricular anterior wall at the second week is reduced, and in the untreated state as it is, the left ventricular wall motion further decreases with time, and the left ventricular wall function is significantly impaired. It can be said that the pressure load on the left ventricular wall also rise
  • left ventricular diameter shortening rate (% Fractional shortening;% FS), left ventricular ejection fraction (EF), left ventricular end-diastolic volume (EDV), left ventricular end systole
  • LV% FS [(LVDd ⁇ LVDs) / LVDd] ⁇ 100
  • LVEF (%) [(LVDd 3 -LVDs 3) / LVDd 3] ⁇ 100
  • EDV (ml) LVIDd 3 ⁇ (0.98 ⁇ LVIDd + 5.9)
  • ESV (ml) LVIDs 3 ⁇ (1.14 ⁇ LVIDs + 4.18)
  • HW / BW Heart weight ratio
  • the heart was removed 8 weeks after the sheet transplantation, and fixed with 10% buffered formalin for 48 hours. After removing both atria, they were embedded in paraffin, sliced and HE stained. Masson trichrome staining was performed to examine left ventricular cavity dilation and left ventricular wall thickness at the infarcted area. The size of the left ventricular cavity was evaluated by the average of the diameters passing through the cavity, and the left ventricular wall thickness was evaluated by the ratio of the left ventricular wall thickness of the infarcted part to the wall thickness of the normal part (left ventricular rear wall). In order to evaluate the myocardial fibrosis rate, Sirius red staining was performed to specifically dye collagen fibers in red.
  • the infarct boundary was observed with an optical microscope at a magnification of 20x, and an image taken in ACT-2U software (NIKON) was analyzed by image analysis software Image J. Evaluation was shown by the fibrosis rate per visual field.
  • Periodic Acid / Schiff reaction (PAS) staining was performed to evaluate the lateral diameter of cardiomyocytes. After staining, observation was performed with an optical microscope at a magnification of 40x, and 100 cells with nuclei were randomly selected at the infarct boundary and the left ventricular posterior wall, and the minor axis crossing the nucleus was defined as the cell transverse diameter. .
  • the section was washed with PBS-T and reacted with a biotinylated labeled anti-Rabbit IgG antibody (Anti-Rabbit Ig, biotinyated species-specific whole antibody, DAKO) as a secondary antibody.
  • Color was developed with DAB (3,3-diaminobenzidine, Sigma, St. Louis, MO, USA) using LSAB (Labeled Streptavidin Biotinyated Antibody) method with HRP-labeled streptavidin (Streptavidin-Horseradish peroxidase conjugate, GE Healthcare).
  • FIG. 10 shows% FS at 2, 4, 6, and 8 weeks after cell sheet transplantation, respectively.
  • EF left ventricular ejection fraction
  • % FS left ventricular diameter shortening rate
  • FIG. 12 shows EDV at 8 weeks after cell sheet transplantation, and 8 weeks after cell sheet transplantation.
  • the ESVs are shown in FIG.
  • EDV the WiDa-rSkM group showed a significant decrease compared to the Control group and the WT-rSkM group.
  • ESV the WiDa-rSkM group showed a significant decrease compared to the Control group and the WT-rSkM group.
  • no significant difference was observed between the Control group and the WT-rSkM group.
  • FIGS. 15 A, B and C.
  • A is a Masson trichrome stained image of the heart, and the scale bar represents 1000 ⁇ m.
  • B is a graph which shows the evaluation result of the left ventricular wall thickness of an infarction part.
  • C is a graph showing the evaluation result of the diameter of the left ventricular cavity.
  • the left ventricular wall at the infarcted site was thinned in the Control group and the WT-rSkM group, whereas the left ventricular wall was kept thick in the WiDa-rSkM group. It was leaning.
  • the left ventricular wall of the infarct was significantly thicker in the WiDa-rSkM group compared to the Control group and the WT-rSkM group (p ⁇ 0.01). There was no significant difference between the Control group and the WT-rSkM group. Further, as is clear from FIGS.
  • the WiDa-rSkM group significantly suppressed the expansion of the left ventricular cavity as compared with the Control group and the WT-rSkM group (p ⁇ 0). .05). There was no significant difference between the Control group and the WT-rSkM group.
  • FIGS. 16 (A) and 16 (B) Histological evaluation of the effect on the myocardial tissue around the infarction
  • Sirius Red staining and the cell lateral diameter by PAS staining Measurements were made.
  • the results of Sirius red stained image and myocardial fibrosis rate are shown in FIGS. 16 (A) and 16 (B).
  • (A) is a Sirius red stained image of the myocardium, and the scale bar represents 100 ⁇ m.
  • (B) is a graph showing the analysis result of the myocardial fibrosis rate.
  • the myocardial fibrosis rate in the WiDa-rSkM group was significantly lower than that in the Control group and the WT-rSkM group (p ⁇ 0.01). There was no significant difference between the Control group and the WT-rSkM group.
  • FIG. 17 (A) and (B) show the PAS-stained image and the measurement results of the cardiomyocyte lateral diameter.
  • (A) is a PAS-stained image of the infarct boundary, and the scale bar represents 50 ⁇ m.
  • (B) is a graph showing the measurement results of the myocardial cell lateral diameter, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary.
  • the myocardial cell transverse diameter at the infarct boundary in the WiDa-rSkM group was significantly lower than that in the Control group and the WT-rSkM group (p ⁇ 0.01). There was no significant difference between the Control group and the WT-rSkM group. There was no significant difference in the cardiomyocyte lateral diameter of the left ventricular posterior wall between any groups.
  • FIGS. 18 Examination of angiogenesis promoting action Immunohistochemical staining was performed using an antibody against Von Willbrand Factor, and the results of counting the number of capillaries are shown in FIGS. 18 (A) and (B).
  • (A) is an immunohistochemically stained image of the infarct boundary, and the scale bar represents 100 ⁇ m.
  • (B) is a graph showing the measurement result of capillary blood vessel density, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary.
  • the number of capillaries at the infarct boundary in the WiDa-rSkM group was significantly increased compared to the Control group and the WT-rSkM group (p ⁇ 0.01). There was no significant difference between the Control group and the WT-rSkM group. Capillary density in the left ventricular posterior wall was not significantly different between any groups.
  • Example 3 (WiDa secretion myoblast cell sheet transplantation), a cell sheet was transplanted two weeks after ligation of the left ventricular descending branch. The second week after ligation of the left ventricular anterior descending branch is a stage in which acute myocardial infarction has progressed and heart failure has occurred. That is, Example 3 evaluates the usefulness of WiDa in the treatment of heart failure.
  • WiDa-secreting myoblasts showed an improvement in cardiac function and an effect of suppressing remodeling in heart failure model rats also at 4, 6, and 8 weeks after the second week after the transplantation. From this, it can be said that WiDa can be a useful therapeutic agent not only for acute myocardial infarction but also for heart failure.
  • Example 4 Evaluation of WiDa secreting myoblast sheet using rat heart failure model II
  • a rat heart failure model was prepared using F344 / NJcl-rnu / rnu rats (8 weeks old, female) in the same manner as in Example 1-1 (1-1). Eight weeks after the transplantation, the heart was removed and fixed with 10% buffered formalin for 48 hours. After removing both atria, they were embedded in paraffin and sliced. In order to examine the distribution of smooth muscle actin (SMA) positive cells, immunohistochemical staining was performed using an anti-smooth muscle actin antibody and observed with an optical microscope.
  • SMA smooth muscle actin
  • FIG. 19 The results are shown in FIG.
  • the upper part of FIG. 19 is a 40 ⁇ observation image, and the scale bar represents 500 ⁇ m.
  • the lower row is a 200-fold observation image, and the scale bar represents 100 ⁇ m.
  • accumulation of SMA positive cells was observed in the infarct region in the WiDa-rSkM group.
  • SMA positive cells such as smooth muscle cells and myofibroblasts have contractility. Therefore, the WiDA peptide secreted from the myoblast cell sheet significantly increases SMA positive cells in the infarct region, and the increased SMA positive cells constrict the infarct wall, thereby significantly improving cardiac function. It is thought that it was connected.

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Abstract

On a découvert qu'un peptide ayant une séquence d'acides aminés représentée par la formule (I), (II), (III), ou (IV) a un effet d'amélioration sur la fonction cardiaque. Le peptide ou un sel de qualité pharmaceutique de celui-ci est utile à titre de principe actif d'un agent thérapeutique destiné à traiter les maladies cardiaques. En outre, quand un feuillet cellulaire pour traiter des maladies cardiaques, qui sécrète le peptide, est transplanté dans un cœur atteint d'une maladie cardiaque, on peut laisser le peptide agir sur le site cible pendant longtemps. X1-X2-Val-Tyr-X5-X6 (I) X2-Val-Tyr-X5-X6-X7 (II) Ser-X2-X3-(Tyr/Phe/Trp)-X5-X6 (III) X2-X3-(Tyr/Phe/Trp)-X5-X6-Arg (IV). Dans les formules (I), (II), (III), et (IV), X1, X2, X3, X5, X6, et X7 sont identiques ou différents et représentent un résidu acide aminé arbitraire.
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JPWO2016084935A1 (ja) * 2014-11-27 2017-10-19 国立大学法人大阪大学 Ｉｉｉ型コラーゲン産生促進剤
WO2018230535A1 (fr) * 2017-06-15 2018-12-20 国立大学法人大阪大学 Accélérateur de réparation de lésion musculaire squelettique
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CN112292157A (zh) * 2018-06-12 2021-01-29 国立大学法人京都大学 细胞移植用组合物和细胞移植方法
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WO2022085791A1 (fr) 2020-10-24 2022-04-28 国立大学法人大阪大学 Agent améliorant la fonction musculaire des muscles squelettiques dont la fonction est dégradée en raison du vieillissement

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