JP2019156739A - Mesenchymal stem cell, disease therapeutic agent, and microparticle - Google Patents

Abstract

To provide a novel therapeutic agent for immunologic disease and liver disease.SOLUTION: A mesenchymal stem cell is characterized by high expression of at least one micro RNA selected from the group consisting of miR-210-3p, miR-6846-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634.SELECTED DRAWING: None

Description

  The present invention relates to mesenchymal stem cells, disease therapeutic agents, and microparticles.

  Heart transplantation has been performed as a treatment for end-stage heart failure, coronary artery disease (CAD), arrhythmia, hypertrophic cardiomyopathy, and congenital heart disease. Similarly, hematopoietic stem cells are used to treat hematological diseases such as blood cancer (leukemia, lymphoma, myeloma) and primary immunodeficiency, aplastic anemia, myelodysplasia, and kidneys are used to treat end stage renal failure. Liver cirrhosis, fulminant hepatic necrosis, hepatocellular carcinoma, biliary atresia and metabolic disease in children, cholestatic diseases such as primary sclerosing cholangitis and non-stasis diseases such as autoimmune hepatitis, liver and respiratory function decline Or for patients with respiratory failure, lungs for diabetic patients, islet or pancreatic islet cells, abdominal wall rupture, Hirschsprung's disease, autoimmune enteritis and other intestinal disorders, enteric thromboembolism or widespread Crohn's disease In patients with malabsorption, the small intestine is transplanted. In addition, skin allografts, cartilage transplants, bone transplants, corneal transplants, and fetal thymus grafts are also performed. As a complication after transplantation of these organs, rejection is a big problem. In particular, hyperacute rejection is a reaction caused by an existing complement-binding antibody against a graft antigen within 48 hours after transplantation, and there is no effective treatment other than graft removal. It is rare.

  On the other hand, autoimmune hepatitis is known as an immune disease. This autoimmune hepatitis has both acute and chronic clinical symptoms, but is an autoimmune disease caused by failure of immunological tolerance to the self-hepatocytes by some mechanism. Involvement of preceding infectious diseases and drugs has been suggested as the cause of the onset. The number of autoimmune hepatitis patients in Japan is about 20,000, of which chronic autoimmune hepatitis accounts for about 1.8% of chronic hepatitis. About 30% of deaths due to autoimmune hepatitis are deaths within half a year from diagnosis, and treatment for acute liver failure at the time of diagnosis is important for improving prognosis (see Non-Patent Document 1). In chronic autoimmune hepatitis, liver regeneration and connective tissue renewal have been repeated due to chronic inflammation, resulting in an increase in extracellular matrix mainly composed of collagen (liver fibrosis) and progress to cirrhosis. . When this cirrhosis progresses from a compensatory period without symptoms to a non-compensated period with higher fibrosis, existing drugs and therapies cannot sufficiently cope with it. Therefore, development of a novel therapeutic agent having a different mechanism of action from the conventional one is desired.

  Mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (1982) (see Non-Patent Document 2). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases ( Patent References 1-2). Recently, it is known that cells having equivalent functions exist in stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, the mesenchymal stem cell may be referred to as a stromal cell (Mesenchymal Stoma Cell).

JP 2012-157263 A Special table 2012-508733 gazette

Liver specialist text, pp. 205-208, Nankodo Co., Ltd., issued March 30, 2013 Pittenger F.M. M.M. et. al. , Science 284, pp. 143-147 (1999)

  An object of the present invention is to provide a novel therapeutic agent and a novel treatment method for immune diseases and liver diseases in the above-described situation.

  As a result of diligent research to solve the above problems, the present inventors have found that miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129- Mesenchymal Stem Cell (MSC) that highly expresses one or more microRNAs selected from the group consisting of 2-3p, miR-129-1-3p, and miR-634, and miR-210 -3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 Mesenchymal stem cell-derived microparticles that highly express one or more microRNAs are used to treat immune and liver diseases Thus, the present invention was completed. According to the present invention, a therapeutic agent effective for the treatment of immune diseases and liver diseases can be provided. That is, the gist of the present invention is as follows.

[1] miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 A mesenchymal stem cell characterized in that one or more microRNAs selected from the group consisting of are highly expressed.
[2] The mesenchymal stem cell according to [1], wherein the microRNA is contained in microparticles derived from mesenchymal stem cells.
[3] The mesenchymal stem cell according to [2], wherein the microparticle is an exosome.
[4] The mesenchymal stem cell according to any one of [1] to [3], which is derived from another family.
[5] The mesenchymal stem cell according to any one of [1] to [4], which is derived from adipose tissue.
[6] A therapeutic agent for a disease comprising the mesenchymal stem cells and / or culture supernatant thereof according to any one of [1] to [5].
[7] A therapeutic agent for diseases containing mesenchymal stem cell-derived microparticles according to any one of [1] to [5].
[8] The disease therapeutic agent according to [6] or [7], wherein the disease is an immune disease or a liver disease.
[9] miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 A microparticle characterized in that one or more microRNAs selected from the group consisting of are highly expressed.

  According to the present invention, a novel therapeutic agent for immune diseases and liver diseases can be provided.

It is a figure which shows the expression of each microRNA in the mesenchymal stem cell origin microparticle of this invention by the comparison with the conventional mesenchymal stem cell origin microparticle. It is a figure which shows the therapeutic effect with respect to the autoimmune hepatitis model mouse of the mesenchymal stem cell of this invention (AST). It is a figure which shows the therapeutic effect with respect to the autoimmune hepatitis model mouse of the mesenchymal stem cell of this invention (ALT). It is a figure which shows the therapeutic effect with respect to the autoimmune hepatitis model mouse of the microparticle of this invention (AST). It is a figure which shows the therapeutic effect with respect to the autoimmune hepatitis model mouse of the microparticles | fine-particles of this invention (ALT).

  Hereinafter, the mesenchymal stem cells, microparticles, and disease therapeutic agents of the present invention will be described in detail.

[Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1. One or more microRNAs selected from the group consisting of -3p and miR-634 are highly expressed.

  MicroRNA (miRNA) is a non-coding RNA gene that is about 22 bases long and does not encode protein. Each miRNA causes expression suppression with respect to complementary or partially complementary messenger RNA, and one type of miRNA is involved in the control of the expression of multiple genes. The gene expression regulation mechanism related to miRAN is considered to be related to various life phenomena, life functions, diseases, etc. (see real-time PCR complete experiment guide, etc. well understood from the separate principle of experimental medicine).

  In the present invention, high expression of microRNA means that the expression level of the microRNA is high compared to cells obtained under conventional culture conditions, preferably 1.5 times or more, More preferably, it is expressed 2 times or more, and more preferably 3 times or more. Here, conventional culture conditions include, for example, culture using 10% FBS-containing DMEM medium.

  Compared with conventional mesenchymal stem cells, the mesenchymal stem cells of the present invention are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-. It is sufficient that one or more microRNAs selected from the group consisting of 2-3p, miR-129-1-3p and miR-634 are highly expressed, preferably two or more, more preferably three or more, Preferably four or more, particularly preferably five or more, more particularly preferably six or more, and most preferably all of these microRNAs are highly expressed. Specifically, the mesenchymal stem cells of the present invention are miR-210-3p, miR-, compared to mesenchymal stem cells obtained under conventional culture conditions (for example, culture in DMEM medium containing 10% FBS). One or more micros selected from the group consisting of 6846-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 RNA is expressed 1.5 times or more, more preferably 2 times or more, and further preferably 3 times or more.

  The mesenchymal stem cell of the present invention contains the microRNA in intracellular microparticles. These microparticles are vesicles of a size that can be confirmed with an electron microscope, produced in mesenchymal stem cells and released from mesenchymal stem cells. As for the size of the fine particles, the average particle diameter is about 1 nm to 1,000 nm, preferably 10 nm to 500 nm, and more preferably 30 nm to 200 nm. Here, the average particle diameter is an average value of the diameters of the respective particles measured by a dynamic light scattering method or an electron microscope. The microparticle can have a lipid bilayer surrounding a biomolecule. Examples of the microparticles include membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (microvesicles, average particle size 30 to 1,000 nm), exosome-like vesicles, exosomes (exosomes, averages) Particle diameters of 30 to 200 nm), ectosome-like vesicles, ectosomes or exovesicles, and the like, preferably exosomes. Microparticles derived from different types of mesenchymal stem cells can be derived from intracellular sources, microparticle density in sucrose, shape, sedimentation rate, lipid composition, protein markers and mode of secretion (ie after signal (inducible) or A distinction is also made based on voluntary (constitutive). For example, the fine particles are fractionated by 1.0 to 1.5 g / mL, preferably 1.1 to 1.3 g / mL, in the density gradient centrifugation method. Furthermore, the microparticle contains any one of phosphatidylserine, cholesterol, sphingomyelin and ceramide as its constituent lipid.

  The microparticles include proteins, fatty acids, other nucleic acids and the like in addition to microRNA.

  Examples of the protein and fatty acid include IL-10, HGF (Hepatocyte Growth Factor), Apolipoprotein A-2 (Apolipoprotein A-2), Pigment epithelium-derived factor (PEDF), SERPINF1 Gin, and SERPINF1 (Haptoglobin), pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, Isoheptadecanic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, nonadecylic acid , Isononadecylic acid, arachidic acid, 11Z-eicosenoic acid, Dihom -Γ-linolenic acid, arachidonic acid, erucic acid, 13Z, 16Z-Docosadienic acid, 13Z, 16Z, 19Z-Docosadienic acid, adrenic acid, Clupanodonic acid, preferably IL-10, HGF, apolipoprotein i-2A A-2), pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid Margaric acid, Isoheptadecanic acid, stearic acid, isos Aric acid, oleic acid, elaidic acid, linoleic acid, arachidic acid, 11Z-eicosenoic acid, arachidonic acid, erucic acid, 13Z, 16Z-Docosadienic acid, more preferably IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A -2), pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, isoheptadecanoic acid, stearic acid, stearic acid, stearic acid, stearic acid, stearic acid Nonadecyl acid, erucic acid, 13Z, 16Z-Docosadienic acid, especially preferred In addition, IL-10, HGF, palmitic acid, margaric acid, stearic acid, oleic acid, 13Z, 16Z-Docosadienic acid are exemplified, and fine particles containing these are preferable.

  In the present invention, the mesenchymal stem cell refers to one or more cells belonging to the mesenchymal system (bone cells, cardiomyocytes, chondrocytes, tendon cells, fat cells, etc.), preferably two or more cells, more preferably three. It means a cell that has the ability to differentiate into the above cells and can proliferate while maintaining the ability. The term mesenchymal stem cell used in the present invention means the same cell as the stromal cell, and does not particularly distinguish them. Moreover, it may be simply described as a mesenchymal cell. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, Examples include dental pulp and tooth germ. For example, an adipose tissue-derived mesenchymal stem cell means a mesenchymal stem cell contained in an adipose tissue, and may be referred to as an adipose tissue-derived stromal cell. Among these, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived from the viewpoint of efficacy and availability for treatment of immune diseases and liver diseases Mesenchymal stem cells and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and adipose tissue-derived mesenchymal stem cells are most preferred.

  The mesenchymal stem cells in the present invention may be derived from the same species as the subject (subject) to be treated, or may be derived from different species. Examples of the mesenchymal stem cell species in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, preferably cells derived from the same species as the subject to be treated (subject). . The mesenchymal stem cell in the present invention may be derived from a subject (subject) to be treated, that is, an autologous cell, or may be derived from another subject of the same type, that is, an autologous cell. An allogeneic cell is preferable.

  Since mesenchymal stem cells are less likely to cause rejection even for allogeneic subjects, the mesenchymal system in the disease therapeutic agent of the present invention is prepared by pre-cultivating donor cells prepared in advance and cryopreserved. Can be used as stem cells. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, commercialization is easy, and from the viewpoint that a certain effect can be easily obtained stably, the mesenchymal stem cells in the present invention, More preferably, it is allogeneic.

  In the present invention, the mesenchymal stem cell means any cell population containing mesenchymal stem cells. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are mesenchymal stem cells.

  The adipose tissue in the present invention means a tissue containing adipocytes and stromal cells including microvascular cells and the like, for example, a tissue obtained by surgical excision or aspiration of mammalian subcutaneous fat. Adipose tissue can be obtained from subcutaneous fat. The adipose tissue-derived mesenchymal stem cells described later are preferably obtained from the same animal as the subject of administration, and considering administration to humans, human subcutaneous fat is more preferable. An individual supplying subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably a tissue collected from a living individual. In the case of collecting from an individual, liposuction is exemplified by PAL (power assist) liposuction, Erconia laser liposuction, or body jet liposuction. From the viewpoint of maintaining the state of cells, ultrasound is used. It is preferable not to use.

  In the present invention, the umbilical cord is a white tubular tissue connecting the fetus and the placenta, and is composed of umbilical vein, umbilical artery, collagenous tissue (Wharton's Jelly), umbilical matrix itself, etc., and mesenchymal stem cells Including many. The umbilical cord is preferably obtained from the same animal as the subject (administration subject) using the disease therapeutic agent of the present invention, and more preferably in consideration of administering the disease therapeutic agent of the present invention to a human. The umbilical cord.

  In the present invention, bone marrow refers to soft tissue filling the bone lumen, and is a hematopoietic organ. Bone marrow fluid is present in the bone marrow, and the cells present therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, and the like. Bone marrow cells can be collected, for example, from human iliac bone, long bone, or other bone.

  In the present invention, the tissue-derived mesenchymal stem cells such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells, respectively. It means any cell population containing mesenchymal stem cells derived from each tissue such as stem cells and bone marrow-derived mesenchymal stem cells. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are mesenchymal stem cells derived from each tissue such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

  The mesenchymal stem cells in the present invention are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-. In addition to high expression of one or more selected from the group consisting of 1-3p and miR-634, for example, growth characteristics (eg, population doubling ability from aging to aging, doubling time), karyotype analysis ( For example, normal karyotype, maternal or neonatal line), surface marker expression by flow cytometry (eg FACS analysis), immunohistochemistry and / or immunocytochemistry (eg epitope detection), gene expression profiling (eg Gene chip array; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression May be characterized by protein profiling (eg, plasma clotting analysis, ELISA, cytokine array), metabolites (metabolomic analysis), other methods known in the art, etc. .

(Method for preparing mesenchymal stem cells and mesenchymal stem cell-derived microparticles)
The group consisting of miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 The method for preparing mesenchymal stem cells and mesenchymal stem cell-derived microparticles in which one or more selected from the above are highly expressed is not particularly limited, and can be prepared, for example, as follows. That is, mesenchymal stem cells are isolated and cultured from adipose tissue, umbilical cord, bone marrow, and other tissues according to a method known to those skilled in the art, and miR-210-3p, miR-6646-5p, miR-6757-3p, miR Using an antibody that specifically binds to any of -23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634, miR-210-3p, miR- One or more selected from the group consisting of 6846-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 are high. Cells and microparticles that are expressed can be obtained by separating them with a cell sorter, magnetic beads, ultracentrifugation, reagents, or the like. Moreover, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-2 in mesenchymal stem cells can be obtained by culturing using a specific medium. By inducing the expression of one or more microRNAs selected from the group consisting of 3p, miR-129-1-3p and miR-634, miR-210-3p, miR-684-5p, miR-6757-3p , MiR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 are highly expressed mesenchymal stem cells And mesenchymal stem cell-derived microparticles can also be obtained. In the cell population and microparticles obtained by this induction, 50% or more of the cell population and microparticles are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5, One or more microRNAs selected from the group consisting of miR-129-2-3p, miR-129-1-3p, and miR-634 are preferably highly expressed, and more than 70% are miR-210-3p, one or more selected from the group consisting of miR-6846-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 It is more preferable that the microRNA is highly expressed, and 80% or more is miR-210-3p, miR-684-5p, m High expression of one or more microRNAs selected from the group consisting of R-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 More preferably, 90% or more is miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129 It is particularly preferred that one or more microRNAs selected from the group consisting of 1-3p and miR-634 are highly expressed, substantially miR-210-3p, miR-6646-5p, miR-6757-3p. , MiR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 Most preferably one or more micro RNA selected from the group is a homogeneous cell population, and trace particles high expression that. MiR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 A method for preparing mesenchymal stem cells and microparticles that are highly expressed in one or more microRNAs selected from the group consisting of:

Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. Below, the preparation method of an adipose tissue origin mesenchymal stem cell is demonstrated as an example. Adipose tissue-derived mesenchymal stem cells may be obtained, for example, by the production method described in US Pat. No. 6,777,231. For example, the adipose tissue-derived mesenchymal stem cells can be produced by a method including the following steps (i) to (iii). it can:
(I) obtaining a cell suspension by digesting adipose tissue with an enzyme;
(Ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not show binding to the solid surface.

  The adipose tissue used in step (i) is preferably washed. Washing may be performed by sedimentation with vigorous stirring using a physiologically compatible saline solution (eg, phosphate buffered saline (PBS)). This is because impurities (also called debris, such as damaged tissue, blood, and red blood cells) contained in the adipose tissue are removed from the tissue. Accordingly, washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. Since the remaining cells exist as lumps of various sizes, in order to dissociate them while minimizing damage to the cells themselves, the washed cell lumps are made to have an enzyme (eg, an enzyme that weakens or breaks cell-cell junctions). It is preferable to treat with collagenase, dispase or trypsin. The amount of such enzyme and the duration of treatment vary depending on the conditions used, but are known in the art. Instead of or in combination with such enzyme treatment, the cell mass can be broken down by other treatment methods such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal cell damage In order to suppress it, it is preferable to carry out only by enzyme treatment. When an enzyme is used, in order to minimize harmful effects on cells, it is desirable to deactivate the enzyme using a medium or the like after a suitable period of time.

  The cell suspension obtained by the step (i) includes a slurry or suspension of aggregated cells and various contaminated cells such as erythrocytes, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the cells in the aggregated state and these contaminated cells may be separated and removed, but they can be removed by adhesion and washing in step (iii) to be described later. May be. When contaminating cells are separated and removed, this can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically compatible solvent. Suspended cells may contain erythrocytes, but erythrocytes are excluded by selection by adhesion to the individual surface described later, and thus a lysis step is not always necessary. As a method for selectively lysing erythrocytes, for example, a method well known in the art such as incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride can be used. After lysis, the lysate may be separated from the desired cells, for example, by filtration, centrifugation, or density fractionation.

  In step (ii), in order to increase the purity of the mesenchymal stem cells in the suspended cells, the cells may be washed once or continuously several times, centrifuged, and resuspended in the medium. Alternatively, cells may be separated based on cell surface marker profile or based on cell size and granularity.

  The medium used in the resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium includes basal medium added with serum and / or albumin, transferrin, fatty acid, One or more serum substitutes such as insulin, sodium selenite, cholesterol, collagen precursor, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, and the like may be added. In addition to these media, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc. are added as necessary. May be.

  Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, Ham's F16 medium, Examples include a medium, Fischer's medium, MCDB201 medium, and a mixed medium thereof.

  Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum and the like. . When using serum, 5 v / v% to 15 v / v%, preferably 10 v / v% may be added to the basal medium.

  Examples of the fatty acid include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of the lipid include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are particularly exemplified, but the polysaccharide is not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF) Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. From the viewpoint of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a (xenofree) medium that does not contain components derived from different species such as serum. Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, and Roto. Has been.

  Subsequently, in step (iii), the appropriate cell density and the appropriate cell medium are used on the solid surface without differentiating the cells in the cell suspension obtained in step (ii). Culture under culture conditions. In the present invention, the “solid surface” means any material that enables binding and adhesion of the adipose tissue-derived mesenchymal stem cells in the present invention. In a particular embodiment, such a material is a plastic material that has been treated to promote the attachment and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish or a flask is preferably used. The cells are washed after incubation to remove unbound cells and cell debris.

  In the present invention, cells that finally remain in a state of being bonded and adhered to the solid surface can be selected as a cell population of adipose tissue-derived mesenchymal stem cells.

  In order to confirm that the selected cells are adipose tissue-derived mesenchymal stem cells in the present invention, surface antigens may be analyzed by a conventional method using flow cytometry or the like. Further, the ability to differentiate into each cell line may be examined, and such differentiation can be performed by conventional methods.

Mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics;
(1) It exhibits adhesiveness to plastic under the culture conditions in a standard medium.
(2) Surface antigens CD44, CD73, CD90 are positive, CD31, CD45 are negative, and (3) Differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.

  From the mesenchymal stem cells obtained by the above step (iii), miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p , MiR-129-1-3p and miR-634 can be obtained by selective separation by immunological techniques using a cell sorter, magnetic beads or the like. In addition, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 By culturing in a specific medium capable of inducing expression, each miRNA expression in mesenchymal stem cells is induced and efficiently miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c MiR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 highly expressing mesenchymal stem cells and mesenchymal stem cell-derived microparticles can also be obtained. Furthermore, by culturing the mesenchymal stem cells obtained by the above step (iii) under specific culture conditions such as hypoxic culture, each miRNA expression in the mesenchymal stem cells is induced, and miR is efficiently performed. Between -210-3p, miR-6844-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 Leaf stem cells and mesenchymal stem cell-derived microparticles can also be obtained. As the conditions for the low oxygen culture, the oxygen concentration is preferably about 0% to 5%, more preferably 0.2% to 3%, further preferably 0.5% to 2%, and particularly preferably around 1%.

The cell suspension obtained by treating the prepared mesenchymal stem cells with a trypsin / EDTA solution or the like is centrifuged (room temperature, 400 G, 5 minutes) to remove the supernatant. Add Staining Buffer (1% BSA-PBS) to the cells to prepare 1 × 10 ⁶ cells / 500 uL, homogenize the cell suspension concentration by pipetting, and then add 50 uL to a new 1.5 mL microtube. Dispense one by one. A primary antibody (Mouse anti human TFPI, manufactured by Sekisui Diagnostics, ADG4903) is added to the dispensed cell suspension at a concentration of 5 to 20 μg / mL, and then suspended for 30 minutes to 1 hour under light shielding and refrigeration. React. After washing 3 times with 1 mL of Staining Buffer, Staining Buffer was added to make 50 uL, and a secondary antibody (Anti Mouse IgG alexar 488, manufactured by Thermofisher Scientific, A21202) was added at a concentration of 1 to 10 μg / mL, and suspended. React for 30 minutes to 1 hour in the dark and under refrigeration. After washing 3 times with 1 mL of Staining Buffer, PI Buffer (prepared by adding 28.8 μL of Propium iodide solution (manufactured by SIGMA, P4864) to 14.4 mL of Staining buffer) and adding 300 uL of suspension well, with tube with cell strainer The separation can be performed by fluorescence activated cell sorting (FACS).

(Cryopreservation of mesenchymal stem cells)
The mesenchymal stem cell according to the present invention may be a cell that has been appropriately cryopreserved and thawed as long as it has a disease therapeutic effect. In the present invention, cryopreservation can be performed by suspending and cooling mesenchymal stem cells in a cryopreservation solution well known to those skilled in the art. The suspension can be performed by detaching the cells with a release agent such as trypsin, transferring the cells to a cryopreservation container, treating them appropriately, and then adding a cryopreservation solution.

  The cryopreservation solution may contain DMSO (dimethylsulfoxide) as a frost damage protective agent, but DMSO has a property of inducing differentiation of mesenchymal stem cells in addition to cytotoxicity. Is preferably reduced. As an alternative to DMSO, glycerol, propylene glycol or polysaccharides are exemplified. When DMSO is used, it contains a concentration of 5% to 20%, preferably a concentration of 5% to 10%, more preferably a concentration of 10%. In addition, additives described in WO2007 / 058308 may be included. As such a cryopreservation solution, for example, cryopreservation provided by Bioverde, Nippon Genetics, Reprocell, Xenoac, Cosmo Bio, Kojin Bio, Thermo Fisher Scientific, etc. A liquid may be used.

  When the above-mentioned suspended cells are cryopreserved, they can be stored at a temperature between −80 ° C. and −100 ° C. (eg, −80 ° C.), and any freezer that can achieve this temperature is used. obtain. Although not particularly limited, in order to avoid a sudden temperature change, the cooling rate may be appropriately controlled using a program freezer. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.

  The upper limit of the storage period is not particularly limited as long as the cells cryopreserved under the above conditions are thawed and retain the same properties as before freezing, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing it at a lower temperature, it may be stored by transferring to a gas phase (about −150 ° C. or lower to −180 ° C. or lower) on liquid nitrogen. When storing in a gas phase on liquid nitrogen, it can be performed using a storage container well known to those skilled in the art. Although not particularly limited, for example, when storing for 2 weeks or more, it is preferable to store in a gas phase on liquid nitrogen.

The thawed mesenchymal stem cells may be appropriately cultured before the next cryopreservation. Culture of mesenchymal stem cells is performed using a medium capable of culturing the mesenchymal stem cells described above is not particularly limited, about 30 to 40 ° C., preferably at a culture temperature of about 37 ° C., the CO ₂ containing air It may be performed under an atmosphere. CO ₂ concentration is about 2-5%, preferably about 5%. In culture, after reaching the appropriate confluency for the culture container (for example, cells may occupy 50% to 80% of the culture container), the cells are detached with a release agent such as trypsin. Alternatively, the culture may be continued by seeding in a separately prepared culture vessel at an appropriate cell density. When seeding cells, typical cell densities are 100 cells / cm ^{2 to} 100,000 cells / cm ² , 500 cells / cm ^{2 to} 50,000 cells / cm ² , 1,000 to 10,000 cells. / ^cm 2, such as 2,000 to 10,000 cells / cm ² is exemplified. In a particular embodiment, the cell density is 2,000 to 10,000 cells / cm ^2. It is preferable to adjust the period until reaching appropriate confluency from 3 days to 7 days. During culture, the medium may be changed as necessary.

  Thawing cryopreserved cells can be performed by methods well known to those skilled in the art. For example, the method performed by standing or shaking in a 37 degreeC thermostat or a hot water bath is illustrated.

  The mesenchymal stem cell of the present invention may be a cell in any state, for example, a cell recovered by detaching a cell in culture or a cell frozen in a cryopreservation solution Good. Use of the same lot of cells obtained by expanding and culturing in the same lot is preferable in that the same action and effect can be stably obtained and the handling property is excellent. The mesenchymal stem cells in a cryopreserved state may be thawed immediately before use and directly mixed with a solution such as an infusion solution or a medium while being suspended in the cryopreservation solution. Alternatively, the cryopreservation solution may be removed by a method such as centrifugation and then suspended in a solution such as an infusion solution or a medium. Here, the “infusion” in the present invention refers to a solution used in human treatment, and is not particularly limited. For example, physiological saline, JP physiological saline, 5% glucose solution, JP Glucose injection, Ringer solution, JP Ringer solution, Lactated Ringer solution, Ringer acetate solution, No. 1 solution (starting solution), No. 2 solution (dehydration replenishment solution), No. 3 solution (maintenance solution), No. 4 solution (postoperative recovery solution) Etc.

[Mesenchymal stem cell-derived microparticles]
The microparticles derived from mesenchymal stem cells of the present invention are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR. One or more selected from the group consisting of -129-1-3p and miR-634 is highly expressed.

  Compared with other microparticles, the mesenchymal stem cell-derived microparticles of the present invention have miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR- It is sufficient that one or more selected from the group consisting of 129-2-3p, miR-129-1-3p, and miR-634 is highly expressed. Specifically, it is derived from the mesenchymal stem cell of the present invention. The microparticles are miR-210-3p, miR-684-5p, miR-6757-3p, miR-, compared to microparticles obtained under conventional culture conditions (for example, culturing with DMEM medium containing 10% FBS). It is sufficient that at least one selected from the group consisting of 23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 is highly expressed. Preferably, it is expressed 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to mesenchymal stem cells obtained under conventional culture conditions. In addition, the mesenchymal stem cell-derived microparticles of the present invention are miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, compared to other microparticles. It is sufficient that at least one selected from the group consisting of miR-129-2-3p, miR-129-1-3p and miR-634 is highly expressed, preferably two or more, more preferably three or more More preferably, four or more, particularly preferably five or more, more particularly preferably six or more, and most preferably all of these microRNAs are highly expressed.

  The microparticles in the present invention are microparticles obtained from mesenchymal stem cells and are produced by mesenchymal stem cells. The mesenchymal stem cell-derived microparticles in the present invention may be contained in a mesenchymal stem cell, may be contained in a mesenchymal stem cell culture supernatant, and may be contained in a mesenchymal stem cell or mesenchymal stem cell. It may be a microparticle isolated from the culture supernatant. That is, the mesenchymal stem cell-derived microparticles in the present invention may be in any form, not only the microparticles themselves, but also mesenchymal stem cells having the ability to contain or secrete microparticles. May be. Examples of the method for isolating microparticles include ultracentrifugation, microfiltration, antibody capture, and use of a microfluidic system. The isolated microparticles may contain cells such as mesenchymal stem cells and may contain mesenchymal stem cell culture medium.

The mesenchymal stem cell-derived microparticles in the present invention can be recovered from a culture supernatant obtained by culturing mesenchymal stem cells using the above-mentioned medium. When recovering the culture supernatant, for example, the mesenchymal stem cells are brought into a subconfluent or confluent state in a culture vessel, replaced with a new medium, and further cultured for 1 to 5 days. The supernatant can be collected. Although this culture supernatant can be used as a therapeutic agent for liver disease or immunology of the present invention, microparticles are separated by ultracentrifugation, density gradient centrifugation, various microparticle separation kits, etc. It can also be used as a material for the therapeutic agent for immune disease or therapeutic agent for liver disease of the invention.
The therapeutic agent for immune diseases and liver diseases of the present invention may contain mesenchymal stem cells themselves that include or secrete the microparticles in addition to the microparticles. In addition, when the therapeutic agent for immune disease and liver disease includes mesenchymal stem cells that include or secrete the microparticles, the microparticles are included at the same time by including the mesenchymal stem cells. It can be interpreted that the above requirement will be satisfied.

  The specific description of the mesenchymal stem cell-derived microparticles in the present invention applies the description of the microparticles contained in the cells of the mesenchymal stem cells in the above-mentioned section of the mesenchymal stem cells of the present invention. be able to.

[Immune disease therapeutic agent and liver disease therapeutic agent]
The therapeutic agents for immune diseases and hepatic diseases of the present invention are the miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR- of the present invention described above. A mesenchymal stem cell (MSC) that highly expresses one or more microRNAs selected from the group consisting of 129-2-3p, miR-129-1-3p, and miR-634, or miR From the group consisting of -210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 It contains mesenchymal stem cell-derived microparticles that highly express one or more selected microRNAs. According to the therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention, the immune disease and liver disease can be improved. For the mesenchymal stem cells and mesenchymal stem cell-derived microparticles contained in the therapeutic agent for immune disease and the therapeutic agent for liver disease of the present invention, the description in the above-mentioned section of mesenchymal stem cells and mesenchymal stem cell-derived microparticles is applied. it can.

  The therapeutic agent for immune disease and the therapeutic agent for liver disease of the present invention are within the range that does not impair the effects of the present invention, in addition to the mesenchymal stem cells or mesenchymal stem cell-derived microparticles, depending on their use and form. According to a conventional method, a pharmaceutically acceptable carrier or additive may be contained. Examples of such carriers and additives include isotonic agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents. , Oily base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizer, dispersant, emulsifier, buffer, solubilizer , Antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances or refreshing agents, but are not limited thereto. Examples of typical components include the following carriers and additives.

  Examples of the carrier include aqueous carriers such as water and hydrous ethanol; examples of the isotonic agent (inorganic salt) include sodium chloride, potassium chloride, calcium chloride and magnesium chloride; examples of the polyhydric alcohol include , Glycerin, propylene glycol, polyethylene glycol and the like; Examples of the thickener include carboxyvinyl polymer, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, alginic acid, polyvinyl alcohol (completely or partially saponified product), polyvinylpyrrolidone, macrogol Examples of sugars include cyclodextrin and glucose; Examples of sugar alcohols include xylitol, sorbitol, mannitol, and the like (which may be any of d-form, l-form, or dl-form). Examples of the antiseptic, bactericidal agent or antibacterial agent include dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid , Potassium sorbate, trometamol, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, hydrochloric acid Polyhexanide (polyhexamethylene biguanide, etc.), glowkill (trade name, manufactured by Rhodia), etc .; examples of pH regulators include hydrochloric acid, boric acid, Noethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, di Isopropanolamine, sulfuric acid, magnesium sulfate, phosphoric acid, polyphosphoric acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconolactone, ammonium acetate, etc .; For example, dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (Longalite), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glyceryl monostearate, Sodium hydrogen sulfate, sodium sulfite, and the like; Examples of the oily base include vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, and cottonseed oil; and medium chain fatty acid triglycerides; Examples of gel bases include, for example, carboxyvinyl polymer, gums, etc .; Examples of surfactants include polysorbate 80, hydrogenated castor oil, glycerin fatty acid ester, sorbitan sesquioleate, and the like; Examples thereof include honey beeswax and various surfactants, gum arabic, gum arabic powder, xanthan gum, soybean lecithin, and the like. Examples of binders include hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose sodium. Excipients such as sucrose, lactose, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like; lubricants such as sucrose fatty acid esters, Magnesium stearate, talc, etc .; Examples of disintegrants include low-substituted hydroxypropylcellulose, crospovidone, croscarmellose sodium, etc .; Examples of foaming agents include sodium bicarbonate; Examples thereof include sodium aluminate metasilicate and light anhydrous silicic acid.

  The therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention can be provided in various forms according to the purpose, for example, various dosage forms such as a solid preparation, a semi-solid preparation, and a liquid preparation. For example, solid preparations (tablets, powders, powders, granules, capsules, etc.), semi-solid preparations [ointment (hard ointment, ointment etc.), creams, etc.], liquid preparations (lotions, extracts, suspensions) Suspensions, emulsions, syrups, injections (including infusions, implants, continuous injections, injections prepared at the time of use), dialysis agents, aerosols, soft capsules, drinks, etc.], patches It can be used in the form of an agent, a poultice and the like. In addition, the therapeutic agent for immune disease and the therapeutic agent for liver disease of the present invention can be used in the form of a solution or emulsion in an oily or aqueous vehicle. Furthermore, the immune disease and liver disease therapeutic agent of the present invention can be applied to the affected area by spraying, and the immune disease therapeutic agent and liver disease therapeutic agent of the present invention are gelled or sheeted in the affected area after spraying. Can also be used. The therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention can also be applied to the affected area after the mesenchymal stem cells are made into a sheet or a three-dimensional structure.

  The therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention are physiological saline, JP physiological saline solution, 5% glucose solution, JP glucose injection solution, Ringer solution, JP Ringer solution, lactated Ringer solution, acetate Ringer solution, bicarbonate. Use Ringer's solution, No. 1 solution (starting solution), No. 2 solution (dehydration replenisher), No. 3 solution (maintenance solution), No. 4 solution (postoperative recovery solution), or cell culture media such as DMEM. Suspended or diluted, preferably with physiological saline, 5% glucose solution, No. 1 solution (starting solution), more preferably with 5% glucose solution, No. 1 solution (starting solution). It can be used turbid or diluted.

  When the immune disease therapeutic agent and liver disease therapeutic agent of the present invention are liquids, the pH of the immune disease therapeutic agent and liver disease therapeutic agent is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. However, as an example, there is a range of 2.5 to 9.0, preferably 3.0 to 8.5, more preferably 3.5 to 8.0. Can be mentioned.

  When the therapeutic agent for immune disease and the therapeutic agent for liver disease of the present invention are liquids, the osmotic pressure of the therapeutic agent for immune disease and the therapeutic agent for liver disease is not particularly limited as long as it is within the acceptable range for the living body. As an example of the osmotic pressure ratio of the composition of the present invention, a range of preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and still more preferably 0.9 to 1.4. The osmotic pressure can be adjusted by a method known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, saccharides and the like. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 15th revised Japanese Pharmacopoeia. Measure by referring to the freezing point method. The standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution) was dried in sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 ° C. for 40 to 50 minutes, and then in a desiccator (silica gel). The mixture is allowed to cool and 0.900 g is accurately weighed and dissolved in purified water to make exactly 100 mL, or a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) is used.

  The administration route to the subject of the therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention is oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, sublingual administration. Administration, rectal administration, vaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implant, direct administration by spraying on the surface of organs and sticking sheets, etc., and the immunity of the present invention From the viewpoint of the effectiveness of a therapeutic agent for diseases and a therapeutic agent for liver diseases, it is preferably an implant, intrahepatic arterial administration, intravenous administration, and direct administration by spraying on the surface of an organ or sticking a sheet, etc., to reduce the burden on the subject. In view of the above, intravenous administration is more preferable.

In the therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention, the dose (dosage) is the patient's condition (body weight, age, symptoms, physical condition, etc.), and the therapeutic agent for immune disease and liver disease treatment of the present invention. The amount may vary depending on the dosage form of the agent, but from the viewpoint of achieving the therapeutic effect of a sufficient immune disease therapeutic agent and liver disease therapeutic agent, a larger amount tends to be preferable, while side effects are suppressed. From the viewpoint, the smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 1 × 10 ^{3 to} 1 × 10 ¹² cells / time, preferably 1 × 10 ^{4 to} 1 × 10 ¹¹ cells / time, more preferably 1 × 10 ^{5 to} 1 × ^{10 10} cells / time, and even more preferably 5 × ¹⁰ 5 ^{6 to 1} × 10 ⁹ pieces / time. The dose per body weight of the patient is 1 × 10 ⁵ to 10 × ^{10 10} cells / kg, preferably 1 × 10 ^{2 to} 5 × 10 ⁹ cells / kg, more preferably 1 × 10 ^{3 to} 5 × 10 ⁸ cells / kg, and further preferably 1 × 10 ^{4 to} 5 × 10 ⁵ . ⁷ / kg. In addition, this dose may be administered multiple times as a single dose, or this dose may be administered in multiple doses.

  The therapeutic agent for immune disease and therapeutic agent for liver disease of the present invention may be administered together with one or more other drugs. Other agents include any agent that can be used as a therapeutic for immune disorders, such as abciximab, adalimumab, alemtuzumab, basiliximab, cetuximab, daclizumab, efalizumab, gemtuzumab, ibritumomab, infliximab, mepolizumab, Monoclonal antibodies such as OKT3, omalizumab, palivizumab, pexelizumab, rituximab, tositumomab, trastuzumab, fusion proteins such as alefacept, denileukin diftitox, etanercept, soluble cytokine receptors such as anakinra, IFN-α, IFN-β, IFN-γ , IL-2, IL-11, erythropoietin, G-CSF, GM-CSF and other cytokines, cyclosporine, azathioprine, mizoribine, tacroli Scan hydrate, gusperimus hydrochloride, mycophenolate mofetil, everolimus, and the like. Examples of other drugs include drugs that can be used as therapeutic drugs for the liver, such as hepatitis B drugs (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon α, interferon, etc.). α-2b, interferon β, peginterferon α-2a, peginterferon α-2b, etc.), hepatitis C drug (ribavirin, telapyrevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), corticosteroid (prednisolone, methyl) Prednisolone sodium succinate, etc.), anticoagulants (dry concentrated human antithrombin III, gabexate mesylate, thrombomodulin α, etc.), antidote (calcium disodium edetate hydrate, gluta ON, dimethicaprol, sodium thiosulfate hydrate, sugamadesk sodium, etc., human serum albumin, liver extract, ursodeoxycholic acid, glycyrrhizic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine, L Isoleucine, L-leucine, L-valine, L-threonine, L-serine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and their salts Etc.), vitamins (tocopherol, flavin adenine dinucleotide, thiamine phosphate disulfide, pyridoxine, cyanocobalamin and their salts), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride) ), And the like.

  The mesenchymal stem cell or mesenchymal stem cell-derived microparticle of the present invention can be used for various immune diseases and immune disorders. Specific diseases include graft-versus-host disease (GVHD), secondary immune deficiency. Disease, primary immunodeficiency disease, B cell deficiency, T cell deficiency, B and T cell complex deficiency, NK cell deficiency, phagocytic deficiency, complement deficiency in classical pathway, complement deficiency in MBL pathway, complement in alternative pathway Immunodeficiency diseases such as body deficiency, complement regulatory protein deficiency, complement receptor deficiency, telangiectasia ataxia, Chediak-East syndrome, chronic granulomatous disease, general variant immunodeficiency, Di George syndrome , High IgE syndrome, high IgM syndrome, IgA deficiency, leukocyte adhesion deficiency, severe combined immunodeficiency, transient hypogammaglobulinemia, Viscott-Oldrich syndrome, X-linked Gamma globulinemia (Breton's disease), X-linked lymphoproliferative syndrome (Duncan syndrome), ZAP-70 deficiency, angioedema, hereditary angioedema, allergic rhinitis, food allergy, anaphylaxis, autoimmune disease And allergic diseases such as drug hypersensitivity and mastocytosis and hypersensitivity diseases.

The mesenchymal stem cells or mesenchymal stem cell-derived microparticles of the present invention can be used for various liver diseases and liver disorders. Specific diseases include autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, virus Hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver (liver NA) , Liver fibrosis, cirrhosis, liver cancer, fatty liver, drug allergic liver disorder, hemochromatosis, hemosiderosis, Wilson disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver Abscess, chronic activity Examples include liver diseases such as dynamic hepatitis and chronic persistent hepatitis.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Example 1
[Preparation and culture of adipose-derived mesenchymal stem cells]
Adipose-derived mesenchymal stem cells (Lonza Japan Co., Ltd., P #: PT-5006) were cultured at 37 ° C. under low oxygen concentration (1%) for 72 hours, and low oxygen concentration cultured adipose tissue-derived mesenchymal stem cells were Obtained. From the culture supernatant, microparticles derived from mesenchymal stem cells derived from low-oxygen concentration culture-treated adipose tissue were obtained. When the content of microRNA in the microparticles was measured by the microarray method, miR-210-3p, miR-6844-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p , MiR-129-1-3p and miR-634 content was found to be higher than that of mesenchymal stem cell-derived microparticles cultured under normal oxygen concentration (atmospheric concentration) . The results are shown in FIG.

(Example 2)
[Confirmation of therapeutic effect using autoimmune hepatitis model mice]
Autoimmune hepatitis was induced by a single administration of 20 mg / kg of concanavalin A (sigma-aldrich) to mice (BALB / cA, CLEA Japan). This autoimmune hepatitis-induced mouse was treated with miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129- obtained in Example 1. 2 × p, miR-129-1-3p and miR-634 highly expressed adipose-derived mesenchymal stem cells (hASC-H) and normal adipose-derived mesenchymal stem cells (hASC) 1 × 10 ⁶ cells Intravenous administration was performed to evaluate liver damage. In addition, the animal (Control) and the healthy animal (Normal) which do not administer a cell were provided as a comparison object. The measurement results of the liver injury marker are shown in FIG. 2 (AST) and FIG. 3 (ALT). AST and ALT were measured with a transaminase measurement kit.

  As shown in FIGS. 2 and 3, AST and ALT, which are markers of liver damage, were elevated by administration of concanavalin A (Control) as compared with healthy mice (Normal). In contrast, administration of normal adipose-derived mesenchymal stem cells (hASC) significantly suppressed the increase, but miR-210-3p, miR-684-5p, miR-6757-3p, By administering adipose-derived mesenchymal stem cells (hASC-H) that highly expressed miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634, Furthermore, hepatitis suppression effect increased. From the above, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 It was revealed that the adipose-derived mesenchymal stem cells that highly expressed the expression of autoimmune hepatitis significantly increased compared to the normal adipose-derived mesenchymal stem cells.

(Example 3)
[Preparation of microparticles and examination of autoimmune hepatitis treatment effect]
The culture supernatant of adipose-derived mesenchymal stem cells prepared in the same manner as described above was collected, and the collected culture supernatant was filtered with a filter (0.22 μm, Merck Millipore), and then centrifuged (35,000 rpm, 70 minutes, 4 minutes Microparticles were collected by BECKMAN Optima XE-90 at 0 ° C. The average particle diameter of the obtained fine particles was 145 ± 4.5 nm (average ± standard error, n = 3).

  The obtained miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 Adipose-derived mesenchymal stem cells (hASC-H) and microparticles derived from normal adipose-derived mesenchymal stem cells (hASC), Exo (hASC-H) and Exo (hASC), respectively, as in Example 2. In addition, 0.4 μg was administered to each mouse in which autoimmune hepatitis was induced, and liver damage was evaluated. In addition, as a comparison object, an animal (Control) and a healthy animal (Normal) to which no microparticles were administered were provided. The measurement results of the liver injury marker are shown in FIG. 4 (AST) and FIG. 5 (ALT). AST and ALT were measured with a transaminase measurement kit.

  As shown in FIGS. 4 and 5, AST and ALT, which are markers for liver damage, were elevated by the administration of concanavalin A (Control), as compared with healthy mice (Normal). In contrast, the administration of fat-derived mesenchymal stem cell-derived microparticles Exo (hASC) significantly suppressed the increase, but miR-210-3p, miR-684-5p, miR-6757- 3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634-derived adipose-derived mesenchymal stem cell-derived microparticles Exo (hASC-H) The inhibitory effect was further increased by administering. From the above, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 It has been clarified that fat-derived mesenchymal stem cell-derived microparticles that highly expressed phenoline significantly increase the therapeutic effect on autoimmune hepatitis compared to normal fat-derived mesenchymal stem cell-derived microparticles.

  From the above results, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR Adipose-derived mesenchymal stem cells highly expressing -634, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, It was suggested that adipose-derived mesenchymal stem cell-derived microparticles that highly expressed miR-129-1-3p and miR-634 have significant therapeutic effects on immune diseases and liver diseases.

  MiR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR- of the present invention Adipose-derived mesenchymal stem cells highly expressing 634, miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR The adipose-derived mesenchymal stem cell-derived microparticles that highly express -129-1-3p and miR-634 are suitably used as a therapeutic agent exhibiting a significant therapeutic effect against immune diseases and liver diseases.

Claims (9)

  The group consisting of miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 A mesenchymal stem cell, wherein one or more microRNAs selected from the above are highly expressed.   The mesenchymal stem cell according to claim 1, wherein the microRNA is contained in a microparticle derived from a mesenchymal stem cell.   The mesenchymal stem cell according to claim 2, wherein the microparticle is an exosome.   The mesenchymal stem cell according to any one of claims 1 to 3, which is derived from another family.   The mesenchymal stem cell according to any one of claims 1 to 4, which is derived from adipose tissue.   The therapeutic agent for a disease containing the mesenchymal stem cell of any one of Claim 1 to 5, and / or its culture supernatant.   The disease therapeutic agent containing the microparticle derived from the mesenchymal stem cell of any one of Claim 1 to 5.   The disease therapeutic agent according to claim 6 or 7, wherein the disease is an immune disease or a liver disease.   The group consisting of miR-210-3p, miR-684-5p, miR-6757-3p, miR-23c, miR-195-5p, miR-129-2-3p, miR-129-1-3p and miR-634 A microparticle characterized in that one or more selected microRNAs are highly expressed.

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