WO2015023165A1 - Composite de régulation d'une inflammation et cellules souches mésenchymateuse stabilisées ayant une fonction optimisée de régulation de l'immunité par blocage de la molécule de signalisation stat3 - Google Patents

Composite de régulation d'une inflammation et cellules souches mésenchymateuse stabilisées ayant une fonction optimisée de régulation de l'immunité par blocage de la molécule de signalisation stat3 Download PDF

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WO2015023165A1
WO2015023165A1 PCT/KR2014/007640 KR2014007640W WO2015023165A1 WO 2015023165 A1 WO2015023165 A1 WO 2015023165A1 KR 2014007640 W KR2014007640 W KR 2014007640W WO 2015023165 A1 WO2015023165 A1 WO 2015023165A1
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mesenchymal stem
stem cells
srage
disease
metformin
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PCT/KR2014/007640
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English (en)
Korean (ko)
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조미라
양철우
박민정
이성희
이정아
문영미
서현범
김은경
이선영
박성환
문수진
곽승기
정정희
양은지
김석중
이은정
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가톨릭대학교 산학협력단
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Priority claimed from KR1020140104574A external-priority patent/KR101659158B1/ko
Application filed by 가톨릭대학교 산학협력단 filed Critical 가톨릭대학교 산학협력단
Priority claimed from KR1020140106718A external-priority patent/KR101636139B1/ko
Publication of WO2015023165A1 publication Critical patent/WO2015023165A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a sRAGE-overexpressing mesenchymal stem cells excellent in immunomodulatory activity and stability, and a cell therapeutic composition for preventing or treating immune diseases including the same.
  • the present invention also relates to a mesenchymal-mesenchymal mesenchymal stem cells treated with metformin, and a cell therapeutic composition for preventing or treating immune diseases including the same.
  • Immunity is one of the body's self-protection systems against all foreign polymers (antigens) that invade or are injected into living tissue.
  • the main component of the immune system is lymphocytes, which are white blood cells that are made in the bone marrow and circulate along the blood into lymph tissues and organs, mainly lymph nodes, spleen, and tonsils.
  • lymphocytes When stimulated by an appropriate antigen, B cells multiply rapidly to form clones that produce specific antibodies (immunoglobulins) that neutralize the antigen.
  • Antibodies produced by B cells circulate in body fluids to perform humoral immunity. T cells are made in the thymus and travel to lymphoid tissue, responsible for cell-mediated immunity that directly attacks antigens.
  • one of the most important traits of all normal individuals is that they do not deleteriously react with the antigenic substances that make up self, whereas many non-self antigens can recognize and react to eliminate them.
  • Is to have the ability to The non-response of the body to autoantigens is called immunologic unresponsiveness or tolerance.
  • an immune response occurs to autoantigens, and as a result, various autoimmune diseases occur while attacking one's own tissues.
  • the main method for treating such autoimmune diseases is mainly used drugs that inhibit the autoimmune function.
  • drugs that inhibit the autoimmune function.
  • cell therapies for the treatment of immune diseases have been developed, and studies to use stem cells as cell therapies are increasing.
  • the mesenchymal stem cells of the stem cells has not yet been revealed the exact mechanism of action on the immunomodulatory ability and there is little research on the production of stem cells having excellent immune disease treatment effect.
  • an object of the present invention is to provide mesenchymal stem cells exhibiting excellent immunomodulatory ability and stability by overexpressing the soluble receptor for advanced glycation endproducts (sRAGE) gene.
  • sRAGE advanced glycation endproducts
  • Another object of the present invention is to provide a cell therapy composition for the prevention or treatment of immune diseases, including mesenchymal stem cells having the immunomodulatory ability of the present invention as an active ingredient.
  • Another object of the present invention is to provide a method for producing mesenchymal stem cells having a mass-produced human transforming growth factor-beta (TGF- ⁇ and immunomodulatory capacity) comprising the step of culturing metformin in mesenchymal stem cells To provide.
  • TGF- ⁇ and immunomodulatory capacity human transforming growth factor-beta
  • another object of the present invention is to provide a mesenchymal stem cell having a mass production of human transforming growth factor-beta (TGF- ⁇ ) produced by the method of the present invention and having immunomodulatory capacity.
  • TGF- ⁇ human transforming growth factor-beta
  • Another object of the present invention to provide a cell therapy composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cells having an immunomodulatory ability according to the present invention as an active ingredient.
  • the present invention provides mesenchymal stem cells having immunomodulatory capacity to which nucleotide sequences encoding soluble receptor for advanced glycation endproducts (sRAGE) are introduced.
  • sRAGE advanced glycation endproducts
  • the sRAGE may have an amino acid sequence of SEQ ID NO: 2.
  • the nucleotides encoding the sRAGE may have a nucleotide sequence of SEQ ID NO: 1.
  • the nucleotide sequence encoding the sRAGE may be introduced by a vector expressing sRAGE in mesenchymal stem cells.
  • the vector is a recombinant vector containing a nucleotide sequence encoding sRAGE, as long as it can overexpress sRAGE in mesenchymal stem cells, the type thereof is not limited.
  • the mesenchymal stem cells may be derived from peripheral blood or adipose tissue, but is not limited thereto.
  • the mesenchymal stem cells exhibit excellent immunomodulatory ability by overexpression of sRAGE, which can be achieved by controlling the expression of various immune or inflammation-related factors.
  • overexpression of sRAGE in mesenchymal stem cells induces increased expression of any one or more factors selected from the group consisting of IDO, TGF-beta, HGF and IL-10.
  • overexpression of sRAGE in the mesenchymal stem cells induces decreased expression of any one or more factors selected from the group consisting of IL-1 ⁇ , IL-6 and HMGB-1.
  • the overexpression of sRAGE in the mesenchymal stem cells induces increased expression of any one or more factors selected from the group consisting of CCR1, CCR3, CCR4, CCR7, CXCR1 and CXCR4.
  • the mesenchymal stem cells may be stabilized by overexpression of sRAGE.
  • sRAGE is essential for stabilizing stem cells themselves.
  • the present invention also provides a cell therapy composition for the prevention or treatment of immune diseases, including mesenchymal stem cells overexpressing the sRAGE as an active ingredient.
  • the composition may be prepared or used to be administered in a number of 2.5 ⁇ 10 5 to 2.5 ⁇ 10 7 cells per kg of body weight of the administered object.
  • the immune disease is osteoarthritis, Rheumatoid Arthritis, Asthma, Dermititis, Psoriasis, Cystic Fibrosis, Late organ transplantation and chronic Post transplantation late and chronic solid organ rejection, Multiple Sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma (scleroderma), Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Mal Malohns disease ), Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angiopl asty restenosis, Chronic obstructive pulmonary diseases (COPD), Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease ), Angina, cancer
  • the present invention provides a method for producing mesenchymal stem cells having a mass production of human transforming growth factor-beta (TGF- ⁇ ), comprising the step of treating metformin to mesenchymal stem cells.
  • TGF- ⁇ human transforming growth factor-beta
  • the mesenchymal stem cells are CD105, CD29 and CD44 is positive and CD34, CD45 and HLA-DR may be a method for producing mesenchymal stem cells, characterized in that it has a negative immunological characteristics. .
  • it may be a method for producing mesenchymal stem cells, characterized in that it further comprises the step of culturing the metformin-treated mesenchymal stem cells for 14 to 21 days at a temperature of 28 ⁇ 42 °C. .
  • the mesenchymal stem cells may be a method for producing mesenchymal stem cells, characterized in that separated from peripheral blood or adipose tissue.
  • the metformin may be a method for producing mesenchymal stem cells, characterized in that treated with a concentration of 0.2 ⁇ 2mM based on the number of mesenchymal stem cells 2 ⁇ 10 5 ⁇ 1 ⁇ 10 6 cells. .
  • the mesenchymal stem cells may be increased by the expression of any one or more factors selected from the group consisting of IDO, TGF-beta and IL-10 by the treatment of metformin.
  • the present invention provides a mesenchymal stem cell having a mass production of human transforming growth factor-beta (TGF- ⁇ ) and immunomodulatory capacity produced by the method of the present invention.
  • TGF- ⁇ human transforming growth factor-beta
  • the present invention also provides a cell therapy composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cells of the present invention as an active ingredient.
  • the composition may be administered mesenchymal stem cells having immunomodulatory capacity in the number of 2 ⁇ 10 5 to 2.5 ⁇ 10 7 cells per kg body weight of the subject.
  • the immune disease is osteoarthritis, Rheumatoid Arthritis, Asthma, Dermititis, Psoriasis, Cystic Fibrosis, Late organ transplantation and chronic Post transplantation late and chronic solid organ rejection, Multiple Sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma (scleroderma), Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Mal Malohns disease ), Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angiopla sty restenosis, Chronic obstructive pulmonary disease (COPD), Graves disease, Gastrointestinal allergy, Conjunctivitis, Atherosclerosis, Coronary artery disease ), Angina,
  • COPD chronic obstructive
  • Mesenchymal stem cells of the present invention exhibits excellent stability by overexpressing sRAGE, and have an effect of increasing the expression of genes and cell migration-related genes with immunomodulatory capacity, and simultaneously inhibit immunosuppression while inhibiting proliferation of inflammatory cytokines and inflammatory cells.
  • Proliferation of T cells has the effect of promoting activity has the effect that can be usefully used as a cell therapy for the treatment of immune diseases.
  • the present invention is a mass production of human transforming growth factor-beta (TGF- ⁇ ) by treating metformin in mesenchymal stem cells having CD105, CD29 and CD44 is positive and CD34, CD45 and HLA-DR have negative immunological characteristics
  • TGF- ⁇ human transforming growth factor-beta
  • the present invention relates to a method for producing mesenchymal stem cells having immunomodulatory capacity, and a mesenchymal stem cell prepared by the above method and a cell therapeutic composition for treating or preventing immune diseases containing the mesenchymal stem cells.
  • Mesenchymal stem cells with immunomodulatory activity have increased expression of any one or more factors selected from the group consisting of immunomodulatory factor IDO, TGF-beta and IL-10, and increase autophagy and mitochondrial activity. Has the effect that can be usefully used as a cell therapy for the treatment of immune diseases.
  • Figure 1 shows a schematic diagram of the recombinant vector pcDNA.3-HA-hsRAGE containing a human-soluble sRAGE (soluble receptor for advanced glycation endproducts) gene prepared according to an embodiment of the present invention.
  • Figure 2 shows the ELISA (left photo) and Western blot (right) to measure the expression level of sRAGE expressed in mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mesenchymal stem cells transformed with mock vector not containing sRAGE. Photo) shows the results.
  • FIG. 3 shows Th1 (IFN- ⁇ ), Th17 (IL-17), and Th2 (IL-) of mesenchymal stem cells transformed with sRAGE-containing recombinant vectors and mesenchymal stem cells transformed with mock vectors not containing sRAGE. 4) and Foxp3 + Tregs cell numbers were analyzed by flow cytometry.
  • Figure 4 shows the expression of immunoregulatory related genes and cell migration-related genes in mesenchymal stem cells transformed with sRAGE-containing recombinant vectors and mesenchymal stem cells transformed with mock vectors not containing sRAGE through real-time PCR. The analysis results are shown.
  • 5 shows the results of analyzing the effect of sRAGE overexpression on T cell differentiation in mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mesenchymal stem cells transformed with mock vector not containing sRAGE.
  • Figure 6 shows the analysis of allogeneic response control effect of mesenchymal stem cells transformed with sRAGE-containing recombinant vector, showing the expression of CFSE expression.
  • Figure 7 shows the results of analyzing allogeneic response control of mesenchymal stem cells transformed with sRAGE-containing recombinant vector, showing the results of analysis of IFN-r, TH17 (IL-17) and Foxp3 + Tregs.
  • Figure 8 shows the results of examining the effects on the cytokine expression of mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mesenchymal stem cells transformed with mock vector not containing sRAGE under homologous reaction conditions.
  • FIG. 9 is a photograph showing observation of the expression level of IDO and IL-10 by fluorescence microscope in mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mesenchymal stem cells transformed with mock vector not containing sRAGE. will be.
  • FIG. 10 shows a photograph of the mesenchymal stem cells transformed with the RAGE-containing recombinant vector and the mesenchymal stem cells transformed with the mock vector not containing sRAGE.
  • FIG. 11 shows the results of analysis of rheumatoid arthritis symptom improvement and treatment effects of sRAGE-overexpressing mesenchymal stem cells of the present invention in a mouse model induced arthritis.
  • Figure 12 shows the results of the improvement and treatment effect of rheumatoid arthritis symptoms of sRAGE-overexpressing mesenchymal stem cells of the present invention through immunochemical staining and flow cytometry.
  • Figures 13 and 14 show the results of examining the immunomodulatory ability of the sRAGE-overexpressing mesenchymal stem cells of the present invention on lupus animal model cells.
  • Figure 15 shows the results of microscopic observation of the cell shape of mesenchymal stem cells isolated from peripheral blood, and the results of analyzing the expression of cell markers of mesenchymal stem cells.
  • 16 is a graph showing the results of analyzing the degree of IDO and TGF-beta expression in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood.
  • FIG. 17 analyzes the expression changes of HMGB-1, IL-6, and IL-1beta, genes related to inflammatory and tumor factors, in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood. One result is graphed.
  • FIG. 18 is a photograph showing the results of observing the activity of autophagic vacuoles and mitochondria through electron microscopy of mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood.
  • 19 is a graph showing the results of analysis of IL-10 and TGF-beta expression levels in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from adipose tissue.
  • FIG. 20 shows photographs of IL-10 and IDO expression in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from adipose tissue by DAPI staining.
  • Figure 21 is a group injected with human adipose tissue-derived mesenchymal stem cells prepared by treating metformin in a mouse model induced osteoarthritis (Met-MSC), a group injecting only metformin untreated mesenchymal stem cells into osteoarthritis mice (MSC) And it shows the results of pain analysis in the osteoarthritis mouse group (MIA; no treatment group) (A), and the degree of destruction of cartilage was observed by staining with India ink staining (B).
  • MIA no treatment group
  • Figure 22 is a group injected with human adipose tissue-derived mesenchymal stem cells prepared by treating metformin in the mouse model induced inflammatory growth disease (MSC + Metformin), the group injected only with mesenchymal stem cells (MSC; metformin untreated group ), Weight change (A), IBD disease activity (B), and length and thickness change (C) of the untreated group (IBD; inflammatory bowel disease group).
  • Figure 23 is a group injected with human adipose tissue-derived mesenchymal stem cells prepared by treating metformin in the mouse model induced inflammatory growth disease (MSC + Metformin), the group injected only with mesenchymal stem cells (MSC; metformin untreated group ), Intestinal inflammatory cell infiltration and changes in cell tissue morphology (A) by H & E staining in inflammatory bowel disease group (IBD) and no treatment group (WT) and TNF-a observation by immunochimistry staining (A) B).
  • Figure 24 is a group injected with human adipose tissue-derived mesenchymal stem cells prepared by treating metformin in the arthritis model IL-1Ra Knockout mouse (Met-MSC), the group injected only with mesenchymal stem cells (MSC; untreated group metformin And arthritis index in the group treated with nothing (ie arthritis invention group; IL-1Ra Knockout mouse).
  • FIG. 25 shows human adipose tissue-derived mesenchymal stem cells (Met-MSC), mesenchymal stem cells (MSC; metformin untreated mesenchymal stem cells) and nothing treated in rheumatoid arthritis animal models.
  • Figure 26 is a group injected with human adipose tissue-derived mesenchymal stem cells prepared by treating metformin in a mouse model induced osteoarthritis (Met-MSC), a group injecting only metformin untreated mesenchymal stem cells into osteoarthritis mice (MSC)
  • Met-MSC mouse model induced osteoarthritis
  • MSC osteoarthritis mice
  • H & E staining, Toluidin staining, and saffranin O staining were analyzed to analyze osteoarthritis and cartilage destruction in the osteoarthritis mice group (MIA; no treatment group).
  • the present inventors confirmed through experiments that sRAGE-overexpressing mesenchymal stem cells are stable and can effectively treat immune diseases, while studying to develop a cell therapy that can effectively treat immune diseases.
  • the present invention can provide sRAGE-overexpressing mesenchymal stem cells with immunomodulatory ability, and can provide a cell therapeutic composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cells with immunomodulatory activity as an active ingredient.
  • a cell therapeutic composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cells with immunomodulatory activity as an active ingredient.
  • RAGE interacts with endogenous ligands, unlike TLRs that interact with exogenous pathogens, and is involved in the development of a variety of diseases caused by chronic inflammation producing a variety of endogenous ligands.
  • mRAGE membrane-bound form of RAGE
  • mRAGE consists of three domains: an extracellular site, a hydrophobic trans-membrane-spanning domain and a short cytoplasmic domain, which play an important role in post-RAGE signaling.
  • RAGE's binding to endogenous ligands includes reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and janus kinase (JAK) / signal converters, and the STAT pathway.
  • ROS reactive oxygen species
  • MAPK mitogen-activated protein kinase
  • PI3K phosphoinositide 3-kinase
  • JAK janus kinase
  • soluble RAGE (hereinafter referred to as sRAGE) has an extracellular site such as RAGE, while it does not have a trans-membrane domain and cytoplasmic domain.
  • sRAGE is formed due to selective splicing of RAGE mRNA or deletion of the membrane portion, capable of binding to RAGE ligands in extracellular space prior to mRAGE, and sRAGE to the extent that mRAGE binds to their ligands It is known to play a role of competitively inhibiting the.
  • mesenchymal stem cells prepared by the treatment of metformin is significantly more effective in treating immune diseases than mesenchymal stem cells not treated with metformin. Excellent was confirmed through experiments.
  • the present invention is characterized in that the metformin of the formula 1 (Metformin) is treated to mesenchymal stem cells, metformin is N, N-Dimethylimidodicarbonimidic diamide by IUPAC nomenclature, the molecular formula is C 4 H 11 N 5 , molecular weight is 129.16364 .
  • the present invention can provide a mesenchymal stem cell having an immunomodulatory ability treated with metformin, a cell therapeutic composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cell having the immunomodulatory capacity as an active ingredient Can provide.
  • the mesenchymal stem cell is a stem cell isolated from bone marrow, blood, dermis, and periosteum, and can differentiate into various cells, such as adipocytes, chondrocytes, bone cells, and the like. Pluripotent or multipotent cells.
  • the mesenchymal stem cells used in the present invention may be animal mesenchymal stem cells, preferably mammalian, more preferably human mesenchymal stem cells.
  • the mesenchymal stem cells of the present invention may be derived from bone marrow, adipose tissue, peripheral blood, liver, lung, amniotic fluid, placenta, chorionic membrane or cord blood, and preferably derived from adipose tissue or peripheral blood.
  • mesenchymal stem cells can be obtained from a variety of sources as described above.
  • the process of obtaining mesenchymal stem cells will be described in detail as follows: (1) a mammal including a human or a mouse, preferably, a human Separating the mesenchymal stem cells from the mesenchymal stem cell source of, for example, blood or bone marrow or adipose tissue; (2) culturing the isolated cells in a suitable medium; And (3) can be obtained through the step of removing the floating cells in the culturing process and passaging the cells attached to the culture plate to obtain the finally constructed mesenchymal stem cells.
  • the medium used in the above process any medium commonly used for culturing stem cells can be used.
  • the medium contains serum (eg fetal calf serum, horse serum and human serum).
  • serum eg fetal calf serum, horse serum and human serum.
  • Medium that can be used in the present invention is, for example, RPMI series, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), ⁇ -MEM (Stanner, CP et al., Nat New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp.
  • the medium may include other components such as antibiotics or antifungal agents (eg, penicillin, streptomycin, etc.), glutamine, and the like.
  • identification of the mesenchymal stem cells isolated and cultured can be performed through flow cytometry.
  • flow cytometry is performed using specific surface markers of mesenchymal stem cells.
  • the mesenchymal stem cells that can be used for the production of mesenchymal stem cells having immunomodulatory capacity in the present invention are CD105, CD29 and CD44 are positive, and CD34, CD45 and HLA-DR are negative cells. Can be.
  • a new mesenchymal stem cell having an immunomodulatory ability transformed into a vector capable of overexpressing sRAGE was prepared for use in treating mesenchymal stem cells derived from various sources for immune diseases.
  • a new mesenchymal stem cell having immunomodulatory capacity was prepared by treating metformin.
  • the sRAGE introduced into the mesenchymal stem cells in the present invention is composed of a gene consisting of a nucleotide sequence represented by SEQ ID NO: 1, and has an amino acid sequence of SEQ ID NO: 2 encoded from the nucleotide sequence of SEQ ID NO: 1.
  • the base sequence encoding the sRAGE may be introduced into a cell by a method known in the art, for example, the sequence may be introduced in its own sequence or vector. Methods of introducing nucleic acid sequences into cells are known in the art, and can be accomplished by methods including, for example, electroporation, methods using calcium phosphate, gene guns, and liposome methods. In addition, the introduction can be made using a virus as a carrier.
  • the base sequence encoding the sRAGE may be integrated in the genome of the cell or exist in the cell separately from the genome.
  • vector refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. From the standpoint of nucleic acid sequences that mediate the introduction of specific genes, in the present invention, a vector is interpreted to be used interchangeably with a nucleic acid construct and a cassette. Vectors include, for example, plasmids or viral derived vectors. Plasmid refers to a circular double stranded DNA ring to which additional DNA can be linked.
  • Vectors used in the present invention include, for example, plasmid expression vectors, virus expression vectors (eg, SV40, replication defective retroviruses, adenoviruses, and adeno-associated viruses) and viral vectors capable of performing their equivalent functions. However, it is not limited to these.
  • the present inventors conducted experiments to confirm the possibility of use as a therapeutic agent for immune diseases in the new mesenchymal stem cells having the immunomodulatory ability.
  • sRAGE overexpressing mesenchymal stem cells are related to the immunomodulatory ability in the cells.
  • the expression of genes such as TGF-beta, IDO, HGF and IL-10 were significantly increased compared to mesenchymal stem cells without sRAGE overexpression, and expression of genes related to cell migration was also significantly increased. .
  • TGF-beta transforming growth factor beta
  • TGF ⁇ 1 comprises 390 amino acids
  • TGF ⁇ 2 and TGF ⁇ 3 each comprise 412 amino acids.
  • TGF ⁇ since TGF ⁇ has growth inhibitory activity against most cells, TGF ⁇ also has growth inhibitory activity against cancer cells expressing the normal TGF ⁇ receptor (Twardzik et al., J. Natl. Cancer Institute, 81: 1182-). 1185, 1989).
  • tumor cells may escape from growth inhibition by TGF ⁇ by inhibiting its expression of TGF ⁇ receptor.
  • the expression of Foxp3 in the presence of TGF-beta is induced from naive T cells and thus induced Tregs have the same immunosuppressive function.
  • deficiency of the TGF-beta gene leads to severe autoimmune diseases because it does not control the activity of inflammatory immune cells.
  • mesenchymal stem cells of the present invention overexpressing sRAGE also have excellent autophagy and mitochondrial activity.
  • the autophagy is a process in which bacteria decompose their organelles or cellular components, and the polymer is extracellular. It is a distinct action from heterophages that are taken by intracellular intake such as negative or phagocytic.
  • cells act to break down their proteins or to remove unnecessary cellular components in the process of rebuilding the cells.
  • the cellular components are surrounded by membranes derived from the endoplasmic reticulum, forming a follicle. It is fused with cotton to form a child pagolyosomal to decompose.
  • autophagy action can control the beneficial or harmful effects of immunity and inflammation and can prevent infectious diseases
  • autoimmunity and inflammatory diseases mesenchymal stem cells overexpressing sRAGE in the present invention are autophagy. It can have more effects in the treatment of immune and inflammatory diseases. In situations where mitochondrial activity is not regulated, more severe inflammation occurs, which is why mitochondrial adverse events are very important in the development of immunoinflammatory diseases. When mitochondrial activity is lowered, the control system of pathogenic cells becomes unstable, which causes excessive cell activity or proliferation.
  • the mesenchymal stem cells of the present invention in the mesenchymal stem cells overexpressing sRAGE, an increase in autophagy vesicles confirmed that the autophagy action was increased compared to the mesenchymal stem cells without sRAGE overexpression and the increase in mitochondrial activity was confirmed. It was found that the mesenchymal stem cells of the present invention can be used as a cell therapy for effective immune disease treatment.
  • the mesenchymal stem cells overexpressing sRAGE with immunomodulatory ability may be used as mesenchymal stem cells isolated from peripheral blood or adipose tissue, and the immune including mesenchymal stem cells having the immunomodulatory ability as an active ingredient. It can be used as a cell therapy composition for the prevention or treatment of diseases.
  • the cell therapeutic agent refers to a method of proliferating or screening live autologous, allogenic, or xenogenic cells in vitro or restoring biological characteristics of cells in order to restore the function of cells and tissues.
  • These cell therapies can be broadly classified into two fields. The first is stem cell therapy for tissue regeneration or long-term function recovery, and the second is immunization for the regulation of immune responses such as suppressing the immune response or enhancing the immune response in vivo. Can be classified as a cell therapy.
  • the route of administration of the cell therapy composition of the present invention may be administered via any general route as long as it can reach the desired tissue.
  • Parenteral administration for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration may be, but is not limited thereto.
  • composition may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy.
  • a pharmaceutical carrier generally used for cell therapy.
  • 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human.
  • Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • composition may also be administered by any device in which the cell therapy agent can migrate to the target cell.
  • the cell therapy composition of the present invention may include a therapeutically effective amount of cell therapy for the treatment of a disease.
  • therapeutically effective amount means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician. This includes amounts that induce alleviation of the symptoms of the disease or disorder being treated.
  • the cell therapy agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy content can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of other components contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient.
  • the cell therapeutic composition of the present invention may be included so that mesenchymal stem cells having immunomodulatory capacity are administered at a number of 2.5 ⁇ 10 5 to 2.5 ⁇ 10 7 cells per kg of body weight.
  • the present invention also provides a method for preventing or treating an immune disease comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the present invention.
  • mammal refers to a mammal that is the subject of treatment, observation or experiment, preferably human.
  • the cell therapy agent included in the composition is 1 ⁇ 10 4 to 1 ⁇ 10 8 per kg of body weight of the administered individual. It is preferable to include the cell number.
  • the composition comprising the cell therapy of the present invention as an active ingredient is rectal, intravenous (iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal. Administration can be by conventional routes.
  • Metformin is an oral antihyperglycemic drug that is currently being used in the treatment of non-insulin dependent diabetes mellitus (NIDDM) by lowering both basal plasma glucose and postprandial plasma glucose. Improve glucose tolerance in NIDDM patients.
  • NIDDM non-insulin dependent diabetes mellitus
  • metformin has been widely used as a type 2 diabetes treatment, and a clinical approach is being taken for polycystic ovary syndrome, weight loss, and cancer treatment.
  • the present inventors have treated the metformin in the mesenchymal stem cells.
  • the expression of related factors, TGF-beta, IDO and IL-10, was found to be markedly increased, especially when metformin was treated to peripheral blood-derived mesenchymal stem cells compared to the group not treated with metformin. Appears to increase about 10-fold (see FIGS. 16 and 18).
  • the present inventors confirmed that the mesenchymal stem cells of the present invention prepared by treating metformin have excellent autophagy and mitochondrial activity.
  • the metformin treatment to the mesenchymal stem cells through the experiment it was confirmed that the increase in autophagy vesicles can be confirmed that the autophagy action was increased compared to the case without the treatment with metformin and metformin treatment because the increase in mitochondrial activity was also confirmed
  • the mesenchymal stem cells can be seen to be more effective in treating immune diseases.
  • the present invention can provide a method for producing mesenchymal stem cells having a mass production of human transforming growth factor-beta (TGF- ⁇ ) and immunomodulatory capacity, wherein CD105, CD29 and CD44 are positive and CD34.
  • CD45 and HLA-DR may include the step of treating metformin to mesenchymal stem cells having negative immunological characteristics, incubating for 14 to 21 days at a temperature of 28 ⁇ 42 °C.
  • Mesenchymal stem cells that can be treated with metformin in the present invention may be used mesenchymal stem cells isolated from peripheral blood or adipose tissue, the metformin is based on the number of mesenchymal stem cells 2 ⁇ 10 5 ⁇ 5 ⁇ 10 5 cells 0.2 It can be treated at a concentration of ⁇ 2 mM.
  • the present invention can provide a cell therapy composition for the prevention or treatment of immune diseases, including mesenchymal stem cells prepared by treating the metformin having the immunomodulatory capacity as an active ingredient.
  • the immune disease to be treated by mesenchymal-treated mesenchymal stem cells prepared by metformin treatment and cell therapy composition for treating immune diseases including the cells is not limited thereto, osteoarthritis, rheumatoid arthritis (Rheumatoid Arthritis), Asthma, Dermititis, Psoriasis, Cystic Fibrosis, Post transplantation late and chronic solid organ rejection, Multiple Sclerosis, Systemic lupus erythematosus Systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia , Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Dese, Crohns disease, autoimmunity Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-ang
  • the present inventors first prepared an expression vector operably linked with the sRAGE gene for the production of mesenchymal stem cells overexpressing sRAGE.
  • the sRAGE gene represented by SEQ ID NO: 1 was cloned into a pcDNA 3.0-HA vector to prepare a recombinant vector of FIG. 1.
  • PBMCs Peripheral Blood Mononuclear Cells
  • FBS Fetal Bovine Serum
  • the medium was changed at 3 days intervals, and the cells were removed from the culture plate and cultured according to the degree of cell proliferation, and the culture was performed three times (passage 3) to obtain mesenchymal stem cells from peripheral blood.
  • Cell markers were checked to determine whether they were mesenchymal stem cells. As a result, CD105, CD29 and CD44 were positive, and CD34, CD45 and HLA-DR were negative to confirm that the isolated cells were mesenchymal stem cells.
  • Example ⁇ 1-2> The mesenchymal stem cells isolated from Example ⁇ 1-2> were transformed with the recombinant vector prepared in Example ⁇ 1-1> to prepare mesenchymal stem cells overexpressing sRAGE.
  • the expression level of sRAGE in the mesenchymal stem cells transformed with the vector containing no sRAGE and the mesenchymal stem cells produced in the present invention was determined by ELISA ( Enzyme-linked immunosorbent assay and western blot were performed.
  • the inventors of the present invention show real-time PCR (polymerase chain reaction) changes in the expression of IDO, TGF-beta and HGF, genes related to immunomodulatory activity in mesenchymal stem cells, by overexpression of sRAGE in sRAGE-overexpressing mesenchymal stem cells. Analyzed using the method. As a result, in the mesenchymal stem cells overexpressing sRAGE, the expression of IDO, TGF-beta and HGF was significantly increased (about 2 to 6 times) compared to mesenchymal stem cells without sRAGE overexpression (Fig. 4A). ).
  • CCR1, CCR3, CCR4, CCR7, CXCR1 and CXCR4 which are factors related to the movement of mesenchymal stem cells.
  • sRAGE overexpression of sRAGE
  • the expression of genes related to cell migration is significantly increased compared to mesenchymal stem cells without sRAGE overexpression, and thus, sRAGE-overexpressing mesenchymal stem cells of the present invention are more effectively treated for inflammatory and immune diseases. It can be used as a cell therapy for.
  • Primer sequences used in PCR to analyze the expression level of each of the factors are as described in Table 1 below.
  • Real-time PCR was used to observe gene expression when an inflammatory environment in mesenchymal stem cells was applied.
  • Mesenchymal stem cells 5 ⁇ 10 5 cells were plated in 6-well plates and incubated at 37 ° C. for 3 days while stimulating with Lg 1ug / ml.
  • LPS-treated mesenchymal stem cells were used as a control, and expression levels of inflammatory and tumor factors in LPS-treated mesenchymal stem cells were analyzed using real-time PCR.
  • IL-1 ⁇ IL-6
  • VEGF Vascular Endothelial Growth Factor
  • HMGB-1 High-Mobility Group protein B1
  • Primer sequences used in the PCR are as described in Table 2 below.
  • sRAGE Overexpression of sRAGE was induced in adipose tissue-derived mesenchymal stem cells and cultured for 3 days, and then the degree of autophagy vesicle formation was observed by electron microscopy.
  • 10 is a photograph of the mesenchymal stem cells transformed with the RAGE-containing recombinant vector and the mesenchymal stem cells transformed with the mock vector not containing the sRAGE through electron microscopy. Stem cells can be observed to increase the number of autophagic vacuoles compared to the control group.
  • the sRAGE-overexpressing mesenchymal stem cells of the present invention prepared in Example 1 were analyzed for changes in cytokine expression levels in mesenchymal stem cells due to overexpression of sRAGE. To this end, the sRAGE-overexpressing mesenchymal stem cells were co-cultured with human CD4 + T cells, and then the amounts of IFN- ⁇ , IL-17, IL-4 and Foxp3 + Treg were measured.
  • Th1 IFN- ⁇
  • Th17 IL-17
  • Th2 IL-4
  • Foxp3 + were significantly decreased.
  • Tregs tended to increase slightly (FIG. 3).
  • the present inventors observed cytokine levels in the culture medium under the above experimental conditions, that is, alloresponse conditions, and showed that the sRAGE-MSC of the present invention effectively reduced the production of inflammatory cytokines compared to other groups (Fig. 8).
  • IL-1Ra knockout mouse was produced according to a method published by the Y. Iwakura team, which lacks the IL-1 receptor gene.
  • the IL-1 receptor antagonist (IL-1Ra) acts directly on the IL-1 receptor, preventing IL-1 ⁇ and IL-1 ⁇ from acting on the receptor, thereby naturally causing autoimmune arthritis diseases.
  • Arthritis-induced mice were injected with sRAGE-overexpressing mesenchymal stem cells prepared in the present invention to the diseased mouse, sRAGE-overexpressing mesenchymal stem cells were injected iv into the mouse at a cell number of 2 ⁇ 10 6 .
  • mice treated with sRAGE-overexpressing mesenchymal stem cells showed improvement in arthritis symptoms compared to the group treated with mesenchymal stem cells only (Fig. 11B), and the Th2 type IgG1 and IgG3 acting on the immune response. The production of was all significantly reduced (see FIG. 11C).
  • joints of each group were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, and tissue sections were made and attached to slides.
  • xylen xylen
  • ethanol was hydrated from high to low concentrations. The staining process was hematoxylin and eosin staining.
  • the mesenchymal stem cells that did not overexpress sRAGE showed arthritis inhibitory effect, but the sRAGE-overexpressing mesenchymal stem cells were injected, but the infiltration of inflammatory cells and cartilage destruction were significantly reduced (Fig. 11D). ).
  • cytokines IFN- ⁇ , IL-17, IL-4 and Foxp
  • Th17 cells which are pathogenic cells
  • Treg cells were observed to increase significantly (FIGS. 12A, B).
  • Th1 IFN- ⁇
  • Th17 IL-17
  • sRAGE-overexpressing mesenchymal stem cells prepared according to the present invention on the lupus animal model cells was investigated.
  • Cells were isolated from Roquin mice, a lupus animal model, co-cultured with mesenchymal stem cells at a ratio of 1:10 for 3 days, and the amount of IL-17 present in the culture was measured by ELISA.
  • the sRAGE-overexpressing mesenchymal stem cells showed a further decrease in the amount of IL-17 in the culture medium and increased the activity of immunoregulatory T cells compared to the mesenchymal stem cell group without overexpressing sRAGE. (FIG. 13).
  • the pathogenic cells of the lupus model can be regulated by the sRAGE-overexpressing mesenchymal stem cells of the present invention.
  • the effect of sRAGE overexpressing mesenchymal stem cells prepared in the above example was co-cultured with human CD4 + T cells and analyzed on T cell differentiation using a flow cytometer.
  • sRAGE-overexpressing mesenchymal stem cells were found to increase Foxp3 + Tregs more than control (MSCs without sRAGE overexpression), and Th1 and Th17 cells were significantly decreased. And Th2 cells did not show a significant difference (Fig. 5).
  • PBMC peripheral blood
  • a-MEM medium containing 20% FBS for 5 days
  • the cells floating on the culture medium were removed.
  • the medium was changed at intervals of 3 days, and the cells were removed from the culture plate and cultured according to the degree of cell proliferation, and the culture was performed three times (passage 3) to obtain mesenchymal stem cells from peripheral blood.
  • Cell markers were identified to confirm whether they were mesenchymal stem cells, and as a result, CD105, CD29, and CD44 were positive, and CD34, CD45, and HLA-DR were negative (see FIG. 15A and 100 times on the left in FIG. 15A).
  • Enlarged picture, the right picture is a 40x magnification).
  • the adipose tissue obtained by liposuction or the adipose tissue obtained after surgery was washed 10 times or more with PBS containing 10% penicillin-streptomycin to remove blood and foreign matter, and then the tissue was chopped to 0.2 to 0.3 g. . 0.2% collagenase (Roche, Sandhofer Strasse, Mannheim, Germany) in a solution was reacted for 1 hour at 100 °C water bath, 37 °C. After separating the solution layer and the undecomposed fragments by the collagenase using a 100 ⁇ m mesh, the same amount of PBS was added to the separated collagenase solution. Subsequently, centrifugation was performed at 4 ° C.
  • MSC basal medium (Cambrex, Walkersville, MD, USA), mesenchymal growth aid (Cambrex, Walkersville, MD, USA), 4 mM to remove remaining collagenase solution from submerged MSC L-glutamine and penicillin (0.025 unit / 500 ml) / streptomycin (0.025 mg / 500 ml)] was added again centrifuged for 5 minutes at 4 °C, 1200rpm.
  • MSCGM is a medium based on Dulbecco's modified Eagle's medium (DMEM) containing fetal calf serum. Subsequently, the supernatant was removed, and the obtained MSC was inoculated in a culture plate and incubated in a 37%, 5% CO 2 incubator with MSCGM. Incubate while replacing the culture medium every other day.
  • DMEM Dulbecco's modified Eagle's medium
  • mesenchymal stem cells derived from peripheral blood were treated with metformin at a concentration of 1 mM based on the number of 2 ⁇ 10 5 cells and cultured at 37 ° C. to prepare mesenchymal stem cells treated with metformin.
  • the cells were treated with metformin at a concentration of 1 mM for 2 ⁇ 10 5 cell numbers and cultured at 37 ° C. to prepare mesenchymal-treated mesenchymal stem cells.
  • mesenchymal stem cells that were not treated with metformin were used as controls, and the expression levels of the immunomodulatory genes were analyzed in metformin-treated mesenchymal stem cells.
  • TGF-beta was expressed in about 10 The expression was increased by doubled (see FIG. 16).
  • the present inventors were able to confirm the above results through the DAPI staining method, that is, in the mesenchymal stem cells treated with metformin through fluorescence microscopy after staining using DAPI (4 ', 6-diamidino-2-phenylindole).
  • DAPI 4- ', 6-diamidino-2-phenylindole.
  • the expression levels of IL-10 and IDO were analyzed.
  • HMGB-1 mesenchymal stem cells isolated and cultured in Examples ⁇ 7-1> and ⁇ 7-2>
  • HMGB-1, IL-6, and IL which are genes related to inflammatory and tumor factors, respectively
  • IL which are genes related to inflammatory and tumor factors, respectively
  • the expression change of -1beta was analyzed.
  • the adipose tissue-derived mesenchymal stem cells were treated with metformin at a concentration of 1 mM for 5 ⁇ 10 5 cell numbers, and then cultured under 37 ° C. temperature to prepare mesenchymal-treated mesenchymal stem cells.
  • the mesenchymal stem cells not treated with metformin were used as a control group, and the expression levels of inflammatory and tumor factor genes in the metformin-treated mesenchymal stem cells were analyzed using realtime PCR.
  • the present inventors found that the mesenchymal stem cells prepared by the treatment with metformin significantly increased the expression of IDO, IL-10, and TGF-beta, which have immunomodulatory capacity, compared with the treatment without metformin. It was confirmed that the expression of HMGB-1, IL-6 and IL-1beta, genes related to inflammatory and tumor factors, was significantly reduced.
  • Electron microscopy was performed on mesenchymal stem cells prepared by treating metformin to the mesenchymal stem cells derived from peripheral blood isolated and cultured in Example 7 and those of mesenchymal stem cells derived from peripheral blood not treated with metformin. Autophagy activity and mitochondrial activity change were observed.
  • the autophagy can regulate the beneficial or detrimental effects of immunity and inflammation and can prevent infectious diseases, autoimmunity and inflammatory diseases. Therefore, the present inventors have found that the mesenchymal stem cells of the present invention treated with metformin are autologous. It is intended to check whether it is possible to treat immunity and inflammatory diseases because it has phagocytosis.
  • autophagy is known to be involved in the degradation of microorganisms (viruses, bacteria, parasites, etc.) invaded into cells, and is known to inhibit inflammatory reactions as well as to participate in the actual immune response and its regulation. This shows a very important dynamic for various pathophysiological processes, and in particular can regulate the self-defense (immune) system. Recently, it is emphasized that the regulation of autophagy balance is important in the treatment of inflammatory bowel disease and various autoimmune diseases.
  • the mesenchymal-mesenchymal stem cells treated with metformin were found to have increased numbers of autophagic vacuoles compared to the group not treated with metformin (yellow arrow), and mitochondrial activity. Increased by metformin (red arrow points to mitochondria).
  • metformin-treated mesenchymal stem cells prepared according to the present invention are actually useful for the therapeutic effect of immune diseases.
  • the therapeutic effects of the osteoarthritis-induced animal models were first examined.
  • osteoarthritis-induced mouse animals were prepared by injecting 50 mg of 3 mg monosodium iodoacdtate (Sigma, ST. Louis, MO) using an intra-articular 26.5G syringe for each osteoarthritis.
  • osteoarthritis-induced mice were injected with 2 ⁇ 10 6 adipose tissue-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells treated with metformin at a concentration of 1 mM once a week, twice a total of iv. Then, after 7 days, pain was measured, which is an indicator for evaluating the effect on the behavior of osteoarthritis-inducing animals, and the destruction of cartilage was analyzed by india ink staining.
  • the present inventors analyzed the degree of arthritis and cartilage destruction of osteoarthritis-induced animals after staining using H & E staining, Toluidin blue staining, and saffranin O staining.
  • mice injected with mesenchymal stem cells significantly reduced the infiltration of inflammatory cells and cartilage destruction. Confirmed.
  • test animals used C57BL / 6 (H-2kb) mice, which were infused with 3.5% dextran sulfate sodium (DSS) water for 1 week. Inflammatory bowel disease induction animal models were constructed. After that, the adipose tissue-derived mesenchymal stem cells and metformin-treated adipose tissue-derived mesenchymal stem cells were injected 2 times a week with iv into a cell number of 2 ⁇ 10 6 cells. (Weight, colon length, DAI (disease activity index)) was confirmed.
  • the present inventors analyzed the degree of TNF-a expression through intestinal inflammatory cell invasion, cell tissue morphology, and immunochemistry staining using H & E staining method in tissues in inflammatory bowel disease animal model.
  • intestinal inflammatory cell infiltration and tissue structure of the intestinal inflammatory cells were maintained similar to those of normal mice in the case of the mesenchymal stem cells treated with metformin as compared to the group treated with the adipose tissue-derived mesenchymal stem cells alone
  • TNF-a an inflammatory cytokine in the intestine
  • mesenchymal stem cells treated with metformin were similar to those of normal intestinal tissues compared to those treated with mesenchymal stem cells alone. It was confirmed that the amount of expression of TNF-a is reduced to an extent.
  • the present inventors performed the following experiment to confirm whether the metformin-treated mesenchymal stem cells have a therapeutic effect on arthritis.
  • IL-1Ra knockout mouse was produced according to a method published by the Y. Iwakura team, which lacks the IL-1 receptor gene.
  • the IL-1 receptor antagonist (IL-1Ra) acts directly on the IL-1 receptor, preventing IL-1 ⁇ and IL-1 ⁇ from acting on the receptor, thereby naturally causing autoimmune arthritis diseases. Way.
  • mice with arthritis-induced mesenchymal-treated mesenchymal stem cells prepared in the present invention were injected into the diseased mouse.
  • Metformin-treated mesenchymal stem cells were injected into the mice at a cell count of 2 ⁇ 10 6 for iv to the mouse for one week. A total of three injections were made, and arthritis dimensions of each of these mice were measured one week later.
  • a group of arthritis-induced mice and a group of arthritis mice injected with only adipose tissue-derived mesenchymal stem cells were used as a control group.
  • the arthritis symptom was improved in the group of mice treated with metformin-treated mesenchymal stem cells compared to the group treated with mesenchymal stem cells only (see FIG. 24).
  • DBA / 1J mice were mixed with type II collagen (CII) and CFA (adjuvant) 1: 1, injected 100ul of CII per mouse at 50ul dose into the tail base, and 2 weeks later.
  • CII and IFA 1: 1 was injected secondly at 100 ug / 50 ul.
  • arthritis index For the measurement of arthritis index, three observers who did not know the contents of the experiment were evaluated three times a week and evaluated the severity of joint inflammation for up to 10 weeks. At this time, the evaluation of arthritis is based on the average arthritis index by Rossolinec et al., The average score divided by 3 is obtained by adding the scores according to the following scales on the three legs except the legs to which CII / CFA was administered at the time of the second dose per horse. The mean obtained by summing the values obtained by three observers in the animal model was used. The scores and criteria according to arthritis evaluation are as follows.
  • the best arthritis index per maridang is 4, so the best disease index per rat is 16.
  • arthritis was further suppressed in the mouse group injected with metformin-treated mesenchymal stem cells as compared with the mouse group injected with mesenchymal stem cells (see FIG. 25).
  • the results of the above examples indicate that the mesenchymal stem cells of the present invention prepared by treatment with sRAGE have an activity that can effectively regulate the immune response, and thus can be usefully used as a novel cell therapy for the treatment of immune diseases, especially autoimmune diseases. Shows.
  • the present inventors found that mesenchymal stem cells prepared by treating metformin have an activity that can effectively regulate the immune response, and thus can be usefully used as a novel cell therapy for the treatment of immune diseases, especially autoimmune diseases.
  • metformin mesenchymal stem cells prepared by treating metformin have an activity that can effectively regulate the immune response, and thus can be usefully used as a novel cell therapy for the treatment of immune diseases, especially autoimmune diseases.

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Abstract

La présente invention concerne des cellules souches mésenchymateuses surexprimées par sRAGE stables, ayant une excellente fonction de régulation de l'immunité, ainsi qu'une composition d'agent de traitement cellulaire les contenant pour la prévention ou le traitement d'une maladie immunitaire. La présente invention concerne également : un procédé de préparation de cellules souches mésenchymateuses capable de production de masse du facteur de croissance transformant bêta (TGF-β) humain et ayant la fonction de régulation de l'immunité, comprenant une étape de traitement, avec de la metformine, d'une cellule souche mésenchymateuse ayant les caractéristiques immunologiques des CD105, DC29, et CD44 positives et des CD34, CD45, négatives et du HLA-DR, et sa culture pendant 14 à 21 jours à une température de 28 à 42°C. La présente invention concerne également les cellules souches mésenchymateuses préparées par ledit procédé ; et la composition d'agent de traitement cellulaire contenant les cellules souches mésenchymateuses pour le traitement ou la prévention de maladies immunitaires.
PCT/KR2014/007640 2013-08-16 2014-08-18 Composite de régulation d'une inflammation et cellules souches mésenchymateuse stabilisées ayant une fonction optimisée de régulation de l'immunité par blocage de la molécule de signalisation stat3 WO2015023165A1 (fr)

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KR10-2013-0102155 2013-08-28
KR1020140104574A KR101659158B1 (ko) 2013-08-16 2014-08-12 메트포민이 처리된 면역조절능을 갖는 간엽줄기세포 및 이를 포함하는 면역질환의 예방 또는 치료용 세포치료제 조성물
KR10-2014-0104574 2014-08-12
KR1020140106718A KR101636139B1 (ko) 2013-08-28 2014-08-18 면역조절능이 우수한 sRAGE 과발현 간엽줄기세포 및 이를 포함하는 면역질환의 예방 또는 치료용 세포치료제 조성물
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112190596A (zh) * 2020-09-14 2021-01-08 陕西佰傲干细胞再生医学有限公司 一种用于治疗关节炎的间充质干细胞制剂及其制备方法
CN113881707A (zh) * 2021-10-25 2022-01-04 中国人民解放军军事科学院军事医学研究院 调控脐带间充质干细胞免疫抑制作用的产品、方法及用途
CN117143812A (zh) * 2023-10-31 2023-12-01 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070039981A (ko) * 2004-08-03 2007-04-13 트랜스테크 파르마, 인크. Ｒａｇｅ 융합 단백질 및 이의 사용 방법
US7485697B2 (en) * 2001-03-19 2009-02-03 Japan As Represented By President Of Kanazawa University Soluble rage protein
WO2011049291A2 (fr) * 2009-10-23 2011-04-28 가톨릭대학교 산학협력단 CELLULE SOUCHE MÉSENCHYMATEUSE À SÉQUENCE NUCLÉOTIDIQUE CODANT TGFβ, ET UTLISATIONS
WO2013103688A1 (fr) * 2012-01-03 2013-07-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs humains solubles pour produits finaux de glycation avancée (srage), procédé de préparation de srage humain et traitement et procédés utilisant un srage

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7485697B2 (en) * 2001-03-19 2009-02-03 Japan As Represented By President Of Kanazawa University Soluble rage protein
KR20070039981A (ko) * 2004-08-03 2007-04-13 트랜스테크 파르마, 인크. Ｒａｇｅ 융합 단백질 및 이의 사용 방법
WO2011049291A2 (fr) * 2009-10-23 2011-04-28 가톨릭대학교 산학협력단 CELLULE SOUCHE MÉSENCHYMATEUSE À SÉQUENCE NUCLÉOTIDIQUE CODANT TGFβ, ET UTLISATIONS
WO2013103688A1 (fr) * 2012-01-03 2013-07-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs humains solubles pour produits finaux de glycation avancée (srage), procédé de préparation de srage humain et traitement et procédés utilisant un srage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEE, SANG-WON ET AL.: "Soluble receptor for advanced glycation end products alleviates nephritis in (NZB/NZW)F1 mice", ARTHRITIS & RHEUMATISM, vol. 65, no. 7, July 2013 (2013-07-01), pages 1902 - 1912 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112190596A (zh) * 2020-09-14 2021-01-08 陕西佰傲干细胞再生医学有限公司 一种用于治疗关节炎的间充质干细胞制剂及其制备方法
CN113881707A (zh) * 2021-10-25 2022-01-04 中国人民解放军军事科学院军事医学研究院 调控脐带间充质干细胞免疫抑制作用的产品、方法及用途
CN117143812A (zh) * 2023-10-31 2023-12-01 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用
CN117143812B (zh) * 2023-10-31 2024-01-26 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用

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