KR20170000802A - Composition for preventing or treating osteoarthritis comprising mesenchymal stem cell treated STAT3 inhibitor - Google Patents

Abstract

The present invention relates to composition for preventing or treating osteoarthritis comprising mesenchymal stem cell treated with an STAT3 inhibitor. The mesenchymal stem cell treated with the STAT3 inhibitor can inhibit the expression of proinflammatory factors effectively, increase the expression of anti-inflammatory factors effectively and perform outstanding strengths in expressing chemokine receptors. Accordingly, the mesenchymal stem cell treated with the STAT3 inhibitor can be used as the composition for preventing or treating immune system disorders such as osteoarthritis which results from abnormality of various immune responses.

Description

TECHNICAL FIELD The present invention relates to a cell therapy composition for preventing or treating osteoarthritis comprising an mesenchymal stem cell treated with STAT3 inhibitor as an active ingredient.

The present invention relates to a cell therapy composition for preventing or treating osteoarthritis, which comprises mesenchymal stem cells treated with a STAT3 inhibitor as an active ingredient.

Immunity is one of the biological self-protection systems for all external polymeric substances (antigens) that enter or are injected into living tissue. T cells are one of the cells that play a central role in the immune system. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation with each other.

Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, autoimmune diseases may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

Immunosuppressants that block the signal transduction pathway in T cells are the most commonly used therapeutic agents for immune diseases. Such immunosuppressants are toxic, infectious, lymphoid, diabetes, tremor, headache, diarrhea, hypertension, nausea , And a disorder of renal function.

In addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, a method of regulating the amount of cytokine secreted from the immune cells and a method of using the antibody targeting the cytokine secreted from the immune cells have been developed . However, this method has a problem in that it takes much time to actually apply to patients through clinical experiments, and that the method using antibody is too expensive in the process of antibody production.

Therefore, the development of a new effective immunotherapeutic agent has been studied and developed. Recently, a cell therapy agent for the treatment of immune diseases has been developed, and studies for using stem cells as a cell therapy agent are increasing.

In particular, mesenchymal stem cells in stem cells are stem cells existing in the whole body including bone marrow that can differentiate into various cell lines such as adipocytes, bone cells and chondrocytes. Recently, mesenchymal stem cells In vitro and in vivo T lymphocyte activity is suppressed and immunoregulatory ability is demonstrated. However, the mechanism of action involved in the immunoregulatory activity of mesenchymal stem cells against T lymphocytes has been fully elucidated It does not exist. Therefore, it is urgent to develop a novel cell therapy agent which has no side effects and is inexpensive and has excellent therapeutic effect.

1. Korean Patent Publication No. 10-2014-0032925

It is an object of the present invention to provide a cell therapy composition for preventing or treating osteoarthritis, which comprises an mesenchymal stem cell treated with STAT3 (signal transducer and activator of transcription 3) inhibitor as an active ingredient.

It is still another object of the present invention to provide a method for preparing mesenchymal stem cells for treating osteoarthritis, which comprises treating a mesenchymal stem cell with a STAT3 (signal transducer and activator of transcription 3) inhibitor in vitro.

In order to achieve the above object, the present invention provides a cell therapy composition for preventing or treating osteoarthritis, which comprises mesenchymal stem cells treated with STAT3 (signal transducer and activator of transcription 3) inhibitor as an active ingredient.

The term " STATs (Signal Transducer and Transcriptions) "of the present invention is a transcription factor having seven subunit forms of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. STATs are those that comprise the N-terminal domain (ND), the coiled-coil domain (CCD), the DNA-binding domain (DBD), the linker domain (LD), the Src homology 2 (SH2) It shares six distinct structural domains, among which STAT3 has important C-terminal tyrosine and serine residues (Tyrosine 705, Serine 727 residues for STAT3) that are phosphorylated by activation. In particular, STAT3 is continuously activated through various pathways to promote tumorigenesis, and STAT3 is also involved in various cytokine and growth factor-interferon, epidermal growth factor, platelet-derived growth factor, interleukin 5, interleukin 6, It is activated through phosphorylation of two residues of tyrosine 705 and serine 727 in response to hepatocyte growth factor, leukemia inhibitory factor (LIF), bone formation genetic protein 2, hormone leptin, carcinogens such as Src and Ras. STAT3 is a transcriptional regulator that is involved in the transcription of several genes in human cells. In normal cases, STAT3, which is present in the cytoplasm in response to cytokine or growth factors signal from the outside, migrates into the nucleus, It regulates genes for differentiation, growth, survival, neovascularization and immune function. However, when STAT3 is abnormal, STAT3 plays an important role in the development of cancer. A large number of malignant tumors, animal model experiments, activated STAT3 were found in cancer patients, and STAT3 controls the expression of various genes that induce and develop cancer. STAT3 is known to inhibit the expression of mediators necessary for immune activation against tumor cells, and the activity of STAT3 promotes the production of STAT3-activating factors in various immune cells while changing the gene expression program, .

The term "mesenchymal stem cells" in the present invention refers to stem cells isolated from bone marrow, blood, dermis and periosteum, and pluripotent stem cells capable of differentiating into various cells such as adipocytes, chondrocytes, Or multipotent cells.

As used herein, "mesenchymal stem cells" means mesenchymal stem cells isolated from osteoarthritis patients. The mesenchymal stem cells may be an animal, preferably a mammal, more preferably a human mesenchymal stem cell. The process of isolating and culturing mesenchymal stem cells is well known in the art and is disclosed, for example, in U.S. Patent No. 5,486,359, which is incorporated herein by reference. In addition, the mesenchymal stem cells can be obtained by separating the mesenchymal stem cells from the hematopoietic stem cells of bone marrow according to a known method, and then proliferating without losing their differentiation ability.

More specifically, it isolates mesenchymal stem cells from a mammalian animal, preferably a human mesenchymal stem cell source, such as blood or bone marrow, including human or mouse. The bone marrow can be extracted from the tibia, femur, spinal cord or iliac bone. Cells are then obtained from the bone marrow, and these cells are cultured in a suitable medium. In the course of culturing, the floating cells are removed and the cells attached to the culture plate are subcultured to finally obtain established mesenchymal stem cells. As the medium used in the above process, any medium generally used for culturing stem cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum). Mediums which can be used in the present invention include, for example, RPMI series, Eagles ' s MEM (Eagle's minimum essential medium, Eagle, H. Science 130: Dulbecco's modification of Eagle's medium, Dulbecco, R., et al., J. Exp. Med., 147: 923 (1978)), Iscove's MEM (Iscove, N. et al. et al., VirProcy 8: 396 (1959)), but are not limited thereto. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin) and glutamine. A general description of media and culture is provided in R. Ian Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York (1984), which is incorporated herein by reference.

In addition, identification of mesenchymal stem cells can be performed, for example, through flow cytometry. Such flow cytometry analysis is carried out using a specific surface marker of mesenchymal stem cells. For example, mesenchymal stem cells are positive for CD44, CD29 and / or MHC class I. According to a preferred embodiment of the present invention, the mesenchymal stem cells used in the present invention are positive for the surface markers CD44, CD29 and CD105. For CD34, CD45 and HLA-DR (Human Leukocyte Antigen-antigen D Related) Negative reaction. A term used to refer to stem cells and surface markers refers to the way in which antibodies against surface markers are specifically bound to surface antigens of stem cells when treated with stem cells.

In one embodiment of the present invention, the STAT3 inhibitor is selected from the group consisting of STA-21 (8-hydroxy-3-methyl-3,4-dihydrotetraphene-1,7,12 3- (8,8-dipropyl-3-azaspiro [4.5] decan-3-yl) -2-cyano-3- -yl) -N, N-diethylpropan-1-amine, Auranofin, 3,4,5-triacetyloxy-6- (acetyloxymethyl) oxane- (2-chloro-6-methylphenyl) -2 - [[6- [4- (2-hydroxyethyl) -1-piperazinyl] -2 (3,4-dihydroxy-phenyl) -ethyl ester, CAPE (3- (3,4-dihydroxy-phenyl) -acrylic acid 2- But are not limited to, stathic 6-nitro-1,1-dioxide-benzo [b] thiophene, calcium 2,5-dihydroxybenzenesulfonate, ethanol, NCX- (3S, 4aS, 8aS) -N-tert-butyl-2 - [(2R, 3R) -2-hydroxy- hydroxy-2-methylphenyl) formamido] -4- (phenylsulfanyl) butyl] decahydroisoquinoline-3-carboxamide), PDP (phosphododecapeptide), PS-341 (Bortezomib; 2-ylcarbonyl) amino] propanoyl} amino) butyl] boronic acid, R115777 (tipifarnib (R) (1H-imidazol-5-yl) methyl] -4- (3-chlorophenyl) -1-methylquinolin-2 (N-Hexyl-2- (1-naphthalenyl) -5 - [[4- (phosphonooxy) phenyl], statin compound, sodium salicylate, caffeic acid 3- 4-Dihydroxyphenyl) -2-propenoic acid 3,4-Dihydroxy-cinnamic acid trans-Caffeate 3,4-Dihydroxy-trans- cinnamate), Emodin (1,8-Dihydroxy- , 10-anthracenedione, 3-beta-3-hydroxy-urs-12-ene-28-oic acid, resveratrol, 3,5,4'-trihydroxy- Retinoic acid (2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid ), EGCG (Epigallocatechin gallate; [(2R, 3R) -5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl) chroman-3-yl] 3,4,5-trihydroxybenzoate), Sr11302 , E, Z, E) - 2-cyclohexen-1-yl) -2,4,6,8-nonatetraenoic acid, Tanshinone IIA (2-methyl- ; 1,6,6-Trimethyl-6,7,8,9-tetrahydrophenanthro [1,2-b] furan-10,11-dione and curcumin (1E, 6E) -hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione).

The STAT3 inhibitor can be obtained by treating mesenchymal stem cells at a concentration of 1 x ^{10 5} to 1 x 10 ⁷ cells at a concentration of 0.1 μM to 100 μM and culturing the cells at 37 ° C. for 24 to 72 hours to obtain mesenchymal stem cells excellent in the ability to treat immune diseases.

Among the STAT3 inhibitors, the "statin compounds" include compounds such as Cilastatin, Nystatin, Lovastatin, Somatostatin, Pravastatin, Simvastatin, Fluvastatin, Or atorvastatin, cervastatin, ulinastatin or rosuvastatin. The term " atorvastatin "

In one embodiment of the present invention, the STAT3 inhibitor-treated mesenchymal stem cells are positive for CD44, CD29, and CD105, and the CD34, CD45, and HLA-DR (Human Leukocyte Antigen-antigen D Related) Lt; / RTI >

In one embodiment of the present invention, the STAT3 inhibitor-treated mesenchymal stem cells are selected from the group consisting of Interleukin-6 (IL-6), VEGF (Vascular Endothelial Growth Factor) or Interleukin-1 beta -1?) Can be suppressed.

In one embodiment of the present invention, the mesenchymal stem cells may be isolated from peripheral blood or adipose tissue.

The term "treatment" of the present invention means, unless otherwise stated, reversing, alleviating, inhibiting the progress of, or preventing the disease or condition to which the term applies, And the term " treatment " as used herein refers to an act of treating. The therapeutic or therapeutic treatment of an immune disorder in a mammal therefore may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development;

(2) preventing the spread of immune diseases, i.e., preventing metastasis;

(3) relieving immune diseases;

(4) preventing recurrence of immune disorders; And

(5) palliating symptoms of immune disorders

The composition for the prevention or treatment of osteoarthritis according to the present invention may comprise a mesenchymal stem cell treated with a STAT3 inhibitor in a pharmaceutically effective amount, alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of an immune disease.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating osteoarthritis according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be appropriately determined depending on the route of administration, And the severity of the patient, and the composition for preventing or treating osteoarthritis according to the present invention may be administered in combination with a known compound having an effect of preventing, ameliorating or treating the symptoms of osteoarthritis And preferably parenterally. When administered parenterally, the pharmaceutical composition of the present invention can be administered by intravenous injection, subcutaneous injection, muscle injection, and intraperitoneal injection.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is preferably 1 x 10 ³ to 1 x 10 ¹² cells / kg per day.

Also, the present invention provides a method for preparing mesenchymal stem cells for treating osteoarthritis, which comprises treating a mesenchymal stem cell with a STAT3 (signal transducer and activator of transcription 3) inhibitor in vitro.

The STAT3 inhibitor-treated mesenchymal stem cells according to the present invention are capable of effectively inhibiting the expression of inflammatory factors, effectively increasing the expression of anti-inflammatory agents, and enhancing the expression of chemokine receptors.

In addition, the MS-derived mesenchymal stem cells can further improve cartilage protection and osteoarthritic inflammation control efficacy of stem cells, and can minimize side effects such as immune rejection and the like, as compared with the case of using normal stem cells. However, in the case of mesenchymal stem cells derived from a patient, stem cells are often aged stem cells, and their functions are deteriorated. Therefore, a composition containing the mesenchymal stem cells can be more effectively used as a therapeutic agent for immune diseases.

Therefore, the mesenchymal stem cells treated with the STAT3 inhibitor can be effectively used as a cell therapy composition composition capable of preventing or treating immunological diseases such as osteoarthritis induced by abnormal regulation of various immune responses.

Fig. 1 shows the results of analysis of the expression level of inflammatory inducers in mesenchymal stem cells treated with STAT3 inhibitor (STA21). A is the amount of IL-6, VEGF and IL-1β protein measured by ELISA analysis, and B is the amount of IL-1β gene expression analyzed by real-time PCR.
FIG. 2 shows the results of real-time PCR analysis of the expression levels of IL-10, TGF-beta and HGF genes, which are anti-inflammatory agents in STAT3 inhibitor (STA21) -treated mesenchymal stem cells .
FIG. 3 shows the results of real-time PCR analysis of expression amounts of CCR2, CCR3, CCR7 and CCR9, which are chemokine receptors in STAT3 inhibitor (STA21) -treated mesenchymal stem cells.
FIG. 4 shows the results of confirming whether there is a change in the markers expressed in the mesenchymal stem cells treated with the STAT3 inhibitor (STA21).
FIG. 5 shows the results of real-time PCR analysis of expression of the molecules TIMP1 and TIMP3 associated with cartilage synthesis metabolism by STAT3 inhibitor (STA21) treated mesenchymal stem cells.
FIG. 6 shows the results of intraarticular injection of mesenchymal stem cells treated with STAT3 inhibitor (STA21) in an animal model of osteoarthritis induced by MIA (monosodium iodoacetate) (P), withdrawal latency (B), and withdrawal threshold (C). MIA: group treated with MIA; OA-MSC: group injected with mesenchymal stem cells without STAT3 inhibitor; Nor-MSC: group not treated with mesenchymal stem cells; And STA21 OA-MSC: STAT3 inhibitor (STA21) treated mesenchymal stem cells.
FIG. 7 shows the results of the intraarticular injection of mesenchymal stem cells treated with STAT3 inhibitor (STA21) in an animal model of osteoarthritis induced by MIA (monosodium iodoacetate) Paw withdrawal latency (s), 8 days; B: paw withdrawal latency (s), 16 days; C: paw withdrawal threshold (g) The results are shown in Fig. Normal: normal animal model group; MIA: group treated with MIA; OA-MSC: group injected with mesenchymal stem cells without STAT3 inhibitor; And STA21-MSC: STAT3 inhibitor (STA21) treated mesenchymal stem cells.
FIG. 8 shows the results of intraarticular injection of a STAT3 inhibitor (STA21) -mediated mesenchymal stem cell in an osteoarthritis animal model induced by MIA (monosodium iodoacetate), and the degree of cartilage destruction was measured in the femur and tibia And observed with a microscope. Normal: normal animal model group; MIA: group treated with MIA; MSC: group injected with mesenchymal stem cells without STAT3 inhibitor; And STA21-MSC: STAT3 inhibitor (STA21) treated mesenchymal stem cells.
FIG. 9 is a graph showing the results of an intravenous injection of a STAT3 inhibitor (STA21) -mediated mesenchymal stem cell into an osteoarthritis animal model induced by MIA (monosodium iodoacetate) (P), withdrawal latency (B), and withdrawal threshold (C).
FIG. 10 is a graph showing the results of intravenous injection of a STAT3 inhibitor (STA21) treated mesenchymal stem cells in an osteoarthritic animal model induced by MIA (monosodium iodoacetate) and showing the degree of cartilage destruction to the femur and tibia And observed with a microscope. WT: normal animal model group; MIA: group treated with MIA; OA-MSC: group injected with mesenchymal stem cells without STAT3 inhibitor; And STA21 OA-MSC: STAT3 inhibitor (STA21) treated mesenchymal stem cells.
FIG. 11 shows the results obtained by injecting STAT3 inhibitor (STA21) -mediated mesenchymal stem cells into an osteoarthritic animal model induced by MIA (monosodium iodoacetate) by intravenous injection and measuring TRPV1 (transient receptor Potential cation channel subfamily V member 1) The result of confocal microscopy after staining the pain factor (blue: stained with DAPI, red: stained with TRPV1). MIA: group treated with MIA; OA-MSC: group injected with mesenchymal stem cells without STAT3 inhibitor; And STA21 OA-MSC: STAT3 inhibitor (STA21) treated mesenchymal stem cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

<Reference example>

Mesenchymal stem cell ( mesenchymal  stem cell, MSC ) And culture

The present inventors obtained mesenchymal stem cells through the following procedure. First, in osteoarthritis patients, the fat tissue obtained after surgical operation was washed with PBS containing 10% penicillin-streptomycin more than 10 times to remove blood and foreign matter, and then the tissue was cut to 0.2-0.3 g. 0.2% collagenase (Roche, Sandhofer Strasse, Mannheim, Germany), and reacted at 100 rpm for 1 hour in a water bath at 37 ° C. The solution layer decomposed by the collagenase was separated from the undegraded fragments using a 100 μm mesh, and then an equal volume of PBS was added to the separated collagenase solution. Then, the mixture was centrifuged at 4 ° C and 1200 rpm for 5 minutes to remove lipids and fat layers, and the collagenase supernatant was removed. (Cambrex, Walkersville, MD, USA), a mesenchymal stem cell growth medium (Cambrex, Walkersville, MD, USA), and a mesenchymal stem cell growth medium 4 mM L-glutamine and penicillin (0.025 unit / 500 ml) / streptomycin (0.025 mg / 500 ml)] and centrifuged at 4 ° C and 1200 rpm for 5 minutes. MSCGM is a medium based on DMEM (Dulbecco's modified Eagle's medium) containing fetal bovine serum. Subsequently, the supernatant was removed, and the obtained mesenchymal stem cells were inoculated on a culture dish and cultured in MSCGM at 37 DEG C in a 5% CO ₂ incubator. The cultures were incubated while changing the culture medium once every two days.

&Lt; Example 1 >

STAT3  When the inhibitor is treated Mesenchymal stem cell ( mesenchymal  stem cell, MSC Inhibition of Inflammatory Inducing Factor

STAT3 inhibitor-treated mesenchymal stem cells (STA-21) (8-hydroxy-3-methyl-3,4- interleukin-6 (IL-6), VEGF (vascular endothelial growth factor) and interleukin-1 beta (interleukin-1 beta , IL-1?) Expression was determined by ELISA in the culture medium. Specifically, the STA-21 drug was treated with 5 μM based on the mesenchymal stem cells of the reference example of 2 × 10 ⁵ cells, and cultured at 37 ° C. to prepare STAT3 inhibitor-treated mesenchymal stem cells. As a control group, mesenchymal stem cells without STAT3 inhibitor were used.

In addition, the amount of mRNA expressed in the cells was analyzed by real-time PCR, and the primer sequences used in the PCR for analyzing the expression levels of the respective genes are shown in Table 1.

primer order IL-1? Forward primer TTT GGG TCT TCC CAG AAC C (SEQ ID NO: 1) IL-1? Reverse primer TCG TTA TCC CAT GTG TCG AA (SEQ ID NO: 2)

As a result, the expression of IL-6, VEGF and IL-1β was suppressed in STAT3 inhibitor-treated mesenchymal stem cells compared with the control group (FIG. 1A), and the expression of IL-1β was significantly reduced by about 2 times (Fig. 1B).

Based on these results, the present inventors have found that the STAT3 inhibitor-treated mesenchymal stem cells can be used as a cell therapy agent for the treatment of immune diseases.

&Lt; Example 2 >

STAT3  When the inhibitor is treated In mesenchymal stem cells Anti-inflammatory factor  Increase effect analysis

STAT3 inhibitor-treated mesenchymal stem cells, STA-21, which is a STAT3 inhibitor, was administered to the mesenchymal stem cells of Reference Example, and then the anti-inflammatory factor interleukin The expression levels of interleukin-10 (IL-10), transforming growth factor-beta (TGF-β) and hepatocyte growth factor (HGF) Specifically, the STA-21 drug was treated with 5 μM based on the mesenchymal stem cells of the reference example of 2 × 10 ⁵ cells, and cultured at 37 ° C. to prepare STAT3 inhibitor-treated mesenchymal stem cells. As a control group, mesenchymal stem cells without STAT3 inhibitor were used.

In addition, the amount of IL-10, TGF-beta and HGF gene expression was analyzed by performing real-time PCR on the amount of mRNA expressed in the cells, and the primer sequences used in PCR for analyzing the degree of expression of each gene were Respectively.

primer order IL-10 forward primer CCA AGC CTT GTC TGA GAT GA (SEQ ID NO: 3) IL-10 reverse primer TGA GGG TCT TCA GGT TCT CC (SEQ ID NO: 4) TGF-β forward primer TGC GGC AGC TGT ACA TTG A (SEQ ID NO: 5) TGF-β reverse primer TGG TTG TAC AGG GCC AGG A (SEQ ID NO: 6) HGF forward primer CCA CCA TAA TCC CCC TCA CA (SEQ ID NO: 7) HGF reverse primer GGC TGG GGC TAC ACT GGA TT (SEQ ID NO: 8)

As a result, as shown in Fig. 2, as a result, expression of IL-10 was about 4 times, expression of TGF-beta was about 2.5 times, and expression of HGF was decreased in STAT3 inhibitor-treated mesenchymal stem cells compared with the control group (Fig. 2).

&Lt; Example 3 >

STAT3  When the inhibitor is treated In mesenchymal stem cells Chemokine ( chemokine Analysis of receptor expression level

STAT3 inhibitor-treated mesenchymal stem cells have cell migration ability and can be transferred to the lesion, STA-21, which is a STAT3 inhibitor, is treated with mesenchymal stem cells of Reference Example, We analyzed the expression levels of CCR2 (CC chemokine receptor type 2), CCR3 (CC chemokine receptor type 3), CCR7 (CC chemokine receptor type 7) and CCR9 (CC chemokine receptor type 9) by real time PCR. Specifically, the STA-21 drug was treated with 5 μM based on the mesenchymal stem cells of the reference example of 2 × 10 ⁵ cells, and cultured at 37 ° C. to prepare STAT3 inhibitor-treated mesenchymal stem cells. As a control group, mesenchymal stem cells without STAT3 inhibitor were used.

As a result, as shown in FIG. 3, expression of CCR2 (about 3.5-fold), CCR3 (about 1.5-fold), and CCR9 (about 6-fold) in the STAT3 inhibitor-treated mesenchymal stem cells compared to the group not treated with the STAT3 inhibitor (Fig. 3).

Thus, the present inventors have found that the ability of the STAT3 inhibitor-treated mesenchymal stem cells to migrate increased.

<Example 4>

STAT3  When the inhibitor is treated Mesenchymal  Phenotypic analysis

A phenotypic analysis was performed to determine whether there was a change in the phenotype of STAT3 inhibitor treated mesenchymal stem cells. Specifically, the mesenchymal stem cells of Reference Example were treated with 5 μM of STA-21 drug, and cultured at 37 ° C. for 3 days. From the cells cultured in the culture medium without the inhibitor, fluorescence associated with mesenchymal stem cell phenotype Fluorescence-activated cell sorting (FACS) analysis was performed using the labeled antibody. CD44, CD29 and CD105 were used as positive markers of mesenchymal stem cells, and CD34, CD45 and HLA-DR (Human Leukocyte Antigen-antigen D Related) were used as negative markers.

 As a result, as shown in FIG. 4, the positive markers CD105, CD29 and CD44 were similarly expressed in the STAT3 inhibitor-treated mesenchymal stem cells, and the negative markers CD45 and HLA-DR (Human Leukocyte Antigen-antigen D ) Were all negative (Fig. 4).

Therefore, it could be shown that treatment with STAT3 inhibitor can enhance the immunoregulatory ability of mesenchymal stem cells without causing changes in the cell phenotype of mesenchymal stem cell itself.

&Lt; Example 5 >

STAT3  When the inhibitor is treated On mesenchymal stem cells  Chondrocytes by chondrocyte ) Activity analysis

To investigate the effect of STAT3 inhibitor-treated mesenchymal stem cells on chondrocyte activity, expression of TIMP1 (Tissue inhibitor of metalloprotease1) and TIMP3 (Tissue inhibitor of metalloprotease3) PCR.

Specifically, human-derived chondrocytes were pretreated for 24 hours in vitro to stimulate cells with 10 ng / ml of IL-1β to stimulate cell activity, and the mesenchymal stem cells of Reference Example were stimulated with STA -21 drug was treated at 5 μM and co-cultured using a transwell plate of 8 μm pore size. As a control group, mesenchymal stem cells without STAT3 inhibitor were used.

 As a result, TIMP1 and TIMP3 expressed in cartilage cells in STAT3 inhibitor treated mesenchymal stem cells were significantly increased (FIG. 5).

Therefore, it was confirmed that the mesenchymal stem cells treated with the STAT3 inhibitor can inhibit the chondrogenesis of the chondrocyte.

&Lt; Example 6 >

STAT3  Inhibitor-treated mesenchymal stem cells on  Of osteoarthritis treatment

To investigate whether STAT3 inhibitor treated mesenchymal stem cells are actually useful for the treatment of immune diseases, the effect of osteoarthritis pain relief on osteoarthritis animal models was examined.

Specifically, 5-week-old male Wistar rats (200-250 g) were incubated at 21-22 ° C for 12 hours in light-dark cycle to produce osteoarthritic animal models. The sterilized water and feed . Osteoarthritis was induced by administering monosodium iodoacetate (MIA, Sigma, St. Louis, MO) at a dose of 3 mg / 50 μl to the right knee of a rat for induction of osteoarthritis. MIA was administered by dissolving in physiological saline solution.

After osteoarthritis induction, mesenchymal stem cells treated with 3.5 × 10 ⁵ cells of mesenchymal stem cells (reference example) and STAT3 inhibitor (5 μM of STA-21) were injected intraarticularly at intervals of 4 days 6 to Fig. 8). In addition, mesenchymal stem cells treated with 3.5 × 10 ⁵ cells of mesenchymal stem cells (reference example) and STAT3 inhibitor (5 μM of STA-21) were injected at an interval of 4 days by intravenous injection (FIGS. 11). Then, after 2 days from the administration of MIA, the degree of pain, which is an index for evaluating the effect of the osteoarthritis-induced animal on the performance, was measured, and the femur and tibia of the rat were collected, and the degree of cartilage destruction was analyzed by india ink staining (Figs. 8 and 10). Control group (OA-MSC) was injected with mesenchymal stem cells without STAT3 inhibitor.

Pain was measured using a dynamic plantar aesthsiometer (Ugo Basile, Comerio, Italy), and the mechanical pain was measured by placing a net plate on the measuring machine and placing a rat in an acrylic animal pouch And then stabbed the right foot into which the drug was injected with the measuring machine. After stabbing, we measured the time (s, second) that the machine automatically took to release the foot, and the amount of foot release (g) when the weight was given.

As a result, the pain response in the osteoarthritic rats injected with STAT3 inhibitor-treated mesenchymal stem cells was recovered to near the normal rat at 8 and 16 days after induction of osteoarthritis (Fig. 7). In addition, the degree of cartilage destruction was restored to normal in osteoarthritic rats injected with STAT3 inhibitor-treated mesenchymal stem cells (Fig. 8).

In addition, TRPV1 (transient receptor potential cation channel subfamily V member 1), known as a pain activator, was identified in the ganglion tissue when injecting the STAT3 inhibitor treated mesenchymal stem cells into the blood vessel. As a result, it was confirmed that expression of TRPV1 was slightly suppressed in rats injected with STAT3 inhibitor-treated mesenchymal stem cells (Fig. 11).

Therefore, STAT3 inhibitor-treated mesenchymal stem cells increase the expression of anti-inflammatory agents, and in particular, they can directly inhibit and regulate the pathogenesis of chondrocyte cells in vitro and in vivo I could confirm.

<110> Industry-Academic Cooperation Foundation of Catholic University <120> Composition for preventing or treating osteoarthritis comprising          mesenchymal stem cell treated STAT3 inhibitor <130> PN1606-204 <150> 10-2015-0089535 <151> 2015-06-24 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 1 tttgggtctt cccagaacc 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 2 tcgttatccc atgtgtcgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 3 ccaagccttg tctgagatga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 4 tgagggtctt caggttctcc 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 5 tgcggcagct gtacattga 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 6 tggttgtaca gggccagga 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 7 ccaccataat ccccctcaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Seqeunce <400> 8 ggctggggct acactggatt 20

Claims (10)

Wherein the stem cell comprises a mesenchymal stem cell treated with STAT3 (signal transducer and activator of transcription 3) inhibitor as an active ingredient. The method according to claim 1,
The STAT3 inhibitor may be selected from the group consisting of STA-21 (8-hydroxy-3-methyl-3,4-dihydrotetraphene-1,7,12 4-dihydrophenyl) -N- (phenylmethyl) -2-propenamide, Atiprimod 3- (8,8-dipropyl-3-azaspiro [4.5] decan- -1-amine, auranofin, 3,4,5-triacetyloxy-6-thiolate, triethylphosphanium, Aurothiomalate (Sodium 2- (auriosulfanyl) -3-carboxypropanoate ), BMS-354825 (Dasatinib; N- (2-chloro-6-methylphenyl) -2 - [[6- [4- (2-hydroxyethyl) -1- piperazinyl] 5-thiazole carboxamide monohydrate, CADPE (3- (3,4-dihydroxy-phenyl) -acrylic acid 2- 2-dihydroxybenzenesulfonate, Ethanol, NCX-4016 (NO-Aspirin), Nelfinavir (3S, 4aS , 8aS) -N-tert-butyl-2 - [(2R, 3R) -2-hydroxy-3 - [(3-hydroxy- ylsulfanyl) butyl] -decahydroisoquinoline-3-carboxamide), PDP (phosphododecapeptide), PS-341 (Bortezomib; 2-ylcarbonyl) amino] propanoyl} amino) butyl] boronic acid, R115777 (tipifarnib (R) (1H-imidazol-5-yl) methyl] -4- (3-chlorophenyl) -1-methylquinolin-2 (N-Hexyl-2- (1-naphthalenyl) -5 - [[4- (phosphonooxy) phenyl], statin compound, sodium salicylate, caffeic acid 3- 4-Dihydroxyphenyl) -2-propenoic acid 3,4-Dihydroxy-cinnamic acid trans-Caffeate 3,4-Dihydroxy-trans- cinnamate), Emodin (1,8-Dihydroxy- , 10-anthracenedione, 3-beta-3-hydroxy-urs-12-ene-28-oic acid, resveratrol, 3,5,4'-trihydroxy- Retinoic acid (2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid ), EGCG (Epigallocatechin gallate; [(2R, 3R) -5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl) chroman-3-yl] 3,4,5-trihydroxybenzoate), Sr11302 , E, Z, E) -3 -Methyl-7- (4-methylphenyl) -9- (2,6,6-trimethyl-1-cyclohexen-1-yl) -2,4,6,8-nonatetraenoic acid), Tanshinone IIA (Tanshinone IIA; 1,6,6-Trimethyl-6,7,8,9-tetrahydrophenanthro [1,2-b] furan-10,11-dione and curcumin (1E, 6E) -hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione). The method according to claim 1,
The mesenchymal stem cells are STAT3 inhibitor-treated mesenchymal stem cells, which are positive for CD44, CD29, and CD105, and CD34, CD45 and HLA-DR (immunohistochemical characteristics of human leukocyte antigen-antigen D) / RTI &gt; The method of claim 3,
The STAT3 inhibitor-treated mesenchymal stem cells are selected from the group consisting of Interleukin-6 (IL-6), VEGF (Vascular Endothelial Growth Factor) and Interleukin-1 beta Wherein the expression of any one or more of the factors is inhibited. The method according to claim 1,
Wherein said mesenchymal stem cells are isolated from peripheral blood or adipose tissue in an osteoarthritic patient. A method of producing mesenchymal stem cells for treating osteoarthritis, comprising the step of treating STAT3 (signal transducer and activator of transcription 3) inhibitor in mesenchymal stem cells in vitro. The method according to claim 6,
The STAT3 inhibitor may be selected from the group consisting of STA-21 (8-hydroxy-3-methyl-3,4-dihydrotetraphene-1,7,12 4-dihydrophenyl) -N- (phenylmethyl) -2-propenamide, Atiprimod 3- (8,8-dipropyl-3-azaspiro [4.5] decan- -1-amine, auranofin, 3,4,5-triacetyloxy-6-thiolate, triethylphosphanium, Aurothiomalate (Sodium 2- (auriosulfanyl) -3-carboxypropanoate ), BMS-354825 (Dasatinib; N- (2-chloro-6-methylphenyl) -2 - [[6- [4- (2-hydroxyethyl) -1- piperazinyl] 5-thiazole carboxamide monohydrate, CADPE (3- (3,4-dihydroxy-phenyl) -acrylic acid 2- 2-dihydroxybenzenesulfonate, Ethanol, NCX-4016 (NO-Aspirin), Nelfinavir (3S, 4aS , 8aS) -N-tert-butyl-2 - [(2R, 3R) -2-hydroxy-3 - [(3-hydroxy- ylsulfanyl) butyl] -decahydroisoquinoline-3-carboxamide), PDP (phosphododecapeptide), PS-341 (Bortezomib; 2-ylcarbonyl) amino] propanoyl} amino) butyl] boronic acid, R115777 (tipifarnib (R) (1H-imidazol-5-yl) methyl] -4- (3-chlorophenyl) -1-methylquinolin-2 (N-Hexyl-2- (1-naphthalenyl) -5 - [[4- (phosphonooxy) phenyl], statin compound, sodium salicylate, caffeic acid 3- 4-Dihydroxyphenyl) -2-propenoic acid 3,4-Dihydroxy-cinnamic acid trans-Caffeate 3,4-Dihydroxy-trans- cinnamate), Emodin (1,8-Dihydroxy- , 10-anthracenedione, 3-beta-3-hydroxy-urs-12-ene-28-oic acid, resveratrol, 3,5,4'-trihydroxy- Retinoic acid (2E, 4E, 6E, 8E) -3,7-dimethyl-9- (2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenoic acid ), EGCG (Epigallocatechin gallate; [(2R, 3R) -5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl) chroman-3-yl] 3,4,5-trihydroxybenzoate), Sr11302 , E, Z, E) -3 -Methyl-7- (4-methylphenyl) -9- (2,6,6-trimethyl-1-cyclohexen-1-yl) -2,4,6,8-nonatetraenoic acid), Tanshinone IIA (Tanshinone IIA; 1,6,6-Trimethyl-6,7,8,9-tetrahydrophenanthro [1,2-b] furan-10,11-dione and curcumin (1E, 6E) -hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione). The method according to claim 6,
STAT3 inhibitor treated mesenchymal stem cells are positive for CD44, CD29 and CD105, and CD34, CD45 and HLA-DR have immunological characteristics of negative. 9. The method of claim 8,
The STAT3 inhibitor-treated mesenchymal stem cells are selected from the group consisting of Interleukin-6 (IL-6), VEGF (Vascular Endothelial Growth Factor) and Interleukin-1 beta Wherein expression of one or more factors is suppressed. The method according to claim 6,
Wherein said mesenchymal stem cells are isolated from peripheral blood or adipose tissue in osteoarthritis patients.

Images