Pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells expressing ptx-3, TIMP1, and bdnf, as active ingredients

Abstract

To provide pharmaceutical compositions for the prevention or treatment of inflammatory diseases or pain.SOLUTION: Provided is a pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells expressing TIMP1, as an active ingredient. Also provided is a method for obtaining mesenchymal stem cells with improved anti-inflammatory or pain-relieving effects, which includes a step of stimulating mesenchymal stem cells so that the mesenchymal stem cells exhibit increased expression of TIMP1.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells as an active ingredient that overexpress or increase the expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF. [Background technology]

[0002] Inflammation is an immune response to harmful stimuli such as external pathogens, toxins, and damaged cells. Such inflammation manifests as swelling, pain, or tissue stiffness, loss of function, and loss of movement. In particular, inflammation in the joints can cause pain, impaired activities of daily living, reduced mobility, and a decline in quality of life. The global market for drugs used to treat inflammation-related conditions was approximately $106.1 billion as of 2020, a size comparable to that of anticancer drugs. Of this market, arthritis drugs accounted for 42.41% of global import sales in 2019 and are expected to grow rapidly by 6.60% between 2020 and 2026.

[0003] In the early stages of osteoarthritis, inflammatory cytokines (e.g., IL-6, TNF-α, and IL-8) are secreted into the synovial membrane and cartilage tissue in response to external stimuli. These secreted cytokines continuously induce inflammation in the joint tissue, causing pain, activating cartilage-degrading enzymes and bone-destructive factors, leading to joint destruction and ultimately to joint deformity (Piotr Wojdasiewicz et al., Mediators Inflamm. 2014: 561459). Nonsteroidal anti-inflammatory drugs (NSAIDs) are primarily used to treat osteoarthritis. Among them, Humira (AbbVie), an anti-TNF agent, recorded the highest sales as a single drug in the United States in 2018. While such drugs may help reduce daily pain, they have side effects such as gastrointestinal disorders and limited long-term efficacy. Therefore, there is a need to develop therapeutic agents that can affect the mechanism of arthritis inflammation and provide a fundamental treatment.

[0004] To address this issue, various research and development efforts are underway to develop stem cell therapeutics. Mesenchymal stem cells can be isolated and cultured from various tissues, such as bone marrow, umbilical cord blood, and adipose tissue, and can be differentiated into cartilage tissue. Therefore, these cells are used as a primary treatment for arthritis. Proteins secreted from mesenchymal stem cells have been reported to be effective in treating various diseases and tissue regeneration due to their anti-apoptotic, immunosuppressive, and anti-inflammatory effects (Mancuso et al., Front Bioeng Biotechnol. 2019 Jan 29;7:9.; DS Jevotovsky et al., Osteoarthritis Cartilage. 2018 Jun;26(6):711-729). In particular, the anti-inflammatory effects of mesenchymal stem cells have been reported to regulate macrophage polarization and the secretion of anti-inflammatory cytokines, fundamentally suppressing the development of inflammatory responses (Meng Sun et al., J Immunol Res. 2019 Jun 19;2019:7059680).

[0005] The above-mentioned therapeutic effects induced by mesenchymal stem cells may be affected by the donor, the tissue from which these cells are derived, the isolation method, the culture method, etc. To solve this problem, it is necessary to find characteristics that can be used to select cells with therapeutic effects. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Piotr Wojdasiewicz et al., Mediators Inflamm., 2014: 561459 [Non-patent document 2] Mancuso et al., Front Bioeng Biotechnol., 2019 Jan 29;7:9 [Non-patent document 3] DS Jevotovsky et al., Osteoarthritis Cartilage. 2018 Jun;26(6):711-729 [Non-patent document 4] Meng Sun et al., J Immunol Res. 2019 Jun 19;2019:7059680 Summary of the Invention [Problem to be solved by the invention]

[0007] As a result of research to obtain mesenchymal stem cells that are effective in the prevention or treatment of inflammatory diseases, the present inventors confirmed that mesenchymal stem cells show increased expression of specific proteins under inflammatory conditions, and that mesenchymal stem cells expressing such proteins have excellent anti-inflammatory and pain-relieving effects, thereby completing the present invention. [Means for solving the problem]

[0008] In order to solve the above problems, one aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells expressing one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF.

[0009] In another aspect of the present invention, there is provided a method for obtaining mesenchymal stem cells with improved anti-inflammatory or pain-relieving effects, the method comprising the step of stimulating mesenchymal stem cells such that the mesenchymal stem cells exhibit increased expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF. [Effects of the Invention]

[0010] The mesenchymal stem cells expressing PTX-3, TIMP1, and BDNF of the present invention can suppress inflammatory responses by reducing the expression of inflammatory cytokines IL-1b, IL-6, IL-8, and TNF-α and increasing the expression of anti-inflammatory cytokines IL-10 and arginase-1 (ARG-1); and when administered to rats with osteoarthritis, the mesenchymal stem cells can alleviate pain caused by osteoarthritis. Therefore, the mesenchymal stem cells expressing PTX-3, TIMP1, and BDNF of the present invention can be effectively used to prevent, alleviate, or treat inflammatory diseases, pain, and inflammation-induced pain. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows a graph showing the increase in the expression levels of PTX-3, TIMP1, and BDNF when SMUP-Cells were co-cultured with a macrophage inflammation model obtained by treating the RAW264.7 cell line with lipopolysaccharide (LPS) to induce an inflammatory response, compared to when SMUP-Cells were cultured alone. [Figure 2]FIG. 2 shows the results obtained by confirming the expression of HLA-A2 in two types of SMUP-Cells co-cultured with a macrophage inflammation model. [Figure 3] FIG. 3 shows graphs depicting the expression levels of IL-6, TNF-α, and IL-10 when SMUP-Cell expressing PTX-3, TIMP1, and BDNF were co-cultured with a macrophage inflammation model. [Figure 4] Figure 4 shows graphs showing the increase in the expression levels of PTX-3, TIMP1, and BDNF when SMUP-Cells were co-cultured with a chondrocyte inflammation model obtained by treating SW1353 cell line with IL-1b to induce an inflammatory response. [Figure 5] FIG. 5 shows graphs showing the expression levels of IL-1b, IL-8, and IL-6 when three types of SMUP-Cells were co-cultured with a chondrocyte inflammation model. [Figure 6] Figure 6 shows a graph showing the expression levels of TNF-α and arginase-1 (ARG-1) when SMUP-Cell, SMUP-Cell C-siR, or SMUP-Cell PTX-3 siR were co-cultured with a macrophage inflammation model. [Figure 7] Figure 7 shows photographs showing stained CD11b (macrophage M1 marker) and CD206 (macrophage M2 marker) and the percentage of stained cells when SMUP-Cell, SMUP-Cell C-siR, or SMUP-Cell PTX-3 siR were co-cultured with a macrophage inflammation model. [Figure 8] Figure 8 shows graphs showing the expression levels of IL-1b, IL-6, IL-8, and TNF-α in chondrocytes when SMUP-Cell, SMUP-Cell C-siR, or SMUP-Cell PTX-3 siR were co-cultured with a chondrocyte inflammation model. [Figure 9]FIG. 9 shows a graph showing the expression levels of PTX-3 in SMUP-Cell, SMUP-Cell C-siR, SMUP-Cell PTX-3 siR, or human umbilical vein endothelial cells (HUVEC). [Figure 10] Figure 10 shows a graph illustrating the results obtained by injecting hyaluronic acid (HA) together with SMUP-Cell, SMUP-Cell C-siR, SMUP-Cell PTX-3 siR, or HUVECs into rats in which arthritic pain had been induced by injection of monoiodoacetic acid (MIA), and then performing an incapacitance test to measure asymmetry in weight bearing. [Figure 11] Figure 11 shows graphs showing the expression levels of IL-1b, IL-6, IL-8, and TNF-α in chondrocytes when SMUP-Cell, SMUP-Cell C-vec, or SMUP-Cell TIMP1 KO were co-cultured with a chondrocyte inflammation model. [Figure 12] FIG. 12 shows graphs showing the expression levels of IL-6 and TNF-α in macrophages when SMUP-Cell, SMUP-Cell C-siR, or SMUP-Cell BDNF siR were co-cultured with a macrophage inflammation model. [Figure 13] FIG. 13 shows a graph illustrating the results obtained by examining changes in inflammatory cytokine levels in SMUP-Cell, SMUP-Cell C-vec, and PTX-3-overexpressing stem cells (SMUP-Cell-PTX3 OE). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will now be described in more detail. In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising mesenchymal stem cells expressing one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF as an active ingredient. Specifically, the pharmaceutical composition may comprise mesenchymal stem cells that show overexpression or increased expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF as an active ingredient.

[0013] PTX-3 (pentraxin 3) is a member of the pentraxin superfamily characterized by a cyclic multimeric structure. PTX-3 is rapidly produced and released by several cell types, particularly mononuclear phagocytes, dendritic cells (DCs), fibroblasts, and endothelial cells, in response to primary inflammatory signals. Furthermore, PTX-3 activates the classical pathway of complement activation and promotes antigen recognition by macrophages and dendritic cells.

[0014] TIMP1 (tissue inhibitor of matrix metalloproteinase 1), a glycoprotein expressed in several tissues, represents an endogenous inhibitor that regulates the action of matrix metalloproteinases (MMPs). Specifically, TIMP1 is a member of the TIMP family, a natural inhibitor of matrix metalloproteinases, a group of peptidases involved in the degradation of the extracellular matrix. In addition to its known inhibitory role against matrix metalloproteinases, TIMP1 can promote cell proliferation in a wide range of cell types and may also have anti-apoptotic functions.

[0015] Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor distributed in the brain. BDNF is known to promote neuronal growth and regulate the synthesis, metabolism, and release of neurotransmitters, as well as neuronal activity.

[0016] Mesenchymal stem cells may express PTX-3, TIMP1, or BDNF. Furthermore, mesenchymal stem cells may express PTX-3 and TIMP1; PTX-3 and BDNF; or TIMP1 and BDNF. Furthermore, mesenchymal stem cells may express PTX-3, TIMP1, and BDNF.

[0017] The present invention is based on the finding that mesenchymal stem cells that express or show increased expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF have excellent inflammation suppression or pain relief capabilities.

[0018] Specifically, in an embodiment of the present invention, in order to enable mesenchymal stem cells to be cultured under inflammatory conditions, 2×10 5 SMUP-Cell cells were co-cultured with RAW264.7 cells, a murine macrophage cell line, in a macrophage inflammation model created by treating the cell line with 1 μg / ml lipopolysaccharide (LPS). The changes in the expression levels of PTX-3, TIMP1, and BDNF in the culture medium after co-culture were then monitored. Furthermore, mesenchymal stem cells expressing any one of the following proteins, including PTX-3, TIMP1, BDNF, and their combinations, were confirmed to reduce the expression of pro-inflammatory cytokines IL-1b, IL-6, IL-8, and TNF-α and increase the expression of anti-inflammatory cytokines IL-10 and arginase-1 (ARG-1), resulting in suppression of the inflammatory response (Figures 1, 3, 5, 6, 8, 11, and 12).

[0019] Mesenchymal stem cells may further express HLA-A2. In one embodiment of the present invention, it was confirmed that mesenchymal stem cells expressing any one selected from the group consisting of PTX-3, TIMP1, BDNF, and combinations thereof further express HLA-A2 (FIG. 2).

[0020] Mesenchymal stem cells may express PTX-3 and HLA-A2, TIMP1 and HLA-A2, or BDNF and HLA-A2. Furthermore, mesenchymal stem cells may express PTX-3, TIMP1, and HLA-A2; PTX-3, BDNF, and HLA-A2; or TIMP1, BDNF, and HLA-A2. Furthermore, mesenchymal stem cells may express PTX-3, TIMP1, BDNF, and HLA-A2.

[0021] HLA-A2 is a serotype belonging to the human leukocyte antigen (HLA)-A serotype group. HLA is a glycoprotein molecule encoded by the human major histocompatibility complex (MHC) genes. HLA-A is expressed on the cell surface of all nucleated cells and platelets and functions to enable cytotoxic T cells to recognize antigens and eliminate virus-infected or tumor cells.

[0022] Mesenchymal stem cells may overexpress one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF. Overexpression can be achieved by genetic manipulation, specifically, overexpression can be achieved by transformation using a vector loaded with a gene encoding any one selected from the group consisting of PTX-3, TIMP1, BDNF, and combinations thereof.

[0023] When subjected to an inflammatory stimulus, the mesenchymal stem cells may exhibit an increased expression level of any one selected from the group consisting of PTX-3, TIMP1, BDNF, and combinations thereof.

[0024] In the present invention, inflammatory stimuli may refer to exposure to an inflammatory environment that mimics an in vivo inflammatory response. Inflammatory stimuli may include, but are not limited to, stimuli caused by substances secreted by inflammatory cells, such as cytokines, exposure to an in vitro or animal inflammation model, or co-culture with proinflammatory cells. Furthermore, the inflammatory condition or inflammatory environment may be an affected area where inflammation has occurred.

[0025] Mesenchymal stem cells can be produced by a method comprising the following steps: (1) Isolate mesenchymal stem cells with a size of 10 μm or less; (2) culturing the isolated mesenchymal stem cells in a calcium-containing medium under hypoxic conditions of 2% to 5% oxygen; and (3) Exposing cultured mesenchymal stem cells to inflammatory stimuli.

[0026] In the above method, isolated mesenchymal stem cells having a size of 10 μm or less have superior proliferation ability to mesenchymal stem cells having other sizes, and such mesenchymal stem cells can exhibit further improved proliferation and differentiation ability when cultured in a calcium-containing medium under hypoxic conditions.

[0027] In the above method, calcium can be contained in the medium at a concentration of 1.5 mM to 3.8 mM. Specifically, in the above method, calcium can be contained in the medium at a concentration of 1.5 mM to 2.0 mM.

[0028] Culturing can be carried out by conventional known methods.For example, mesenchymal stem cells can be cultured using a three-dimensional bioreactor (or spinner), or can be cultured by coating a cell adhesive material on a common adhesive container and a 3D carrier.In addition, mesenchymal stem cells can be cultured using a 3D carrier.

[0029] Mesenchymal stem cells can be derived from umbilical cord blood, bone marrow, fat, muscle, skin, umbilical cord, amniotic fluid, or teeth. Specifically, mesenchymal stem cells can be derived from umbilical cord blood.

[0030] The administration route and dosage of the pharmaceutical composition can be administered in various ways and amounts depending on the condition of the subject and the presence or absence of side effects, and the optimal administration method and dosage can be selected within an appropriate range by those skilled in the art. Furthermore, the pharmaceutical composition can be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known for the disease to be treated, or can be formulated in the form of a combined preparation with other drugs.

[0031] The pharmaceutical composition is a type of cell therapy drug and may further comprise a pharmaceutically acceptable carrier. The carrier may be one commonly used in the preparation of pharmaceuticals, and examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0032] Furthermore, the pharmaceutical compositions of the present invention may further comprise lubricating agents, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and combinations thereof.

[0033] Based on the total weight of the pharmaceutical composition of the present invention, the carrier may be contained in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight, and the pharmaceutically acceptable additive may be contained in an amount of about 0.1% by weight to about 20% by weight.

[0034] The pharmaceutical compositions may be prepared in unit dosage form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers or excipients according to conventional methods. The formulations may be in the form of a solution, suspension, syrup, or emulsion, in an oil or aqueous medium, or in the form of an extract, powder, granules, or capsule. The formulations may further include dispersants or stabilizers.

[0035] Inflammatory disease refers to a disease accompanied by inflammation. Specifically, the inflammatory disease can be osteoarthritis, rheumatoid arthritis, atopy, asthma, allergic rhinitis, Alzheimer's disease, graft-versus-host disease (GVHD), diabetic nephropathy, Crohn's disease, inflammatory bowel disease, post-transplant rejection, bronchopulmonary dysplasia (BPD), or chronic obstructive pulmonary disease (COPD). Pain can be inflammatory pain, and the inflammatory pain can be arthritis pain.

[0036] The individual to which the pharmaceutical composition is administered may be a mammal, specifically a human. The administration route and dosage of the pharmaceutical composition may be administered in various ways and amounts depending on the condition of the subject and the presence or absence of side effects, and those skilled in the art may select the optimal administration method and dosage within an appropriate range. Furthermore, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances known to have a therapeutic effect on the inflammatory disease to be treated, or may be formulated in the form of a combined preparation with other drugs.

[0037] When the pharmaceutical composition is administered parenterally, examples thereof include subcutaneous, ocular, intraperitoneal, intramuscular, oral, rectal, intraorbital, intracranial, intraspinal, intraventricular, intrathecal, intranasal, intravenous, and intra-articular administration. Specifically, the pharmaceutical composition can be administered to an intra-articular site, a joint injury site, or the medial and / or lateral compartments of a joint, where the joint can be a knee, finger, or toe joint. "Intra-articular" administration can include "intra-articular" administration.

[0038] Administration can be one or more times, or one to three times, specifically three times. In the case of repeated administration, administration can be performed at intervals of 1 to 56 days, 7 to 49 days, 14 to 42 days, or 21 to 35 days. Preferably, administration can be performed at 28-day intervals. In the case of high doses, administration can be performed several times a day.

[0039] The dose of mesenchymal stem cells was 1 × 10 5 5 x 10 cells / individual 7 It can be a cell / individual.

[0040] In another aspect of the present invention, there is provided a method for obtaining mesenchymal stem cells with improved anti-inflammatory or pain-relieving effects, the method comprising stimulating mesenchymal stem cells so that the mesenchymal stem cells exhibit increased expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF, wherein the mesenchymal stem cells are as described above for the pharmaceutical composition.

[0041] The stimulating step may include exposing the mesenchymal stem cells to an inflammatory environment. Specifically, the exposing step may include, but is not limited to, stimulation caused by substances secreted by inflammatory cells, such as cytokines, co-culture with pro-inflammatory cells, or exposure to an animal model of inflammation that mimics an in vitro or in vivo inflammatory environment.

[0042] Pro-inflammatory cells can be cells in which inflammation has been induced by treatment with lipopolysaccharide (LPS), a mitogen, a chemokine, or a cytokine.

[0043] The cells may be any one selected from the group consisting of somatic cells, germ cells, and combinations thereof. The somatic cells may be any one selected from the group consisting of muscle cells, hepatocytes, neurons, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells, and combinations thereof. The germ cells may be any one selected from the group consisting of sperm, eggs, and combinations thereof.

[0044] The mesenchymal stem cells can be generated by a method comprising the following steps prior to the stimulation step: (1) isolating mesenchymal stem cells with a size of 10 μm or less; and (2) The isolated mesenchymal stem cells are cultured in a calcium-containing medium under hypoxic conditions of 2% to 5% oxygen.

[0045] In the above method, calcium can be contained in the medium at a concentration of 1.5 mM to 3.8 mM. Specifically, in the above method, calcium can be contained in the medium at a concentration of 1.5 mM to 2.0 mM.

[0046] Steps (1) and (2) are as described above for the pharmaceutical composition.

[0047] In yet another aspect of the present invention, there is provided a method for preventing or treating inflammatory disease or pain, comprising the step of administering the pharmaceutical composition to an individual.

[0048] The individual can be a mammal, including a human, and can be a non-human animal. The term "non-human animal" refers to any vertebrate, and can include mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles.

[0049] Pharmaceutical compositions can be appropriately administered to individuals according to the administration method, administration route and dosage conventionally used in the art as needed.An example of administration route includes parenteral administration.In addition, appropriate dosage and administration frequency can be selected according to methods known in the art.The dosage and administration frequency of the composition actually administered can be appropriately determined according to various factors such as the type of symptoms to be prevented or treated, administration route, sex, health condition, diet, individual age and weight, and severity of disease.

[0050] Mode of Invention The present invention will now be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0051] Production Example 1. Preparation of mesenchymal stem cells (SMUP-CELL) First, to generate mesenchymal stem cells (SMUP-CELL), mononuclear cells were isolated from allogeneic umbilical cord blood using Ficoll-Plaque, and then 5 × 10 6 ~1×10 6 cells / cm 2 The cells were cultured at a concentration of 500–3,000 cells / cm at 37°C, 5% CO2, and 3% O2 ​​for up to 21 days. Cells with a size of 10 μm or less were then isolated and cultured at a density of 500–3,000 cells / cm. 2 The cells were cultured under hypoxic conditions (2%-5%) for up to 7 days in α-MEM medium (SH30265.02, Hyclone) containing 5%-30% fetal bovine serum supplemented with calcium at concentrations of 1.5-3.8 mM.

[0052] Example 1. Confirmation of the anti-inflammatory effect of mesenchymal stem cells in a macrophage inflammation model Example 1.1. Confirmation of increased expression of PTX-3, TIMP1, and BDNF in mesenchymal stem cells under inflammatory conditions The macrophage inflammation model was performed using 1 × 10 mouse macrophage cell line. 5 The RAW264.7 cell line was treated with 1 μg / ml of lipopolysaccharide (LPS). 5 SMUP-Cell cells were co-cultured with macrophage-derived inflammatory models and analyzed. This was compared with SMUP-Cell cells cultured alone. RPMI (22400, Gibco) was used as the basal medium.

[0053] Specifically, the expression levels of PTX-3, TIMP1, and BDNF proteins in the medium in which SMUP-Cells were cultured alone and in the medium in which SMUP-Cells were co-cultured with macrophage inflammatory models were analyzed using a human PTX-3 ELISA kit (DPTX30B, R&D Systems), a human TIMP1 ELISA kit (DTM100, R&D Systems), and a human BDNF ELISA kit (DBD00, R&D Systems). The expression levels of each protein measured in the medium in which SMUP-Cells were cultured alone were set at 100. These expression levels were then converted to the expression levels of each protein measured in the medium in which SMUP-Cells were co-cultured with macrophage inflammatory models, and compared.

[0054] As a result, it was confirmed that the expression levels of PTX-3, TIMP1, and BDNF proteins increased in the medium when SMUP-Cells were co-cultured with the macrophage inflammation model (Figure 1).

[0055] Example 1.2. Confirmation of increased expression of HLA-A2 in mesenchymal stem cells under inflammatory conditions 2×10 5 The SMUP-Cell cells were co-cultured with the macrophage inflammation model prepared in Example 1.1 using the same culture conditions (RPMI (22400, Gibco)), and the expression of HLA-A2 in the SMUP-Cell cells was confirmed.

[0056] Specifically, the co-cultured SMUP-cells were treated with trypsin to dissociate them into single cells, and then washed twice with PBS. The washed SMUP-cells were then reacted with PE-labeled anti-HLA-A2 antibodies, which served as the experimental group. An isotype control-PE was used as a negative control. HLA-A2 expression was checked for the experimental and negative control groups using FACS. Two different donors were used for the co-cultured SMUP-cells.

[0057] As a result, fluorescent signals were measured in the co-cultured SMUP-Cell 1 and SMUP-Cell 2, which were the experimental groups, respectively, confirming that HLA-A2 was expressed in SMUP-Cell 1 and SMUP-Cell 2, which were co-cultured with the macrophage inflammation model (Figure 2).

[0058] Example 1.3. Confirmation of the anti-inflammatory effect of mesenchymal stem cells expressing PTX-3, TIMP1, BDNF, and HLA-A2 After co-culture in Example 1.2, the medium was collected and analyzed by ELISA for mouse IL-6 and mouse TNF-α to confirm the expression levels of pro-inflammatory cytokines. Specifically, analysis was performed using a mouse IL-6 ELISA kit (DY406, R&D Systems) and a mouse TNF-α ELISA kit (DY410, R&D Systems) according to the manufacturer's protocol. Each medium sample after co-culture was diluted 1 to 50 and analyzed. The OD values ​​of the reaction mixtures were then measured using a VERSA max microplate reader (Molecular Devices). Furthermore, the expression levels of anti-inflammatory cytokines were checked by ELISA for mouse IL-10 (DY417, R&D Systems). Here, the levels of mouse IL-6, mouse TNF-α, and mouse IL-10 were compared in the medium cultured alone with the non-inflammatory RAW264.7 cell line and in the macrophage inflammation model, and these were also analyzed by ELISA.

[0059] The results confirmed that IL-6, TNF-α, and IL-10 were all increased in the culture medium used to culture the macrophage inflammation model compared to the culture medium used to culture the RAW264.7 cell line alone. Conversely, the expression levels of IL-6 and TNF-α were decreased and the expression level of IL-10 was increased in the culture medium used to culture SMUP-Cell and the macrophage inflammation model (Figure 3).

[0060] Example 2. Confirmation of the anti-inflammatory effect of mesenchymal stem cells in a chondrocyte inflammation model Example 2.1. Confirmation of increased expression of PTX-3, TIMP1, and BDNF in mesenchymal stem cells under inflammatory conditions The chondrocyte inflammation model was performed using 1 × 10 5The SW1353 cell line (Chon) was generated by treating it with 10 ng / ml of IL-1b. 5 SMUP-Cell cells were co-cultured with the chondrocyte inflammation model and then analyzed. The expression levels of PTX-3, TIMP1, and BDNF were measured as in Example 1.1 and compared with those when SMUP-Cells were cultured alone. Three different types of SMUP-Cells were used, received from three different donors. For each of the three types of SMUP-Cells, the rate of further increase in expression levels in the medium after co-culture was graphed. Numerical calculations were performed using "(Chon + IL-1b + SMUP-Cell) medium / SMUP-Cell medium alone x 100," and only the calculated values ​​were graphed.

[0061] As a result, when chondrocytes were cultured alone or in the chondrocyte inflammation model, PTX-3, TIMP1, and BDNF were hardly expressed. Conversely, when SMUP-Cell was co-cultured with the chondrocyte inflammation model, the expression levels of PTX-3, TIMP1, and BDNF were confirmed to increase (Figure 4).

[0062] Example 2.2. Confirmation of the anti-inflammatory effect of mesenchymal stem cells expressing PTX-3, TIMP1, and BDNF 1×10 5 We investigated whether chondrocyte inflammation could be reduced by co-culturing SMUP-Cells with the chondrocyte inflammation model prepared in Example 2.1. Three different types of SMUP-Cells received from three different donors were used. The experiment was conducted using the following groups: 1) a group in which chondrocytes were cultured alone (Chon), 2) a group in which the chondrocyte inflammation model was cultured (Chon + IL-1b), and 3) a group in which each of the three types of SMUP-Cells was co-cultured with the chondrocyte inflammation model.

[0063] Chondrocytes cultured in each group (1)–(3) were treated with trypsin to dissociate into single cells, and mRNA was isolated from them. Expression levels of the inflammatory cytokines IL-1b, IL-8, and IL-6 were analyzed by qPCR. Next, a comparative analysis was performed with values ​​normalized to GAPDH. qPCR was performed as follows: Cells were treated with TRIzol Reagent (12263026, Invitrogen) according to the manufacturer's protocol, and mRNA was extracted. cDNA was synthesized using the Transcriptor-First-Strand-cDNA Synthesis Kit (4897030001, Roche) and then mixed with primers for the inflammatory markers IL-1b, IL-8, IL-6, and GAPDH. The cDNA products corresponding to specific markers were then measured using a Light Cycler 480 Real-Time PCR System (Roche). Here, the expression levels of the measured cDNA products were normalized to GAPDH expression levels, and the expression levels of the markers in cells cultured under each condition were compared and analyzed. The primers used for qPCR are shown in Table 1. [Table 1] [Table 1]

[0064] As a result, it was confirmed that the expression levels of IL-1b, IL-8, and IL-6 were significantly increased in Group 2), where the chondrocyte inflammation model was cultured, compared to Group 1), where chondrocytes were cultured alone. Conversely, it was confirmed that the expression levels of IL-1b, IL-8, and IL-6 were decreased in Group 3), where each of the three types of SMUP-Cell was co-cultured with the chondrocyte inflammation model (Figure 5).

[0065] Example 3. Confirmation of the anti-inflammatory effect of PTX-3 secreted from mesenchymal stem cells Example 3.1. Confirmation of changes in cytokine expression levels in a macrophage inflammation model caused by knockdown of the PTX-3 gene in mesenchymal stem cells To clarify the correlation between PTX-3 secreted from mesenchymal stem cells and inflammation, we used cells in which PTX-3 expression was knocked down by treating mesenchymal stem cells with PTX-3 siRNA (SMUP-Cell PTX-3 C-siR). Mesenchymal stem cells treated with control siRNA (SMUP-Cell C-siR) were also used as a negative control. The macrophage inflammation model (Example 1.1) and the chondrocyte inflammation model (Example 2.1) were co-cultured with SMUP-Cell or SMUP-Cell PTX-3 C-siR, respectively, and their anti-inflammatory effects were compared and analyzed.

[0066] First, the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine arginase-1 (ARG-1) were measured by ELISA using the culture medium from the macrophage inflammation model. Specifically, analysis was performed using mouse TNF-α ELISA (DY410, R&D Systems) and mouse arginase 1 ELISA (ab269541, Abcam) according to the manufacturer's protocol. Each culture medium sample after co-culture was diluted 1 to 50 and analyzed. Next, the OD value of the reaction mixture was measured using a VERSA max microplate reader (Molecular Devices).

[0067] As a result, compared to the medium in which the macrophage inflammation model was cultured alone, the medium in which the macrophage inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-siR showed a decreased expression level of TNF-α and an increased expression level of ARG-1. Conversely, the medium in which the macrophage inflammation model was co-cultured with SMUP-Cell PTX-3 siR showed an increased expression level of TNF-α and a decreased expression level of ARG-1. This confirmed that PTX-3 expression is involved in regulating the inflammatory response (Figure 6).

[0068] Example 3.2. Confirmation of phenotypic changes in a macrophage inflammation model after PTX-3 gene knockout in mesenchymal stem cells To confirm the phenotypic changes of macrophages after LPS-induced inflammation and co-culture with SMUP-Cell (M1: inflammatory activity, M2: anti-inflammatory activity), inflammation-induced RAW264.7 cell line was fixed and cell staining was performed with mouse antibodies.

[0069] Specifically, fixed cells were incubated with anti-CD11b (Abcam, #ab128797) and mouse MMR / CD206 (R&D Systems, #AF2535) primary antibodies for 12–16 hours under refrigeration. Next, cells were developed with Alexa Fluor 488 (green) and Cy3 (red) secondary antibodies, respectively, and incubated at room temperature for 30 minutes. Unreacted reagents were then washed away. Next, cells were treated with Hoechst 33342 (blue, Invitrogen, H3570) for 5 minutes to stain nuclei. Finally, stained cells were photographed using an LSM 800 confocal microscope (Zeiss).

[0070] As shown in Figure 7, nuclei were stained blue, CD11b (M1 marker) was stained green, and CD206 (M2 marker) was stained red. In RAW264.7 cell lines without induced inflammation, CD11b (M1 marker) and CD206 (M2 marker) were barely stained. Conversely, in RAW264.7 cell lines with induced inflammation by LPS treatment, CD11b staining was confirmed in most cell lines. On the other hand, when SMUP-Cell or SMUP-Cell C-siR was cocultured with inflammatory RAW264.7 cell lines, the intensity of CD11b staining was significantly reduced. Furthermore, in inflammatory RAW264.7 cell lines, CD206 staining was barely detected, whereas the intensity of CD206 staining increased in cells cocultured with SMUP-Cell or SMUP-Cell C-siR. Conversely, when SMUP-Cell PTX-3 siR was co-cultured, the staining intensity of CD11b increased and the staining intensity of CD206 decreased.A similar tendency was also observed in the chondrocyte inflammation model.

[0071] This confirmed that PTX-3 secreted from SMUP-Cells affects the phenotype of macrophages and is involved in regulating inflammatory activity.

[0072] Example 3.3. Confirmation of changes in cytokine expression levels in a macrophage inflammation model after PTX-3 gene overexpression in mesenchymal stem cells To clarify the correlation between PTX-3 secreted from mesenchymal stem cells and inflammation, we used cells obtained by inducing PTX-3 overexpression (OE) in mesenchymal stem cells using CRISPR / CAS9 technology (SMUP-Cell PTX-3 OE). As a negative control, we used SMUP-Cell C-vec, which was obtained by transfecting only with the vector. The macrophage inflammation model described in Example 1.1 was co-cultured with SMUP-Cell, SMUP-Cell C-vec, and SMUP-Cell PTX-3 OE, and their anti-inflammatory effects were compared and analyzed.

[0073] The inflammatory cytokine IL-6 was measured by ELISA using culture medium from the macrophage inflammation model. Specifically, analysis was performed using a mouse IL-6 ELISA (DY406, R&D Systems) according to the manufacturer's protocol. Each medium sample after co-culture was diluted 1-50 and analyzed. The OD value of the reaction mixture was then measured using a VERSA max microplate reader (Molecular Devices).

[0074] As a result, the IL-6 expression level was similarly reduced in the medium in which the macrophage inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-vec compared to the medium in which the macrophage inflammation model was cultured alone. Conversely, the IL-6 expression level was significantly reduced in the medium in which the macrophage inflammation model was co-cultured with SMUP-Cell PTX-3 OE compared to the medium in which the macrophage inflammation model was co-cultured with SMUP-Cell (before overexpression). This confirmed that PTX-3 expression is involved in regulating the inflammatory response (Figure 13).

[0075] Example 3.4. Confirmation of changes in cytokine expression levels in a chondrocyte inflammation model after PTX-3 gene knockdown in mesenchymal stem cells The chondrocyte inflammation model described in Example 2.1 was co-cultured with SMUP-Cell, SMUP-Cell C-siR, or SMUP-Cell PTX-3 siR, and the expression levels of inflammatory cytokines in the chondrocytes were then confirmed by qPCR. qPCR was performed in the same manner as in Example 2.2.

[0076] The results showed that the expression levels of IL-1b, IL-6, IL-8, and TNF-α were reduced in the medium in which the chondrocyte inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-siR compared to the medium in which the chondrocyte inflammation model was cultured alone. Conversely, the expression levels of IL-1b, IL-6, IL-8, and TNF-α were higher in the medium in which the chondrocyte inflammation model was co-cultured with SMUP-Cell PTX-3 compared to the medium in which the chondrocyte inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-siR.

[0077] This confirmed that PTX-3 expression in mesenchymal stem cells is involved in regulating the inflammatory response of chondrocytes.

[0078] Example 4. Confirmation of the pain-reducing effect of PTX-3 secreted from mesenchymal stem cells To create a rat model of arthritis pain, male rats weighing 150-250 g were anesthetized and then injected with 2 mg of monoiodoacetate (MIA) using a syringe for intra-articular injection into the knee. Four days after the MIA injection, 2.5 x 10 sucrose containing 1% hyaluronic acid (HA) was injected into the knee using a syringe for intra-articular injection. 5 MSCs were injected at a concentration of 25 μl per cell. To measure pain, incapacity tests were performed on days 0, 1, 4, 7, 14, 21, and 28 after MIA injection. A control group was injected with 1% HA alone. Furthermore, as negative controls, SMUP-Cells treated with PTX-3 siRNA and human umbilical vein endothelial cells (HUVECs) (CRL-1730, ATCC), which exhibit significantly lower PTX-3 expression, were used to confirm the effect of PTX-3 expression.

[0079] Here, the expression level of PTX-3 in mesenchymal stem cells was measured using an HPTX-3 ELISA kit for each medium co-cultured with SMUP-Cell, SMUP-Cell C-siR, SMUP-Cell PTX-3 siR, and HUVECs in the macrophage inflammation model described in Example 1.1. The PTX-3 expression level in SMUP-Cell was then set to 100%. This expression level was then converted, and the converted values ​​were graphed for identification. The results confirmed that the PTX-3 expression level was reduced in SMUP-Cell PTX-3 siR compared to SMUP-Cell, and significantly reduced in HUVEC compared to SMUP-Cell PTX-3 siR (Figure 9).

[0080] We investigated whether differences in PTX-3 expression levels in each group led to differences in pain-relieving effects. In normal rats, body weight was evenly distributed across both hind limbs (ipsilateral / total body weight (%) = 50). However, in controls administered only MIA or MIA + HA, pain intensified and decreased over time after MIA injection. In this way, an animal pain model was created. Next, mesenchymal stem cells expressing different levels of PTX-3 were injected into arthritic pain-induced rats to confirm their pain-relieving effects.

[0081] As a result, in the groups injected with SMUP-Cell and SMUP-Cell C-siR, which have high levels of PTX-3 expression, weight bearing values ​​increased immediately after injection and were almost fully restored within 24 days of cell administration. Conversely, in the group transplanted with SMUP-Cell PTX-3 siR, weight bearing values ​​did not recover even after cell administration, and in fact, they were confirmed to have decreased. Furthermore, in the group transplanted with HUVEC, weight bearing values ​​initially appeared to recover, but then decreased from 10 days after cell administration, and no tendency for pain reduction was observed.

[0082] This confirmed that PTX-3 secreted from mesenchymal stem cells is involved in the reduction of arthritis pain.

[0083] Example 5. Confirmation of TIMP1 secreted from mesenchymal stem cells and its anti-inflammatory effect To confirm the correlation between TIMP1 secreted from mesenchymal stem cells and inflammation, we performed a test using cells obtained by knocking out TIMP1 expression in mesenchymal stem cells using the clustered regularly interspaced short palindromic repeats (CRISPR) / CAS9 system (SMUP-Cell TIMP1 knockout, KO). As a negative control, we used SMUP-Cell C-vec, which was obtained by transfecting only with the vector.

[0084] The chondrocyte inflammation model of Example 2 was co-cultured with mesenchymal stem cells from each group, and then the chondrocyte inflammation model was treated with trypsin to dissociate into single cells. mRNA was extracted from the cells, and the gene expression patterns of inflammatory cytokines were confirmed by qPCR. qPCR was performed as in Example 2.2.

[0085] The results confirmed that the expression levels of IL-1b, IL-6, IL-8, and TNF-α were reduced in the chondrocyte inflammation model cocultured with SMUP-Cell or SMUP-Cell c-vec compared to normal chondrocytes cocultured with SMUP-Cell or SMUP-Cell c-vec. Conversely, when the chondrocyte inflammation model was cocultured with SMUP-Cell TIMP1 KO, the expression levels of IL-1b, IL-6, IL-8, and TNF-α were higher than when the chondrocyte inflammation model was cocultured with SMUP-Cell or SMUP-Cell c-vec. These results confirm that TIMP1 expression in mesenchymal stem cells is involved in regulating the inflammatory response (Figure 11).

[0086] Example 6. Confirmation of the correlation between BDNF secreted from mesenchymal stem cells and anti-inflammatory properties To confirm the correlation between BDNF secretion from mesenchymal stem cells and inflammation, we performed a test using mesenchymal stem cells treated with BDNF siRNA (SMUP-Cell BDNF C-siR) to knock down BDNF expression. Here, mesenchymal stem cells treated with control siRNA (SMUP-Cell C-siR) were used as a negative control.

[0087] The macrophage inflammation model of Example 1 was co-cultured with mesenchymal stem cells from each group. Next, the expression levels of inflammatory cytokines IL-6 and TNF-α were measured using each medium using an ELISA kit.

[0088] As a result, the expression levels of IL-6 and TNF-α were reduced in the culture medium in which the macrophage inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-siR compared to the culture medium in which the macrophage inflammation model was co-cultured alone. Conversely, the expression levels of IL-6 and TNF-α were higher in the culture medium in which the macrophage inflammation model was co-cultured with SMUP-Cell PTX-3 siR compared to the culture medium in which the macrophage inflammation model was co-cultured with SMUP-Cell or SMUP-Cell C-siR. This confirmed that BDNF expression is involved in regulating the inflammatory response (Figure 12).

Claims

1. A pharmaceutical composition for preventing or treating inflammatory diseases or pain, comprising as an active ingredient mesenchymal stem cells that express one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF.

2. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells express PTX-3.

3. The pharmaceutical composition according to claim 1 , wherein the mesenchymal stem cells express BDNF.

4. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells express TIMP1.

5. Mesenchymal stem cells are cultured through the following steps: (1) Isolating mesenchymal stem cells having a size of 10 μm or less; (2) culturing the isolated mesenchymal stem cells in a calcium-containing medium under hypoxic conditions of 2% to 5% oxygen; and (3) Exposing cultured mesenchymal stem cells to inflammatory stimuli; 10. The pharmaceutical composition of claim 1, made by a method comprising:

6. 6. The pharmaceutical composition of claim 5, wherein calcium is contained at a concentration of 1.5 mM to 3.8 mM.

7. 2. The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells are derived from umbilical cord blood, bone marrow, fat, muscle, skin, umbilical cord, amniotic fluid, or teeth.

8. 2. The pharmaceutical composition of claim 1, wherein the inflammatory disease is osteoarthritis, rheumatoid arthritis, atopy, asthma, allergic rhinitis, Alzheimer's disease, graft-versus-host disease (GVHD), diabetic nephropathy, Crohn's disease, inflammatory bowel disease, post-transplant rejection, bronchopulmonary dysplasia (BPD), or chronic obstructive pulmonary disease (COPD).

9. The pharmaceutical composition according to claim 1, wherein the pain is inflammatory pain.

10. The pharmaceutical composition according to claim 9, wherein the inflammatory pain is arthritis pain.

11. The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells exhibit increased expression levels of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF when subjected to an inflammatory stimulus.

12. A method for obtaining mesenchymal stem cells with improved anti-inflammatory or pain-relieving effects, comprising: A method comprising the step of stimulating mesenchymal stem cells such that the mesenchymal stem cells exhibit increased expression of one or more proteins selected from the group consisting of PTX-3, TIMP1, and BDNF.

13. 13. The method of claim 12, wherein the stimulating step comprises exposing the mesenchymal stem cells to an inflammatory environment.

14. The method of claim 13 , wherein the step of exposing the mesenchymal stem cells to an inflammatory environment comprises co-culturing with pro-inflammatory cells.

15. 15. The method of claim 14, wherein the pro-inflammatory cells are cells in which inflammation has been induced by treatment with lipopolysaccharide (LPS), a mitogen, a chemokine, or a cytokine.

16. The method of claim 15, wherein the cell is any one selected from the group consisting of a somatic cell, a germ cell, and a combination thereof.

17. 17. The method of claim 16, wherein the somatic cells are any one selected from the group consisting of muscle cells, hepatocytes, neurons, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells, and combinations thereof.

18. The mesenchymal stem cells may be subjected to the following steps prior to the stimulation step: (1) isolating mesenchymal stem cells having a size of 10 μm or less; and (2) culturing the isolated mesenchymal stem cells in a calcium-containing medium under hypoxic conditions of 2% to 5% oxygen; The method of claim 12, wherein the polymer is produced by a method comprising:

19. 19. The method of claim 18, wherein calcium is included at a concentration of 1.5 mM to 3.8 mM.

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