KR20150026839A - Mesenchymal stem cells overexpressing sRAGE and cell therapeutic agent for preventing or treating immune disease - Google Patents

Abstract

The present invention relates to soluble receptor for advanced glycation endproducts (sRAGE)-overexpressing mesenchymal stem cells which have immuno-modulatory capacity and are stable; and to a cell therapeutic composition containing the same for preventing or treating immunological diseases and, more specifically, to transgenic mesenchymal stem cells into which the sRAGE gene is introduced and overexpressed; and to a cell therapeutic composition containing the mesenchymal stem cells as an active ingredient for preventing or treating immunological diseases. The mesenchymal stem cells prepared according to the present invention have an effect of increasing the expression of genes having immuno-modulatory capacity and genes associated with cell migration, and have activities to inhibit the proliferation of inflammatory cytokines and inflammatory cells and promote the proliferation of immuno-modulatory T cells, and thus can be favorably used as a cell therapeutic agent for treating immunological diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a mesenchymal stem cell overexpressing sRAGE and cell therapeutic agent for preventing or treating immune disease,

The present invention relates to sRAGE-overexpressing mesenchymal stem cells excellent in immunomodulating ability and stability and a cell therapy composition for preventing or treating immune diseases containing the same.

Immunity is one of the biological self-protection systems for all external polymeric substances (antigens) that enter 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 to the lymphatic tissues or organs, mainly the lymph node splenic tonsil. B cells proliferate rapidly when stimulated by the appropriate antigen, forming a clone that produces a special antibody (immunoglobulin) that neutralizes the antigen. The antibody produced by B cells circulates in body fluids and performs humoral immunity. T cells are made from the thymus and migrate to lymphoid tissue, which is responsible for cell-mediated immunity that directly attacks the antigen.

In addition, one of the most important characteristics of all normal individuals is that they do not react negatively to the antigenic substances that make up the self, while many non-self antigens recognize it and can react to remove it I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance. When problems arise in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, thereby attacking the self-organization, resulting in various autoimmune diseases.

On the other hand, drugs that suppress autoimmune function are mainly used as a main method for treating such autoimmune diseases. However, because of the large number of side effects, it is difficult to use continuously, and the treatment is limited because it does not prevent recurrence sufficiently. Therefore, a new therapeutic agent for immune diseases is being developed more effectively. 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, the mesenchymal stem cells of the stem cells have not yet revealed the precise mechanism of action against the immunoregulatory ability, and research on the production of stem cells having excellent immunological disease treatment has been rarely conducted.

Therefore, the present inventors have found that mesenchymal stem cells overexpressing sRAGE (sRAGE) gene have an excellent immunological disease therapeutic effect when studying an effective cell therapy agent for immunotherapy using stem cells Confirming the fact that the present invention has been completed.

Korean Patent Publication No. 10-2013-0083861

Accordingly, it is an object of the present invention to provide mesenchymal stem cells which exhibit excellent immunoregulatory ability and stability by overexpressing sRAGE (soluble receptor for advanced glycation endproducts) gene.

It is another object of the present invention to provide a cell therapy composition for preventing or treating immune diseases comprising mesenchymal stem cells having immunomodulating ability as an active ingredient of the present invention.

In order to achieve the object of the present invention, the present invention provides mesenchymal stem cells having immunoregulatory ability introduced with a nucleotide sequence encoding sRAGE (soluble receptor for advanced glycation endproducts).

In one embodiment of the present invention, the sRAGE may have the amino acid sequence of SEQ ID NO: 2. Also, in one embodiment of the present invention, the nucleotide encoding the sRAGE may have the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the nucleotide sequence encoding sRAGE may be introduced by a vector expressing sRAGE in mesenchymal stem cells. In one embodiment of the present invention, the vector is a recombinant vector containing a nucleotide sequence encoding sRAGE, and there is no restriction as long as it is capable of overexpressing sRAGE in mesenchymal stem cells.

In one embodiment of the present invention, the mesenchymal stem cells may be derived from peripheral blood or adipose tissue, but are not limited thereto.

In one embodiment of the present invention, the mesenchymal stem cells exhibit excellent immunoregulatory ability by overexpression of sRAGE, which can be achieved by controlling the expression of various immunological or inflammatory factors.

In one embodiment of the present invention, overexpression of sRAGE in mesenchymal stem cells induces an increase in expression of one or more factors selected from the group consisting of IDO, TGF-beta, HGF and IL-10.

In one embodiment of the present invention, the overexpression of sRAGE in mesenchymal stem cells induces a decrease in expression of one or more factors selected from the group consisting of IL-1β, IL-6 and HMGB-1.

In one embodiment of the present invention, the overexpression of sRAGE in mesenchymal stem cells induces an increase in the expression of any one or more factors selected from the group consisting of CCR1, CCR3, CCR4, CCR7, CXCR1 and CXCR4.

In one embodiment of the present invention, the mesenchymal stem cells can be stabilized by overexpression of sRAGE. That is, the expression of sRAGE is an essential factor for the stabilization of the stem cell itself.

In addition, the present invention provides a cell therapy composition for preventing or treating immunological diseases comprising mesenchymal stem cells overexpressing sRAGE as an active ingredient.

In one embodiment of the present invention, the composition may be manufactured or used so as to be administered at a dose of 2.5 x 10 ⁵ to 2.5 x 10 ⁷ cells per kg body weight of the administration subject.

In one embodiment of the present invention, the immunological disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, It is known that post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma inflammatory diseases such as scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammatory bowel diseases, Crohns disease ), Autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty (post-angioplasty) asth restenosis, chronic obstructive pulmonary diseases (COPD), Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease ), Angina, cancer metastasis, small artery disease, and mitochondrial disease.

The mesenchymal stem cells of the present invention overexpress sRAGE, exhibit excellent stability, have an effect of increasing the expression of genes having immunomodulatory ability and cell migration related genes, inhibiting the proliferation of inflammatory cytokines and inflammatory cells, T cell proliferation, and thus can be effectively used as a cell therapy agent for the treatment of immune diseases.

FIG. 1 is a schematic diagram of pcDNA.3-HA-hsRAGE, which is a recombinant vector containing a human-derived sRAGE (soluble receptor for advanced glycation endproducts) gene produced according to an embodiment of the present invention.
FIG. 2 shows ELISA (left photograph) and Western blot (right side) to measure the expression amount of sRAGE expressed in mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mock vector without sRAGE Photo).
FIG. 3 is a graph showing the expression of Th1 (IFN-γ), Th17 (IL-17), Th2 (IL-17) and mRNA 4) and Foxp3 + Treg were analyzed by flow cytometry.
FIG. 4 shows the expression levels of immunoregulatory genes and cell migration-related genes in mesenchymal stem cells transformed with sRAGE-containing recombinant vector and mock vector without sRAGE in real-time PCR The results are as follows.
FIG. 5 shows the results of analysis of 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.
FIG. 6 is an analysis of the effect of regulating mesenchymal stem cells transformed with the sRAGE-containing recombinant vector, showing the amount of CFSE expressed.
FIG. 7 is a graph showing the results of analysis of IFN-r, TH17 (IL-17) and Foxp3 + Treg by analyzing the effect of regulating mesenchymal stem cells transformed with the sRAGE-containing recombinant vector.
Fig. 8 shows the results of examining the effect of mesenchymal stem cells transformed with sRAGE-containing recombinant vector under homogenous reaction conditions on cytokine expression of mesenchymal stem cells transformed with sRAGE-free mock vector.
FIG. 9 is a photograph showing the degree of expression of IDO and IL-10 in a mesenchymal stem cell transformed with a sRAGE-containing recombinant vector and a mock vector without sRAGE in a fluorescent microscope will be.
FIG. 10 is a photograph showing electron microscopic observation of autopatch activity of mesenchymal stem cells transformed with a RAGE-containing recombinant vector and mesenchymal stem cells transformed with a mock vector containing no sRAGE.
FIG. 11 shows the results of analysis of the sRAGE-overexpressing mesenchymal stem cells of the present invention on the improvement of the symptoms of rheumatoid arthritis and the therapeutic effect on the arthritis-induced mouse model.
FIG. 12 shows the results of analysis of the sRAGE-overexpressing mesenchymal stem cells of the present invention by improving the symptoms of rheumatoid arthritis symptoms and treating the disease by immunochemical staining and flow cytometry.
FIG. 13 and FIG. 14 show the results of investigating the immunoregulatory ability of the sRAGE-overexpressing mesenchymal stem cells of the present invention on the cells of animal models of lupus.

The inventors of the present invention have experimentally confirmed that sRAGE-overexpressing mesenchymal stem cells can effectively treat immune diseases while studying to develop a cell therapy agent capable of effectively treating immune diseases.

Accordingly, the present invention provides a cell therapy composition composition for the prevention or treatment of immune diseases, which can provide sRAGE-overexpressing mesenchymal stem cells having immunomodulatory ability and comprising mesenchymal stem cells having immunomodulating ability as an active ingredient .

RAGE, on the other hand, interacts with endogenous ligands unlike TLRs (toll like recepters) that interact with extrinsic pathogens and is involved in the onset of a variety of diseases caused by chronic inflammation, which produces a variety of endogenous ligands. In addition, the 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 plays an important role in post-RAGE signaling. RAGE binds to the endogenous ligand, including reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and JAK (janus kinase) Various intracellular signal cascades begin. Significantly, RAGE signaling increases the rate of nuclear translocation of NF-κB (nuclear factor kappa B).

In addition, soluble RAGE (hereinafter sRAGE) has an extracellular site such as RAGE, while it has no trans-membrane domain and cytoplasmic domain. sRAGE is formed by alternative splicing of RAGE mRNA or excision of the membrane moiety and can bind to the RAGE ligand in the extracellular space prior to mRAGE and sRAGE is the degree to which mRAGE binds to their ligand Which is known to play a role in competitive inhibition.

However, studies on the therapeutic efficacy and mechanism of soluble RAGE for immunological and inflammatory diseases have not been disclosed.

In the present invention, the mesenchymal stem cell (MSC) is a stem cell which is isolated from bone marrow, blood, dermis and periosteum, and is a stem cell differentiated into various cells such as adipocytes, chondrocytes and bone cells Quot; means pluripotent or multipotent cells that can be used.

In particular, the mesenchymal stem cell used in the present invention may be an mesenchymal stem cell of an animal, preferably a mammalian animal, more preferably a human mesenchymal stem cell. The mesenchymal stem cells of the present invention may be derived from bone marrow, adipose tissue, peripheral blood, liver, lung, amniotic fluid, placenta chorioamnion or cord blood, preferably derived from adipose tissue or peripheral blood.

For reference, mesenchymal stem cells can be obtained from various sources as described above. Specifically, the process for obtaining mesenchymal stem cells is as follows: (1) a mammal including a human or a mouse, preferably a human Isolating mesenchymal stem cells from an 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) removing floating cells in the culture process and subculturing the cells attached to the culture plate, thereby obtaining finally formed 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: Proc. Soc. Exp (1986)), 199 Iscove's MEM (Iscove, N. et al., J. Exp. Med. 1963), F12 (Ham, Proc. Natl. Acad < / RTI > Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1963)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 1959) and McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp. Biol. Med. )), But is not limited thereto. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin, etc.) and glutamine.

In addition, identification of isolated mesenchymal stem cells can be performed by flow cytometry. Such flow cytometry analysis is carried out using a specific surface marker of mesenchymal stem cells. Preferably, mesenchymal stem cells which can be used for the preparation of mesenchymal stem cells having immunomodulating ability in the present invention are cells which are positive for CD105, CD29 and CD44, and which have immunologic characteristics of CD34, CD45 and HLA-DR .

In the present invention, in order to use mesenchymal stem cells derived from various sources as therapeutic agents for immune diseases, new mesenchymal stem cells having immunomodulating ability transformed with a vector capable of overexpressing sRAGE were prepared.

The sRAGE introduced into mesenchymal stem cells according to the present invention is composed of the nucleotide sequence of SEQ ID NO: 1 and has the amino acid sequence of SEQ ID NO: 2 encoded from the nucleotide sequence of SEQ ID NO:

The nucleotide sequence encoding sRAGE may be introduced into a cell by a method known in the art. For example, the sequence may be introduced into its own sequence or vector. Methods for introducing nucleic acid sequences into cells are known in the art and can be accomplished, for example, by methods including electroporation, calcium phosphate, gene gun and liposome methods. In addition, the introduction can be carried out using virus as a carrier. The nucleotide sequence encoding sRAGE may be integrated into the genome of the cell or may be present in the cell separately from the genome.

In the present invention, "vector" means a nucleic acid molecule capable of transferring another nucleic acid to which it is linked. In view of the nucleic acid sequence mediating the introduction of a specific gene, the vector in the present invention is interpreted as being capable of being used interchangeably with nucleic acid constructs, and cassettes. The vector includes, for example, a plasmid or a virus-derived vector. Plasmid is a circular double-stranded DNA chain to which additional DNA can be ligated. The vectors used in the present invention include, for example, plasmid expression vectors, virus expression vectors (e.g., SV40, replication defective retroviruses, adenoviruses, and adeno-associated viruses) and viral vectors capable of performing their equivalent functions But are not limited to these.

In addition, the present inventors have conducted an experiment to confirm the possibility of using the novel mesenchymal stem cells having the immunomodulating ability as a therapeutic agent for an immune disease. As a result, sRAGE-overexpressing mesenchymal stem cells Expression of genes such as TGF-beta, IDO, HGF and IL-10, which are factors, was found to be significantly increased compared with that of sSAGE-overexpressed mesenchymal stem cells, and the expression of cell migration related genes was also markedly increased .

Among these TGF-beta, IDO, HGF and IL-10, indoleamine 2,3-dioxygenase (IDO) is known to be a factor for measuring the function and activity of the immune system. And transforming growth factor beta (TGF-beta) is a secreted protein in which three isoforms called TGFbeta1, TGFbeta2, and TGFbeta3 are present. TGFbeta is a large protein precursor Wherein TGF [beta] l comprises 390 amino acids, and TGF [beta] 2 and TGF [beta] 3 contain 412 amino acids, respectively. In particular, since TGFβ has a growth inhibitory activity on most cells, TGFβ has a growth inhibitory activity against cancer cells expressing normal TGFβ receptors (Twardzik et al., J. Natl. Cancer Institute, 81: 1182- 1185, 1989). Therefore, tumor cells may be inhibited from growth inhibition by TGFβ by inhibiting their expression of TGFβ receptor. In addition, the expression of Foxp3 in the presence of TGF-beta is derived from naive T cells, and the Tregs thus induced have the same immunosuppressive function. In addition, the deficiency of the TGF-beta gene does not control the activity of inflammatory immune cells, leading to severe autoimmune diseases.

Furthermore, the present inventors have confirmed that the mesenchymal stem cells of the present invention in which sRAGE is overexpressed are excellent in autophagy and mitochondrial activities. The autophoresis is a process in which bacteria degrade their own organelles or cellular components, Is a function distinct from heterophages that are accepted by intracellular ingestion such as phonation or phagocytosis. The cell functions to decompose its own protein in response to a nutrient deficiency and to remove unnecessary cellular components in the process of rebuilding the cells. The cell component is surrounded by a membrane derived from an endoplasmic reticulum to form a child cell, It fuses with the cotton to form the child's parosomes and break it down.

In addition, since the self-feeding effect can control the beneficial or deleterious effects of immunity and inflammation and has an effect of preventing infectious diseases, autoimmune and inflammatory diseases, the mesenchymal stem cells overexpressing sRAGE in the present invention are self- And may be more useful for the treatment of immune and inflammatory diseases. In situations where the mitochondrial activity is not regulated, more severe inflammation occurs, which makes the mitochondrial adverse reaction to the development of immune inflammatory disease very important. When the mitochondrial activity is decreased, the control system of the diseased cell becomes unstable, thereby causing excessive cell activity or proliferation.

Therefore, in the present invention, in the case of mesenchymal stem cells overexpressing sRAGE in the present invention, it was confirmed that autogenous phagocytosis was increased and mitochondrial activity was increased as compared with mesenchymal stem cells in which sRAGE was not overexpressed. The inventive mesenchymal stem cells can be used as a cell therapy agent for treating an effective immune disease.

In the present invention, mesenchymal stem cells overexpressing sRAGE having immunomodulatory ability may be mesenchymal stem cells isolated from peripheral blood or adipose tissue, and may be immunized with immunodeficient mesenchymal stem cells as an active ingredient And can be used as a cell therapy composition for preventing or treating diseases.

In the present invention, the cell therapeutic agent refers to a substance that alters the biological characteristics of a cell such as autologous, allogenic, and xenogenic cells in vitro, in order to restore the functions of cells and tissues , Which is used for the purpose of treatment, diagnosis and prevention through a series of actions. Since 1993, the United States has been administering cell therapy products as medicines since 2002. These cell treatments can be classified into two categories. The first is stem cell therapy for tissue regeneration or restoration of long-term function, the second is immunization for controlling immune response such as inhibition of in vivo immune response or exaggeration of immune response Cell therapy.

The administration route of the cell therapeutic composition of the present invention can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration.

The composition may be formulated in a suitable form together with a pharmaceutical carrier commonly used in cell therapy. &Quot; Pharmaceutically acceptable " refers to compositions which are physiologically acceptable and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain 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 can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition may also be administered by any device capable of transferring the cell therapy agent to the target cell.

The cell therapeutic composition of the present invention may comprise a therapeutically effective amount of a cell therapy agent for the treatment of diseases. The term therapeutically effective amount refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, This includes an amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the cell therapy agent contained in the composition of the present invention will vary depending on the desired effect. The optimal cell therapy agent content can therefore be readily determined by those skilled in the art and will depend upon a variety of factors including the nature of the disease, the severity of the disease, the amount of other ingredients contained in the composition, the type of formulation, and the age, weight, , The time of administration, the route of administration and the fraction of the composition, the duration of the treatment, the co-administered drug, and the like. It is important to include the amount by which all of the above factors are taken into account so as to obtain the maximum effect in a minimal amount without side effects. For example, the cytotoxic agent composition of the present invention may be administered such that mesenchymal stem cells having immunomodulating ability are administered at a dose of 2.5 × 10 ⁵ to 2.5 × 10 ⁷ cells per kg body weight of an administration subject.

The invention also provides a method of preventing or treating an immune disorder comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the invention.

As used herein, the term mammal refers to a mammal that is the subject of treatment, observation, or experiment, preferably a human.

In the treatment method of the present invention, when the cell therapeutic composition of the present invention is administered to an adult, the cell therapeutic agent contained in the composition is administered at a dose of 1 × 10 ⁴ to 1 × 10 ⁸ It is preferable to include the number of cells.

In the therapeutic method of the present invention, the composition comprising the cell treatment agent of the present invention as an active ingredient can be administered orally, rectally, intravenously, intravenously, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, Lt; RTI ID = 0.0 > route. ≪ / RTI >

In the present invention, the immunological diseases to be treated with the sRAGE-overexpressing mesenchymal stem cells and the cell therapeutic composition for treating an immunological disease comprising the cells include, but are not limited to, osteoarthritis, rheumatoid arthritis, asthma, Dermatitis, psoriasis, cystic fibrosis, posttransplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus (systemic lupus) erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, pernicious anemia, glomerulonephritis glomerulonephritis and pulmonary fibrosis, inflammatory bowel dieses, Crohns disease, autoimmune D diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD) ), Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease, Angina, Cancer metastasis, Small artery disease and mitochondrial diseases Mitochondrial disease).

Hereinafter, the present invention will be described in detail with reference to examples. Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

< Example  1>

According to the invention sRAGE - Overexpression Mesenchymal  making

<1-1> sRAGE  Production of Recombinant Expression Vector Containing the Gene

For the production of mesenchymal stem cells overexpressing sRAGE, the present inventors first constructed an expression vector in which the sRAGE gene is operatively linked. For this, the sRAGE gene shown in SEQ ID NO: 1 was cloned into a pcDNA 3.0-HA vector to prepare the recombinant vector of FIG.

&Lt; 1-2 > Mesenchymal stem cell  Isolation and Culture

After removing 50 cc of blood from normal subjects, PBMC (Peripheral Blood Mononuclear Cell) was separated and cultured in a-MEM medium containing 20% FBS (Fetal Bovine Serum) for 5 days. Subsequently, the medium was replaced at intervals of 3 days. The cells were removed from the culture plate according to the degree of cell proliferation and cultured. The cultivation was carried out three times (passage 3) to obtain mesenchymal stem cells from peripheral blood. Cellular markers were identified to identify mesenchymal stem cells. As a result of the confirmation, it was confirmed that CD105, CD29 and CD44 were positive, and CD34, CD45 and HLA-DR were negative, so that isolated cells were mesenchymal stem cells.

<1-3> sRAGE Over-expressed recombination Mesenchymal  making

The mesenchymal stem cells isolated in Example < 1-2 > were transformed with the recombinant vector prepared in Example <1-1> to produce sRAGE overexpressed mesenchymal stem cells.

In order to confirm whether sRAGE was actually overexpressed in the prepared mesenchymal stem cells, the expression level of sRAGE in the mesenchymal stem cells transformed with the vector not containing sRAGE and the mesenchymal stem cells prepared in the present invention was measured by ELISA enzyme-linked immunosorbent assay) and western blot analysis.

As a result, as shown in FIG. 2, it was confirmed that sRAGE was overexpressed in the sRAGE-overexpressing mesenchymal stem cells prepared in the present invention (left graph is ELISA result and right image is Western blot result).

< Example  2>

sRAGE - Overexpression Mesenchymal  Modulating effect of immunity or inflammation related factors

&Lt; 2-1 > Immunomodulatory molecules and Chemokine  Receptor chemokine receptor ) Expression induction assay

The present inventors performed real-time PCR (polymerase chain reaction) of sRAGE-overexpressing mesenchymal stem cells of the present invention by expressing the expression of IDO, TGF-beta and HGF, which are genes involved in immunoregulatory activity in mesenchymal stem cells, Method. As a result, in the mesenchymal stem cells overexpressing sRAGE, the expression of IDO, TGF-beta and HGF was significantly (about 2 to 6 times) higher than that of mesenchymal stem cells in which sRAGE was not overexpressed ). In addition, fluorescence microscopy revealed that the expression of IL-10 cytokine was also significantly increased by overexpression of sRAGE (FIG. 9). These results indicate that the expression of IDO, IL-10, HGF and TGF-beta is significantly increased in sRAGE-overexpressing mesenchymal stem cells compared to cells not overexpressing sRAGE.

Furthermore, the expression levels of CCR1, CCR3, CCR4, CCR7, CXCR1 and CXCR4, which are related to the migration of mesenchymal stem cells, were analyzed by real time PCR. As a result, the expression of the above factors was markedly increased by overexpression of sRAGE (Fig. 4B). As described above, sRAGE-overexpressing mesenchymal stem cells significantly increase the expression of cell migration-related genes as compared with mesenchymal stem cells overexpressing sRAGE. Therefore, sRAGE-overexpressing mesenchymal stem cells of the present invention are more effective in treating inflammation and immune diseases Can be used as a cell therapy agent.

The primer sequences used in the PCR to analyze the expression levels of the respective factors are as shown in Table 1 below.

<2-2> Analysis of inhibitory effect of inflammation and tumor factor

Real - time PCR was used to observe gene expression when the inflammatory environment of mesenchymal stem cells was added. 5 × 10 ⁵ cells of mesenchymal stem cells were placed on a 6-well plate and cultured for 3 days at 37 ° C. with stimulation with 1 μg / ml of LPS. At this time, mesenchymal stem cells not treated with LPS were used as a control group, and the expression level of inflammatory and tumor factors in LPS - treated mesenchymal stem cells was analyzed using real - time PCR method. IL-6, VEGF (Vascular Endothelial Growth Factor) and HMGB-1 (High-Mobility Group protein B1) were significantly higher in LPS-treated mesenchymal stem cells than in LPS- (Fig. 4C).

The expression of IL-1β, IL-6, and HMGB-1 was significantly reduced by overexpression of sRAGE in these mesenchymal stem cells (FIG. 4D) .

The primer sequences used in the above PCR are as shown in Table 2 below.

Name of the primer order IL-1beta forward GGA CAA GCT GAG GAA GAT GC IL-1beta Reverse TCG TTA TCC CAT GTG TCG AA IL-6 forward AAT TCG GTA CAT CCT CGA CGG IL-6 reverse direction GGT TGT TTT CTG CCA GTG CC HMGB-1 Forward GAT CCC AAT GCA CCC AAG AG HMGB-1 reverse direction TTC GCA ACA TCA CCA ATGA VEGF forward CCA TGA ACT TTC TGC TGT CTT Reverse VEGF ATC GCA TCA GGG GCA CAC AG

<2-3> sRAGE - Overexpression Mesenchymal Self-predation ( autophagy ) Increase in action

The overexpression of sRAGE in adipose tissue - derived mesenchymal stem cells was induced and cultured for 3 days, and the degree of autogenous vesicle formation was observed by electron microscopy. FIG. 10 is a photograph of the mesenchymal stem cell transfected with the RAGE-containing recombinant vector and the mesenchymal stem cell transformed with the mock vector not containing sRAGE, and showing the degree of autopatch activity of the sAGE-overexpressed mesenchymal stem cells The number of autophagic vacuoles increased in stem cells compared to the control group.

<2-4> T cells Pathogenic activity  Inhibition and Immunoregulatory T Cell Increase

The sRAGE-overexpressing mesenchymal stem cells of the present invention prepared in Example 1 were analyzed for the expression level of mesenchymal stem cell cytokine by overexpression of sRAGE. To this end, the sRAGE-overexpressing mesenchymal stem cells were co-cultured with human CD4 + T cells and the amounts of IFN-y, IL-17, IL-4 and Foxp3 + Treg were measured.

Th1 (IFN-γ) and Th17 (IL-17) levels were significantly decreased when sRAGE was overexpressed in mesenchymal stem cells compared with no overexpression of sRAGE, but Th2 (IL-4) and Foxp3 + Tregs were slightly increased (Fig. 3). These results indicate that the sRAGE-overexpressing mesenchymal stem cells of the present invention reduce inflammatory effector T cells such as Th1 and Th17, and do not decrease Treg and Th2 cells, compared with mesenchymal stem cells without sRAGE treatment .

< Example  3>

sRAGE - Overexpression Mesenchymal GVHD ( Graft-versus-host disease ) Analysis of treatment effect

The following experiment was carried out to confirm that the sRAGE-overexpressing mesenchymal stem cells prepared according to the present invention were actually useful for the treatment of immune diseases.

First, in order to observe alloresponse modulating effect by sRAGE-overexpressing mesenchymal stem cells, CFSE (Carboxy Fluorescein Succinimidyl Ester) was co-cultured with labeled CD4 + T cells and CFSE expression was observed, .

As a result, alloreactive T cell proliferation was significantly decreased under the condition of co-culturing of mesenchymal stem cells and sRAGE-mesenchymal stem cell (MSC), and sRAGE-MSC inhibited alloreactive T cell proliferation more significantly 6). As shown in FIG. 7, sRAGE-MSC was further reduced in the Th1 and Th17 cells compared to mesenchymal stem cells in the pathogenesis of T cell differentiation in the allogeneic control by sRAGE overexpression And Treg showed no significant difference.

Further, the present inventors observed the level of cytokine in the culture medium under the above experimental conditions, that is, alloresponse conditions, and the sRAGE-MSC of the present invention showed a more effective reduction of inflammatory cytokine production than the other groups 8).

< Example  4>

sRAGE - Overexpression Mesenchymal  Analysis of treatment effect of arthritis

The following experiment was conducted to confirm that sRAGE-overexpressing mesenchymal stem cells have therapeutic effect on arthritis.

<4-1> sRAGE - Overexpression Mesenchymal Rheumatism  Arthritis treatment effect

In order to prepare an autoimmune arthritis mouse model, a mouse (IL-1Ra knockout mouse) lacking the IL-1 receptor gene was prepared according to the method disclosed by Y. Iwakura, The IL-1 receptor antagonist (IL-1Ra) acts directly on the IL-1 receptor and prevents IL-1α and IL-1β from acting on the receptor, thereby naturally inducing autoimmune arthritis disease Method. The sRAGE-overexpressing mesenchymal stem cells prepared in the present invention were injected into mice having the above-mentioned diseases, and sRAGE-overexpressing mesenchymal stem cells were injected iv into the mice at a density of 2 x 10 &lt; ⁶ & After 10 days, serum was separated from these mice to analyze the amount of IgG, IgG1 and IgG2a present in the serum (FIG. 11A). As a control, the serum of the arthritic mouse injected with only mesenchymal stem cells in which sRAGE was not overexpressed in the mice in which arthritis was induced was used to measure the amount of the immunoglobin. In addition, the treatment effect of arthritis was analyzed by measuring arthritic index in each mouse group.

As a result, arthritic symptoms were improved in the mouse group treated with sRAGE-overexpressing mesenchymal stem cells compared to the group treated with mesenchymal stem cells (Fig. 11B), and Th2 type IgG1 and IgG3 (Fig. 11C). &Lt; tb &gt;&lt; TABLE &gt;

<4-2> sRAGE - Overexpression Mesenchymal stem cells  Inflammatory cell infiltration analysis of injected mouse model

In order to analyze the degree of joint inflammation and cartilage destruction in the mouse joint tissues, the joints of each group were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, and tissue slices were attached to the slides. Before the basic dyeing, xylen was used to pass the deparaffinization process, and then ethanol was allowed to function from a high concentration to a low concentration. The staining procedure proceeded with hematoxylin and eosin staining.

As a result of the analysis, although arthritis inhibitory effect was exhibited by mesenchymal stem cells not overexpressing sRAGE, infiltration of inflammatory cells and destruction of cartilage were remarkably reduced in the group injected with sRAGE-overexpressing mesenchymal stem cells (Fig. 11D ).

<4-3> sRAGE - Overexpression Mesenchymal stem cells  Cytokine &lt; / RTI &gt; of the injected mouse model and Splenocyte  My T cell analysis

Cytokines (IFN-y, IL-17, IL-4 and Foxp) were stained with immunochemical staining from spleens obtained from each mouse and analyzed by optical microscope. Th17 (IL-17) cells, an intrinsic T cell subset of spleen cells of each mouse, were examined by mouse injected with sRAGE overexpressed mesenchymal stem cells compared to the mouse group injected with mesenchymal stem cells , While Treg (Foxp3 +) cells were significantly increased (Fig. 12A, B).

In addition, Th1 (IFN-γ) and Th17 (IL-17) cells, which are pathogenic cells, were observed in the mouse group injected with mesenchymal stem cells , Whereas sRAGE was significantly reduced by mice injected with overexpressing mesenchymal stem cells, whereas Treg (Foxp3 +) cells were significantly increased (Fig. 12C, D).

< Example  5>

sRAGE - Overexpression Mesenchymal Lupus ( lupus ) Analysis of treatment effect

In addition, sRAGE-overexpressing mesenchymal stem cells prepared according to the present invention were examined for their ability to regulate immunity on lupus animal model cells. Cells were isolated from Roquin mouse, a lupus animal model, and cultured for 3 days at a ratio of 1:10 with mesenchymal stem cells. The amount of IL-17 present in the culture solution was measured by ELISA.

As a result, compared with the mesenchymal stem cell group in which sRAGE was not overexpressed, the amount of IL-17 in sRAGE-overexpressing mesenchymal stem cells was further decreased, and the activity of immunoregulatory T cells was further increased by FACs (Fig. 13).

The sRAGE-overexpressing mesenchymal stem cells produced in the culture supernatant showed that the sRAGE-overexpressing mesenchymal stem cells had a decreased level of IgG secretion in the culture medium compared to the mesenchymal stem cells without sRAGE overexpression (Fig. 14).

From these results, it can be confirmed that the sLAGE-overexpressing mesenchymal stem cells of the present invention can regulate the pathogenic cells of the lupus model.

< Example  6>

sRAGE - Overexpression Mesenchymal stem cells  Analysis of effects on human T cell differentiation

The sRAGE-overexpressing mesenchymal stem cells prepared in the above examples were co-cultured with human CD4 + T cells and analyzed for their effect on T cell differentiation using a flow cytometer.

As shown in FIG. 5, sRAGE-overexpressing mesenchymal stem cells were found to further increase Foxp3 + Treg compared to the control (MSC without sRAGE overexpression), and Th1 and Th17 cells were significantly decreased And Th2 cells did not show a large difference (Fig. 5).

The results of the above examples show that the mesenchymal stem cells of the present invention produced by treating sRAGE can be effectively used as a new cell therapy for the treatment of immune diseases, Show.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Mesenchymal stem cells over expressing sRAGE having          immuno-modulating activity and cell therapeutic agent for          preventing or treating immune disease <130> PN1308-284 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> sRAGE DNA sequence <400> 1 atggcagccg gaacagcagt tggagcctgg gtgctggtcc tcagtctgtg gggggcagta 60 gtaggtgctc aaaacatcac agcccggatt ggcgagccac tggtgctgaa gtgtaagggg 120 gcccccaaga aaccacccca gcggctggaa tggaaactga acacaggccg gacagaagct 180 tggaaggtcc tgtctcccca gggaggaggc ccctgggaca gtgtggctcg tgtccttccc 240 aacggctccc tcttccttcc ggctgtcggg atccaggatg aggggatttt ccggtgccag 300 gcaatgaaca ggaatggaaa ggagaccaag tccaactacc gagtccgtgt ctaccagatt 360 cctgggaagc cagaaattgt agattctgcc tctgaactca cggctggtgt tcccaataag 420 gtggggacat gtgtgtcaga gggaagctac cctgcaggga ctcttagctg gcacttggat 480 gggaagcccc tggtgcctaa tgagaaggga gtatctgtga aggaacagac caggagacac 540 cctgagacag ggctcttcac actgcagtcg gagctaatgg tgaccccagc ccggggagga 600 gatccccgtc ccaccttctc ctgtagcttc agcccaggcc ttccccgaca ccgggccttg 660 cgcacagccc ccatccagcc ccgtgtctgg gagcctgtgc ctctggagga ggtccaattg 720 gtggtggagc cagaaggtgg agcagtagct cctggtggaa ccgtaaccct gacctgtgaa 780 gtccctgccc agccctctcc tcaaatccac tggatgaagg atggtgtgcc cttgcccctt 840 ccccccagcc ctgtgctgat cctccctgag atagggcctc aggaccaggg aacctacagc 900 tgtgtggcca cccattccag ccacgggccc caggaaagcc gtgctgtcag catcagcatc 960 atcgaaccag gcgaggaggg gccaactgca ggtgaggggt ttgataaagt cagggaagca 1020 gaagatagcc cccaacacat gtga 1044 <210> 2 <211> 347 <212> PRT <213> Artificial Sequence <220> <223> sRAGE peptide sequence <400> 2 Met Ala Ala Gly Thr Ala Val Gly Ala Trp Val Leu Val Leu Ser Leu   1 5 10 15 Trp Gly Ala Val Val Gly Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu              20 25 30 Pro Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg          35 40 45 Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu      50 55 60 Ser Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro  65 70 75 80 Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly Ile                  85 90 95 Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn             100 105 110 Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp         115 120 125 Ser Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys     130 135 140 Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp 145 150 155 160 Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln                 165 170 175 Thr Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu             180 185 190 Met Val Thr Pro Ala Arg Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys         195 200 205 Ser Phe Ser Pro Gly Leu Pro Arg His His Ala Leu Arg Thr Ala Pro     210 215 220 Ile Gln Pro Arg Val Trp Glu Pro Val Pro Leu Glu Glu Val Gln Leu 225 230 235 240 Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr                 245 250 255 Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met             260 265 270 Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile Leu         275 280 285 Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr     290 295 300 His Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile 305 310 315 320 Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala Gly Glu Gly Phe Asp Lys                 325 330 335 Val Arg Glu Ala Glu Asp Ser Pro Gln His Met             340 345

Claims (12)

A mesenchymal stem cell having immunoregulatory ability introduced with a nucleotide sequence encoding sRAGE (soluble receptor for advanced glycation endproducts). The method according to claim 1,
Wherein the sRAGE has the amino acid sequence of SEQ ID NO: 2. The method according to claim 1,
Wherein the nucleotide sequence encoding the sRAGE has the nucleotide sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the nucleotide sequence encoding sRAGE is introduced by a vector expressing sRAGE in mesenchymal stem cells. The method according to claim 1,
Wherein the mesenchymal stem cells are derived from peripheral blood or adipose tissue. The method according to claim 1,
Wherein the mesenchymal stem cells have increased expression of one or more factors selected from the group consisting of IDO, TGF-beta, HGF and IL-10 by overexpression of sRAGE. The method according to claim 1,
Wherein said mesenchymal stem cells are characterized in that the expression of any one or more of the elements selected from the group consisting of IL-1, IL-6 and HMGB-1 is reduced by overexpression of sRAGE. The method according to claim 1,
Wherein the mesenchymal stem cells have increased expression of at least one factor selected from the group consisting of CCR1, CCR3, CCR4, CCR7, CXCR1 and CXCR4 by overexpression of sRAGE. The method according to claim 1,
Wherein said mesenchymal stem cells are stabilized by overexpression of sRAGE. 10. A cell therapy composition for the prevention or treatment of immune diseases comprising the mesenchymal stem cells according to any one of claims 1 to 9 as an active ingredient. 11. The method of claim 10,
Wherein said composition is administered at a dose of 2.5 x 10 ⁵ to 2.5 x 10 ⁷ cells per kg body weight of the subject. 11. The method of claim 10,
The immunological diseases are selected from the group consisting of osteoarthritis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, posttransplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease Glomerulonephritis and pulmonary fibrosis, inflammatory bowel dieses, Crohns disease, autoimmune diabetes, autoimmune diabetes, autoimmune diabetes, autoimmune diabetes, Diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive cardiomyopathy, Or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis of chronic obstructive pulmonary diseases (COPD), Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease, Metastasis, metastatic disease, metastasis, small artery disease, and mitochondrial disease.

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