KR101516763B1 - Pharmaceutical composition comprising stem cells treated with DNA methyltransferase or culture thereof for prevention and treatment of immune diseases and inflammatory diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating immune diseases or inflammatory diseases, containing stem cells cultured by adding a DNA methyltransferase inhibitor to stem cells, or a culture thereof. The pharmaceutical composition according to the present invention can be economically used as a cellular therapeutic agent for preventing or treating immune diseases and inflammatory diseases like self-immune diseases such as Crohn′s disease, rheumatoid arthritis, atopic dermatitis, and the like, without any side effects, thereby replacing the existing immunosuppressants and anti-inflammatory drugs which are known as having side effects.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating an immunological disease or an inflammatory disease comprising a stem cell treated with a DNA methyltransferase inhibitor or a culture thereof, inflammatory diseases}

The present invention relates to a pharmaceutical composition for the prevention or treatment of immune diseases and inflammatory diseases including stem cells or cultures thereof cultured by adding a DNA methyl group transferase inhibitor to stem cells.

In order to protect the human body from internal and external pathogenic substances, the immune system constructs a defense system consisting of various cells, which is accomplished through the specific function of each cell and signal transmission between the cells. The immune system of the human body can be largely divided into immunity tolerance that controls immune suppression and immunity which promotes immunity. These two immune functions are balanced to form immunological homeostasis . However, if for some reason its balance is broken and the immune response is greater than that of immune tolerance, immune and inflammatory diseases will result.

Immunosuppression for the prevention or treatment of an immune disease or an inflammatory disease includes a specific inhibitor and a nonspecific inhibitor that inhibit the response only. Theoretically, a specific inhibitor should be excellent, but a nonspecific inhibitor is mainly used. Clinically, the most commonly used immunosuppressants are cyclosporine (Neoral, Cipol A), azathioprine (imuran), and prednisolone (a kind of steroid). The above three cases showed less side effects and immunosuppressive effects than when they were taken together. Recently, various immunosuppressants such as FK 506, RATG, OKT3, and Cellcept have been developed and used.

These immunosuppressants inhibit several processes such as antigen phagocytosis by macrophage, antigen recognition by lymphocyte, cell division, division of T cells and B cells, and antibody production during the course from antigen stimulation to antibody production, It causes. Most of them have antitumor activity because they inhibit cell division by mediating DNA disorder or DNA synthesis inhibition.

However, there have been problems such as hypertension and nephrotoxicity (lowered renal function), which are typical side effects, and that the incidence of this side effect is high, so it is necessary to observe the progress sufficiently during use. Other side effects include tremors, seizures, hepatitis, retention of fluid, increased blood uric acid, decreased muscle strength, hypertrichosis, and gingival hypertrophy. Azathioprine, a commonly used inhibitor, may inhibit bone marrow function such as decreased leukocyte levels, anemia, and thrombocytopenia. There may be complications such as pancreatitis, hepatitis, biliary retention, and rarely hair loss and fever. Prednisolone, one of the steroids, is the first of the immunosuppressants to be used and has the broadest inhibitory effect. It increases appetite, increases shoulders and back muscles, and temporarily brings happiness. However, these steroid drugs not only promote atherosclerosis but also cause hypertension, gastric ulcer, diabetes, growth retardation, osteoporosis, cataracts, glaucoma It is a drug to watch out for.

As a therapeutic agent for overcoming the above-mentioned side effects, a biopharmaceutical drug has become popular, and a typical example thereof is a stem cell treatment agent. Although mesenchymal stem cell research has been reported to utilize the differentiation ability of stem cells to treat damaged joint tissues and to treat inflammation and the like (U.S. Patent No. 6,835,377), the therapeutic mechanism has been clarified Therapeutics are a rare reality.

5-azacytidine has been used as an anticancer agent for the treatment of myelodysplastic syndrome. It is inserted into DNA during cell proliferation or inserted into RNA during transcription, methyltransferase), thereby reducing DNA methylation. By such a mechanism, it inhibits the gene expression of the cell, thereby blocking the proliferation of cancer cells. However, it has not been known that pre-treatment of 5-azacytidine with stem cells enhances the expression of inflammatory regulators and the like secreted from stem cells to prevent or treat inflammatory diseases or immune diseases.

Accordingly, the present inventors have completed the present invention by confirming that when 5-azacytidine is added to stem cells, the expression of an inflammatory regulator or a cell migration-related factor secreted from stem cells is significantly increased.

It is an object of the present invention to provide an economical pharmaceutical composition free from side effects that can replace conventional immunosuppressants and inflammation inhibitors, and more particularly to a pharmaceutical composition comprising stem cells cultured in the presence of a DNA methyltransferase inhibitor The present invention provides a pharmaceutical composition for preventing or treating an immunological disease or an inflammatory disease, which comprises a cell or a culture thereof.

Another object of the present invention is to provide a method for treating an immunological disease or inflammatory disease, comprising the step of administering the composition to an individual suffering from an immunological disease or an inflammatory disease.

It is still another object of the present invention to provide a method for inhibiting an immune response or an inflammatory response of a subject comprising the step of administering the stem cell or a culture thereof to a subject.

It is another object of the present invention to provide a method for producing an immunosuppressant or an anti-inflammatory agent.

It is still another object of the present invention to provide an implanted material containing the stem cell and a DNA methyltransferase inhibitor, or an implanted material from which the stem cell has been removed from the implanted material, and a method for producing the implanted material.

It is another object of the present invention to provide a complex comprising the stem cell and a DNA methyltransferase inhibitor.

Yet another object of the present invention is to provide a stem cell or a culture thereof in which a DNA methyl group transferase inhibitor is added to stem cells.

In order to achieve the above object, the present invention provides a method for preventing or treating an immune disease or inflammatory disease including stem cells cultured by treating a stem cell with a DNA methyltransferase inhibitor To provide a pharmaceutical composition.

In the present invention, when a DNA methyltransferase inhibitor, specifically 5-azacytidine, is administered to stem cells, inflammatory cell regulatory factors (COX2, PTGES, IDO , IL-6) and adhesion molecules and receptors (ICAM, VCAM, CXCR2, CXCR4, CXCR6) proteins associated with cell migration are increased and thus control immune and inflammation Respectively. In particular, it was first identified that 5-azacytidine can increase the expression of the factor by hypomethylation or demethylation of the promoter region of the factor. Accordingly, the composition of the present invention can be used as a cell therapy agent for immunomodulation and inflammation control by a stem cell treated with a DNA methyl group transferase inhibitor or a culture thereof.

The term "DNA methyl group transfer enzyme" in the present invention is known as a DNA methylase that adds a methyl group to a DNA base or a DNA demethylase that removes a base methyl group, and exists in many species ranging from bacteria to mammals.

In the present invention, "DNA methyltransferase inhibitor" is a substance that inhibits DNA methylation or DNA demethylation by inhibiting the activity of the enzyme. Representative examples include 5-azacytidine ). In the present invention, 5-azacytidine can increase the expression of the factor by hypomethylation or demethylation of the DNA promoter portion encoding the inflammatory regulator and / -aza 'can be used in combination.

In the present invention, "treatment and cultivation" of a DNA methyl group transferase inhibitor may be specifically performed by adding an inhibitor of the enzyme to a medium of stem cells. Preferably, the inhibitor may be added and cultured at a concentration of 0.01 to 100 μM for 0.1 to 200 hours, more preferably for 1 to 72 hours. Alternatively, it may be cultured after addition of a DNA methyl group transferase inhibitor, followed by culture medium replacement.

In the present invention, "stem cells" are cells having the ability to differentiate into various tissues, i.e., undifferentiated cells, and multipotent stem cells include bone marrow, cord blood, placenta (or placental tissue cells) , Fat (or adipose tissue cells), and the like are pluripotent stem cells derived from various adult cells. For example, mesenchymal stem cells derived from bone marrow have undergone various studies for development as a cell therapy agent by the pluripotency that can be differentiated into adipose tissue, bone / cartilage tissue, and muscle tissue.

In the present invention, the stem cells may be human adult stem cells, human pluripotent stem cells, human pluripotent stem cells, induced pluripotent stem cells, animal embryonic stem cells, or animal adult stem cells Lt; / RTI > On the other hand, adult stem cells can be mesenchymal stem cells, mesenchymal stromal cells derived from human tissue, mesenchymal stem cells derived from human tissues, multipotential stem cells or amniotic epithelial cells, , Mesenchymal stem cells selected from the group consisting of cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. Most preferably, it may be human Umbilical Cord Blood Mesenchymal Stem Cell (hUCB-MSC). The method of obtaining stem cells from each of the strains can be performed by a method known in the art and is not limited to the method of the embodiment of the present invention.

In the present invention, the term "culture product" may include a cell culture solution containing stem cells, a culture supernatant from which stem cells have been removed from the cell culture solution, and a dilution solution thereof. The composition of the culture may further include components necessary for culturing stem cells as well as components that act synergistically with stem cell proliferation. The composition may be easily prepared by those skilled in the art Can be selected.

Preferably, adult stem cells obtained by treating human adult stem cells with 0.01 to 100 μM 5-azacytidine for 0.1 to 200 hours can be used. When 5-azacytidine is treated for less than 0.1 hour, the DNA demethylation pathway may not be sufficiently activated, and since treatment of 5-azacytidine for 200 hours or more is not economically advantageous, May be used for adult stem cells treated with 5-azacytidine for 0.1 to 200 hours, more preferably for 1 to 72 hours, and most preferably for 24 hours.

In addition, the pharmaceutical composition of the present invention may further include a stem cell or a culture thereof cultured by addition of IFN-? And / or TNF-? To the DNA methyltransferase inhibitor.

The DNA methyltransferase inhibitor of the present invention and IFN-? And / or TNF-? May be added at the same time, or IFN-? And / or TNF-? May be added after the addition of the DNA methyltransferase inhibitor, After the addition of? and / or TNF-a, a DNA methyltransferase inhibitor may be added and cultured.

(COX2, PTGES, IDO, IL-6) secreted from the stem cells and cell migration (proliferation) by IFN-y and / or TNF- (ICAM, VCAM, CXCR2, CXCR4, and CXCR6) associated with the migration of the 5-azacytidine was increased, and further, in the case of pretreating 5-azacytidine, the expression of the adhesion molecule and the receptor And the expression of these genes was further increased.

As the medium used for culturing adult stem cells, a conventional medium known to be suitable for stem cell culture can be used. For example, DMEM (Dulbecco's modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) Can be used.

The adult stem cell medium may be supplemented with an additive. (E. G., Serum, e. G., FBS, serum replacement, albumin, or essential amino acids and non-essential amino acids such as glutamine) in an isotonic solution. Furthermore, it has been found that lipid (fatty acid, cholesterol, serum HDL or LDL extract) and other components found in most types of storage medium of this kind (such as insulin or transferrin, nucleoside or nucleotide, pyruvate, G., Glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as? -Mercaptoethanol).

In the examples of the present invention, COX2, PTGES, IDO, and IL-6, which are inflammatory cytokines (inflammatory factors), and adherence related to cell migration in stem cells cultured with 5-azacytidine The expression of adhesion molecules and receptors (ICAM, VCAM, CXCR2, CXCR4, and CXCR6) was increased and the secretion of PGE2 was also increased depending on the concentration of 5-atatidine. PGE2 is known to suppress the secretion of cytokines such as inflammatory cytokines such as interleukin-1 [beta] and TNF- [alpha].

Therefore, the stem cells and the cultures thereof according to the present invention are useful for the prevention and treatment of immune diseases and inflammatory diseases.

The term "inflammatory disease" in the present invention refers to a disease in which inflammation is the main lesion, including but not limited to edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, Rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, nephritis, myositis, hepatitis, cystitis, nephritis, Sjögren's syndrome, inflammatory bowel disease, Syndrome (sjogren's syndrome) or multiple sclerosis.

In the present invention, "autoimmune disease" is a disease that is problematic when a specific immune response occurs. Preferably, the autoimmune disease, graft rejection, graft versus host disease, autoimmune disease, Crohn's disease, Hyperlipidemia and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, hyperlipidemia, hyperlipidemia, hyperlipidemia, rheumatoid arthritis, rheumatoid arthritis, Hashimoto's thyroiditis, May be, for example, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma or ulcerative colitis.

In the present invention, "prevention" includes all actions that inhibit or delay the onset of an immunological disease or inflammatory disease upon administration of the composition, and "treatment" means that the administration of the composition improves or reduces the symptoms of the immune or inflammatory disease Including all acts that

In addition, the composition of the present invention contains 1.0 × 10 ⁵ to 1.0 × 10 ⁹ , preferably 1.0 × 10 ⁶ to 1.0 × 10 ⁸ , more preferably 1.0 × 10 ⁷ cells per 1 ml But is not limited thereto.

The compositions of the present invention can be used without being frozen or frozen for future use. If required to be frozen, standard cryopreservatives (eg, DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) may be added to the pre-freeze cell population.

In addition, the composition may be formulated into a unit dosage form suitable for administration to the body of a patient according to a conventional method in the pharmaceutical field, and the formulation may contain an effective dose by one or several doses do. Suitable formulations for this purpose include injections such as ampoules injected as parenteral dosage forms, injecting agents such as injection bags, and spray agents such as aerosol formulations. The injectable ampoule may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution. The infusion bag may be made of polyvinyl chloride or polyethylene and may be manufactured by Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo, An injection bag of a yarn can be exemplified.

In addition to the active ingredient, the pharmaceutical preparation may contain one or more pharmaceutically acceptable inert carriers such as a preservative, an anhydrous agent, a solubilizing agent or a stabilizer in the case of injection, May further comprise a base, an excipient, a lubricant or a preservative.

The thus-prepared composition or pharmaceutical preparation of the present invention can be administered in the form of a mixture with other stem cells used for transplantation and other uses, or a mixture thereof with such stem cells, using administration methods commonly used in the art , Preferably, it is possible, but not limited, to directly engraft or transplant to the diseased part of the patient in need of treatment, or directly to the abdominal cavity. In addition, the above-mentioned administration is both non-surgical administration using a catheter and surgical administration such as implantation or transplant after incision of a disease site, but non-surgical administration method using a catheter is more preferable. In addition to parenteral administration, for example, to direct lesions according to conventional methods, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible.

The daily dose of the stem cells may be administered once or several times in the range of 1.0 × 10 ⁴ to 1.0 × 10 ¹⁰ cells / kg body weight, preferably 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cells / kg body weight . It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the body weight, age and sex of the patient, The scope of the present invention is not limited thereto.

In another aspect, the present invention provides a method for inhibiting an immune reaction or an inflammatory reaction of a subject, comprising the step of administering a stem cell or a culture thereof cultured by adding a DNA methyl group transferase inhibitor to the stem cell, to provide. Specifically, by administering the stem cell and its culture treated with the DNA methyl group transferase inhibitor of the present invention, the immune response can be suppressed or the inflammation can be controlled.

The term "subject" as used herein includes, but is not limited to, cows, dogs, pigs, chickens, sheep, horses, and mammals including humans. Preferably, the stem cell cultured and cultured by treating with the DNA methyltransferase inhibitor may be administered intraperitoneally or intravenously, directly into a lesion, or in a synovial cavity.

The inhibition of the immune response or the inflammatory response may include the prevention or treatment of an immune or inflammatory disease.

In another aspect, the present invention provides a method for producing an immunosuppressant or an anti-inflammatory agent comprising the step of adding a DNA methyl group transferase inhibitor to a stem cell and culturing.

As described above, the term "immunosuppressive agent" as used herein refers to a stem cell obtained by treating a stem cell with a DNA methyl group transferase inhibitor and culturing the stem cell or a culture thereof, ≪ / RTI >

As described above, the term "anti-inflammatory agent" means a preparation containing a stem cell or a cultured product obtained by treating a stem cell with a DNA methyl group transferase inhibitor and culturing the same, .

In another embodiment, the present invention includes a step of culturing a stem cell in a medium treated with a DNA methyl group transferase inhibitor, wherein the expression of an inflammatory regulator or a cell migration-related factor is increased in the stem cell IL-1 (Interleukin-1?), LIF (Leukemia inhibitory factor), IL-6 (Interleukin-6), COX-2 (Cyclooxygenase-2), Prostaglandin E synthase (PTGES), Indoleamine 2,3- ), ICAM (Intercellular adhesion molecule), VCAM (Vascular cell adhesion molecule), CXCR2 (CXC Chemokine Receptor 2), CXCR4 (CXC Chemokine Receptor 4) and CXCR6 (CXC Chemokine Receptor 6) Provide a production method.

According to an embodiment of the present invention, adult stem cells treated with 5-azacytidine may be administered with COX-2, PTGES, IDO, IL-6, IL-1 ?, LIF, ICAM, VCAM , CXCR2, CXCR4, CXCR6, and the like.

In addition, when IFN-y and / or TNF-? Were added after 5-azacytidine pretreatment, the expression of the factor was further increased than in the group not treated with 5-azacytidine. In the present invention, the protein can be recovered by collecting the medium in which the stem cells have been cultured, removing the cells and suspension by centrifugation and filtration, and recovering the remaining supernatant.

In another aspect, the invention provides an implant comprising stem cells and a DNA methyltransferase inhibitor.

As used herein, the term "implant" is intended to include a material that can be implanted in the human or mammalian body, such as a scaffold for isolating the damaged site from the outside or allowing the grafted cells or secreted therapeutic material to remain. Such grafts include, without limitation, various materials used in the art such as biodegradable synthetic polymers and natural materials as supports for tissue engineering. Since the graft of the present invention contains stem cells and a DNA methyltransferase inhibitor, there is an advantage that it does not cause an immune rejection reaction or an inflammation reaction due to the graft. Therefore, even if there is no need to administer an immunosuppressive agent or an anti-inflammatory agent to inhibit the immune rejection reaction or the inflammatory reaction that may occur when various grafts are transplanted into the body, the grafted graft is stable in vivo without immunodeficiency or inflammation .

In another embodiment, the present invention provides a transplant having a stem cell removed from a stem cell by treating the stem cell with a DNA methyl group transferase inhibitor and culturing the same on a graft.

In another aspect, the present invention provides a method for producing an implant comprising the step of treating stem cells with a DNA methyl group transferase inhibitor and culturing the same on a graft. Preferably, the step of removing the stem cells after the culturing step can further include.

In another aspect, the present invention provides a complex comprising a stem cell and a DNA methyltransferase inhibitor. The complex may be used as a cell therapy agent.

In another embodiment, the present invention provides a culture obtained by treating a stem cell with a DNA methyl group transferase inhibitor.

The culture can exert an effect of preventing or treating an immune disease or an inflammatory disease and can be used for the prevention and treatment of diseases such as COX-2, PTGES, IDO, IL-6, IL-1 ?, LIF, ICAM, VCAM, CXCR2, CXCR4, Inflammatory regulators or cell migration-related factors.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating an immune disease or an inflammatory disease comprising stem cells or culture thereof cultured by treating IFN-? And / or TNF-? With stem cells .

In the present invention, even when DNA methyltransferase inhibitor as well as IFN-γ and / or TNF-α are added to stem cells, inflammatory cell inhibitory factors (COX2, PTGES, IDO (ICAM, VCAM, CXCR2, CXCR4, and CXCR6) associated with cell migration, IL-6, and migration, and thus controls immune and inflammation Respectively. Therefore, stem cells treated with IFN-y and / or TNF-? And cultures thereof can be used as a cell therapy agent for immunomodulation and inflammation control.

By providing a pharmaceutical composition that can be used in the prevention or treatment of the immunological diseases and inflammatory diseases of the present invention, it is possible to produce inflammatory regulatory factors or cell migration-related factors constitutively and simply, economically, and at an improved yield without chemical processes have. The pharmaceutical composition of the present invention is a cell therapy agent which can be used economically and which has no side effects and which can replace existing immunosuppressants and inflammation inhibitors known to have side effects, and is useful as an immunological disease such as Crohn's disease, rheumatoid arthritis, Prevention or treatment.

Fig. 1 (A) is a graph showing the result of treatment of adult stem cell culture with monocyte derived from cord blood (MNC) treated with 5-azacytidine or valproic acid.
FIG. 1B shows the results of treatment of Jurkat cells with adult stem cell culture treated with 5-azacytidine or valproic acid. FIG.
FIG. 1C is a diagram showing Direct MLR results using monocytes derived from cord blood after treating adult stem cells with 5-azacytidine at a concentration. FIG.
FIG. 1D is a graph showing Indirect MLR results using monocytes derived from cord blood after treating adult stem cells with 5-azacytidine at a concentration. FIG.
FIG. 2A is a heat map showing changes in gene expression observed by microarray analysis after treatment of adult stem cells contained in two lots with IFN-γ / TNF-α.
FIG. 2B is a diagram showing the result of analyzing the gene ontology with respect to the increased gene among microarray analysis results.
FIG. 2C is a heat map for the change in promoter methylation through the promoter methylation array after 5-azacytidine treatment of adult stem cells.
FIG. 2D is a Venn diagram showing the number of promoters methylated before and after the 5-azacytidine treatment for the entire promoter that has undergone the test.
FIG. 2E is a table showing the methylation change in the treatment of 5-azacytidine with respect to the factor whose expression was increased by IFN-γ / TNF-α.
FIG. 3 (A) shows the expression levels of COX2, PTEGS, LIF, IL-6, IL-1β, ICAM and VCAM factors by 5-azacytidine treatment on two types of mesenchymal stem cells.
3B and 3C show the treatment of IFN-γ / TNF-α with two kinds of mesenchymal stem cells and the treatment with COX2, PTEGS, LIF, IL-6, IL-1 [beta], ICAM, and VCAM factors.
FIG. 3D shows the expression levels of adult stem cell migration factors CXCR2, CXCR4, and CXCR6 by 5-azacytidine treatment on two kinds of mesenchymal stem cells.
3E and 3F show the treatment of IFN-? / TNF-? With two kinds of mesenchymal stem cells and the treatment with CXCR2, CXCR4, CXCR6 factor Fig.
Fig. 4A is a graph showing the degree of COX2 expression by 5-azacytidine treatment of 5 types of umbilical cord-derived mesenchymal stem cells. Fig.
FIG. 4B is a graph showing the treatment of IFN-? / TNF-? With two kinds of mesenchymal stem cells and the degree of expression of IDO in the case of pre-treatment of 5-azacytidine before the treatment.
FIGS. 5A and 5B show the expression levels of COX2 and PTEGS according to the concentration of 5-azacytidine treated with mesenchymal stem cells. FIG.
FIG. 5C is a graph showing the expression level of PGE2 according to the concentration of 5-azacytidine administered to mesenchymal stem cells.
FIG. 5D is a graph showing the relationship between the inhibitory effect on inflammatory cells of mesenchymal stem cells by 5-azacytidine treatment and the performance of PGE2 aggregation via COX2. FIG.
FIG. 5E shows the relationship between the increase of COX2 and PTGES expression by 5-azacytidine treatment and the demethylation of the promoter.
FIGS. 6A and 6B show the expression levels of CXCR2 and CXCR4 according to the concentration of 5-azacytidine administered to mesenchymal stem cells. FIG.
6C is a graph showing the degree of expression of CXCL8 and SDF-1 by treatment with 5-azacytidine in mesenchymal stem cells.
FIG. 6D is a graph showing a change in mobility of IFN-? / TNF-? By 5-azacytidine treatment on mesenchymal stem cells.
FIG. 6E is a graph showing the relationship between the expression of CXCL2 and CXCL4 by 5-azacytidine treatment and the demethylation of the promoter.
FIG. 7A is a graph showing a change in body weight when an animal model of DSS-induced colitis is administered with 5-azacytidine-treated mesenchymal stem cells.
7B and 7C show changes in colon length when 5-azacytidine-treated mesenchymal stem cells were administered to an animal model of DSS-induced colitis.
FIG. 7D shows the result of H & E staining of a colon tissue section when 5-azacytidine-treated mesenchymal stem cells were administered to a DSS-induced colitis animal model.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Isolation and culture of human cord blood-derived stem cells

Umbilical cord blood samples were obtained from the umbilical vein immediately after delivery with the consent of the supplier and were approved by the Institutional Review Board of the Boramae Hospital and the Seoul National University Clinical Examination Committee (0603 / 001-002-10C4). The umbilical cord blood samples were mixed at a ratio of 5: 1 with Hetasep solution (StemCell Technologies, Vancouver, Canada) and then incubated at room temperature to reduce red blood cell count. The supernatant was collected and centrifuged at 2500 rpm for 20 minutes using a Ficoll density gradient centrifuge (GE healthcare life sciences, Pittsburgh, Pa.). The cells obtained were washed twice with PBS and plated on plates in growth medium consisting of D-medium (formula 78-5470EF; Gibco BRL, Grand Island, NY) containing EGM-2 SingleQuot and 10% FBS And seeded at a density of 2 x 10 ⁵ to 2 x 10 ⁶ cells / cm 2. After 3 days, the non-adherent cells were removed and the adherent cells showed a fusiform shape and formed colonies and proliferated.

Example  2: human Of mononuclear cells (hMNC)  Isolation and Culture

Human mononuclear cells were obtained by isolation from cord blood samples. Umbilical cord blood samples were mixed with Hetasep solution (StemCell Technologies) at a ratio of 5: 1 and then incubated at room temperature to reduce red blood cell count. The supernatant was collected and mononuclear cells were obtained using a Ficoll density gradient centrifuge at 2500 rpm for 20 minutes. The obtained cells were washed twice with PBS and seeded in RPMI 1640 (Gibco BRL) growth medium containing 10% fetal bovine serum (FBS).

Example  3: Mixed leukocyte response ( Mixed Leukocyte Reaction )

The umbilical cord blood-derived mesenchymal stem cells were seeded on a 6-well plate at a concentration of 3 × 10 ⁵ cells / well and treated with 2 μM of 5-azacytidine for 24 hours after 24 hours. The cells were then harvested and co-cultured with human mononuclear cells.

The direct co-cultivation was performed by treating 10 μg / mL mitomycin C for 1 hour with the material-treated stem cells, seeding the cells in 96-well plate at a concentration of 1 × 10 ⁴ cells / well and then using concanavalin A The mononuclear cells were seeded at a concentration of 5 × 10 ⁴ cells / well. After 3 days, the proliferation rate was confirmed by the BrdU assay (bromodeoxyuridine kit, Roche).

In the case of indirect co-culture, a transwell (24-well plate, corning) of 0.4 μm pore size was used. Mononuclear cells stimulated with concanavalin A were seeded at the lower end at a concentration of 1 × 10 ⁶ cells / chamber) was seeded at a concentration of 1 × 10 ⁵ cells / well. After 3 days, the growth rate was confirmed by the same method as above.

In order to confirm MLR as a culture solution of umbilical cord blood-derived pluripotent stem cells, hUCB-MSC was seeded on a 6-well plate at a concentration of 3 × 10 ⁵ cells / well, and then treated with 24-hour 24-hour after 24 hours. After washing three times with PBS, the cells were replaced with RPMI1640 medium without FBS, and cultured for 4 ~ 5 days. Then, the culture solution was collected and used for culturing MNC. MNCs were seeded in 96-well plates at a concentration of 1 × 10 ⁵ cells / well, treated with conA and activated, and 10% FBS was added to the culture to conduct culture. After 3 days, the growth rate was confirmed by the aforementioned BrdU assay method.

Example  4: Western Blat  analysis

Cells were lysed in protein lysis buffer (Pro-PREP, Intron Biotechnologies, Korea), and the lysed proteins were quantitated according to the Lowry method.

CXCR6 (1: 1000, polyclonal, Abcam), CXCR6 (1: 1000, polyclonal, Cayman), PTGES (1: 1000, polyclonal, Santacruz), CXCR2 ^{polyclonal, Millipore), p16 INK4A (} 1: 1000, polyclonal, Abcam, UK), p21 WAF1 / CIP1 (1: 1000, polyclonal, Santa Cruz, CA) , and β-Actin (1: 2000, polyclonal, Abcam, UK) Protein bands were detected using primary antibodies for each and chemiluminescence detection kit (Amersham Pharmacia Biotech, UK) and aFluorChem HD2 imager (Alpha Innotech, San Leandron, Calif.) .

Example  5: RT - PCR  And real-time quantitative PCR

Total intracellular RNA was extracted from the cells using TRIzol reagent (Invitrogen, USA). CDNA was synthesized by adding purified RNA and oligo- dT primers to Accupower RT premix (Bioneer, Korea) and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems, USA). RPL13A was used as an internal control. All amplicons were analyzed using the 7500 Real-Time PCR System (Applied Biosystems, USA) and PCR was performed using Accupower PCR premix (Bioneer). The primer set used in the present invention is shown in Table 1 below.

Gene Sequences SEQ ID NO: COX2 F 5'-TGAGCATCTACGGTTTGCTG-3 ' SEQ ID NO: 1 R 5'-TGCTTGTCTGGAACAACTGC-3 ' SEQ ID NO: 2 IDO F 5'-ACTGTGTCCTGGCAAACTGGAAG -3 ' SEQ ID NO: 3 R 5'-AAGCTGCGATTTCCACCAATAGAG-3 ' SEQ ID NO: 4 ICAM F 5'-TCATCACTGTGGTAGCAGCC-3 ' SEQ ID NO: 5 R 5'-GATAGGTTCAGGGAGGCGTG-3 ' SEQ ID NO: 6 VCAM F 5'-ACGAATGAGGGGACCACATC-3 ' SEQ ID NO: 7 R 5'-TCCAGAGGGCCACTCAAATG-3 ' SEQ ID NO: 8 PTGES1 F 5'-ATG TGA GTC CCT GTG ATG GC-3 ' SEQ ID NO: 9 R 5'-TGA GCC AGA GAG AAG ACT GC-3 ' SEQ ID NO: 10 PTGES2 F 5'-GCC ATC CTC TCC CTG GAA AT-3 ' SEQ ID NO: 11 R 5'-CCT ACT GTG ACC CGA ACC AT-3 ' SEQ ID NO: 12 CXCR2 F 5'-CTC CAG AAG CCA TCA GAC AGG-3 ' SEQ ID NO: 13 R 5'-CTG GGT TGC AAG GGG GAA AT-3 ' SEQ ID NO: 14 CXCR4 F 5'-GGC AGA GGA GTT AGC CAA GA-3 ' SEQ ID NO: 15 R 5'-TAG TGG GCT AAG GGC ACA AG-3 ' SEQ ID NO: 16 CXCR6 F 5'-GAC ACT CTG GCT GGT TTG GA-3 ' SEQ ID NO: 17 R 5'-TCA TCC CCC TTG GTT TCA GC-3 ' SEQ ID NO: 18 LIF F 5'-CCA CCC ATG TCA CAA CAA CC-3 ' SEQ ID NO: 19 R 5'-CCC CCT GGG CTG TGT AAT AG-3 ' SEQ ID NO: 20 RPL13A F 5'-CATCGTGGCTAAACAGGTACTG-3 ' SEQ ID NO: 21 R 5'-GCACGACCTTGAGGGCAGCC-3 ' SEQ ID NO: 22

Example  6: RNA  Interference

When the cells reached 60% confluence, transfection of siRNA into cells was performed. The siRNAs of PTGS2 (siCOX-2, L-004557-00) and the non-target control siControl 1, D-001810-01 were purchased from Dharmacon (Chicago, IL) DharmaFECT1 (Dharmacon) was used as infectious agent and siRNA.

Example  7: Microarray

Total RNA was extracted from human cells using TRI REAGENT (MRC, OH). To remove insoluble material, the cells were homogenized and 1 ml of the solution was added to a 1.5 ml Eppendorf tube and centrifuged at 12,000 g for 10 minutes at 4 ° C. The supernatant containing RNA was collected, mixed with 0.2 ml of chloroform, and centrifuged again at 12,000 g for 15 minutes at 4 ° C. The RNA in aqueous solution was placed in a new tube, precipitated by mixing RNA with 0.5 ml of isopropyl alcohol and re-obtained by centrifugation at 12,000 g for 10 min at 4 ° C. The RNA pellet was washed with approximately 1 ml of 75% ethanol and centrifuged at 7,500 g for 5 minutes at 4 ° C. Finally, the total RNA pellet was dissolved in Nuclease-water and quantitatively and qualitatively analyzed using Agilent bioanalyzer 2100 analysis.

On the other hand, expression of each gene was analyzed using a GeneChip ^® Affymetrix primeview array (Affymetrix, Santa Clara, Calif.) Containing over 530,000 probes representing about 20,000 well known human genes. Each gene in the array on to the 11 pairs of oligonucleotide probes situ .

Biotinylated cRNA was prepared from 250 ng total RNA using a standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix), and 12 μg of aRNA was cut on a GeneChip Human Genome Array at 45 ° C Lt; / RTI > for 16 hours. GeneChips were washed and stained with Affymetrix Fluidics Station 450. GeneChips were scanned using an Affymetrix GeneChip Scanner 3000 7G and analyzed using the Microarray Suite version 5.0 (MAS 5.0) using Affymetrix defect analysis and global scaling as a normalization method. The average target intensity of each array was arbitrarily set at 100. Normalized, modified logarithm values were analyzed using GeneSpring GX 12.5 (Agilent technologies, CA).

Example  8: Promoter array

After 2 uM 5-azacytidine was treated for 24 hours, a sample to be used for the promoter array was prepared using MethylMiner ™ Methylated DNA Enrichment Kit (Applied Biosystems).

The microarray protocol, including labeling, hybridization and hybridization post-washing, was performed by a method known from http://www.agilent.com.

MeDIP (Cyanine 5-dUTP) and reference DNA (Cyanine 3-dUTP) were labeled (Genomic DNA Enzymatic Labeling Kit, Agilent) and washed (Amicon filters, Millipore). Competitive hybridization on a microarray was performed in a SureHyb chamber rotating at 67 [deg.] C for 40 hours (Methylation Hybridization Kit, Agilent). The washed slides were scanned (High-Resolution C Scanner, Agilent) and fluorescence was obtained using Feature Extraction software.

These two signal values were normalized using background subtraction and the signal ratio (MeDIP / input), signal log rate [log2 (MeDIP / input)], P [X] and P value were calculated using the Agilent Genomic Workbench software (Agilent Technologies). The Log2 value is a Cy5: Cy3: Cy3 fluorescence ratio (total input DNA) for each probe and is converted to a log2 scale to show a relative measure of the amount of methylated DNA at each locus. Median and Lowess normalization were applied to raw data and filtered with outlier probes to remove low-level data. The P [X] and P values used in the Methylation IP (MeDIP) array analysis to obtain a binding call were defined as the probability that the X value would be derived from the Gaussian distribution of the X values of the entire genome of the sample. Here, the X value for the probe is obtained as the difference between the input signal and MeDIP after adjusting the equilibrium of its distribution. The value for the probe was calculated as the average of X taking into account the signal for the neighboring probe (within 1kb of the probe). In the present invention, hypermethylation is defined as a case where the ratio of normalized (normalized) log 2 exceeds 1.0.

Example  9: methylation-specific PCR

Methylation-specific PCR for the, Accuprep ^® using (Bioneer USA) genome DNA (genomic DNA) was extracted from the cells. Bisulfite conversion of the genomic DNA was performed using MethyCode ™ (Invitrogen USA). The sodium bisulfite-modified DNA was amplified using Accupower PCR premix (Bioneer, USA). Primers for each promoter were designed through an online website (www.urogene.org/methprimer/) and are shown in Table 2 below.

Gene Sequences SEQ ID NO: COX2 pm1 M F TTG TTG TAT TTT AAT ATG GTA GAA GAG C SEQ ID NO: 23 R ATC ATA AAA ACT CCA TAC TCA CGAA SEQ ID NO: 24 U F TTG TTG TAT TTT AAT ATG GTA GAA GAG TG SEQ ID NO: 25 R AAT CAT AAA AAC TCC ATA CTC ACA AA SEQ ID NO: 26 COX2 pm2 M F GTG GGA GTT GAA TAA TGA GAT TAT AC SEQ ID NO: 27 R TTC TAA AAC ACA TAT ACA AAA CGTA SEQ ID NO: 28 U F GGG AGT TGA ATA ATG AGA TTA TAT GG SEQ ID NO: 29 R TTC TAA AAC ACA TAT ACA AAA CATA SEQ ID NO: 30 PTGES pm1 M F TGT AGT TTT TAA ATT TGT GAT ATC GA SEQ ID NO: 31 R TCA AAA AAC TAA AAC AAA AAA ATC G SEQ ID NO: 32 U F TGT AGT TTT TAA ATT TGT GAT ATT GA SEQ ID NO: 33 R AAA AAA CTA AAA CAA AAA AAT CAC T SEQ ID NO: 34 PTGES pm2 M F TTT TTA GTT TCG AAC GGG ATT C SEQ ID NO: 35 R CCT CTA ACG AAT AAA ACC TAC CGTA SEQ ID NO: 36 U F TTT TTT AGT TTT GAA TGG GAT TTG SEQ ID NO: 37 R CCC TCT AAC AAA TAA AAC CTA CCA TA SEQ ID NO: 38 PTGES2 pm1 M F TAG TTG TAC GTG TTT AAG TTG TCGA SEQ ID NO: 39 R CAT TAA TAA TAA TAA TCC CCC GAA SEQ ID NO: 40 U F TTA GTT GTA TGT GTT TAA GTT GTT GA SEQ ID NO: 41 R CAT TAA TAA TAA TAA TCC CCC AAA SEQ ID NO: 42 PTGES2 pm2 M F TAA TTT TGG GTT TTT AAA GGT AGT C SEQ ID NO: 43 R TAA CCT CAA AAA TTA CTT TCT CGT C SEQ ID NO: 44 U F ATT TTG GGT TTT TAA AGG TAG TTG T SEQ ID NO: 45 R CTA ACC TCA AAA ATT ACT TTC TCA TC SEQ ID NO: 46 CXCR2 pm M F GTTTTTAATTGGTTGATAGGTGTTC SEQ ID NO: 47 R ATAACACTCACTACAAAAATCCGTA SEQ ID NO: 48 U F TGTTTTAATTGGTTGATAGGTGTTT SEQ ID NO: 49 R ATAACACTCACTACAAAAATCCATA SEQ ID NO: 50 CXCR4 pm M F TGAGTAGAGGATAAGTTTTGGTACGA SEQ ID NO: 51 R ACCAATCTAAAATCGCTTTAAACG SEQ ID NO: 52 U F TGAGTAGAGGATAAGTTTTGGTATGA SEQ ID NO: 53 R ACCAATCTAAAATCACTTTAAACATC SEQ ID NO: 54

Example  10: Mobility ( migration ) Assay

The mobility assay can be performed using conventional methods (Hu X et al., American journal of physiology Cell physiology 2011 Aug; 301 (2): C362-372). That is, the mobility of human adult stem cells was evaluated using a transwell chamber containing a flicarbonate membrane filter with a 6.5 mm diameter and a 8 μm pore size. The top was added to cells (5 x 10 ⁴ ) in 200 μl of media (DMEM with 1% FBS) and the bottom was filled with DMEM with 10% FBS as chemoattractants.

For cell migration, the transwell chamber was incubated for 12 hours at 37 ° C and 5% CO ₂ . Cells present on the upper surface of the filter were wiped off with a cotton swab, and the cells that had been moved to the lower end by being drawn on the lower surface of the filter were stained with crystal violet. To determine the number of migrating adult stem cells, three regions per sample were randomly selected and photographed at 40 × magnification (× 40) and measured at least three times with different samples.

Experimental Example  1: 5- Azacitidine  Inflammatory cells by treatment Inhibition  Confirm

5-azacytidine or valproic acid was treated with 2 μM for 24 hours in the adult stem cells (hUBC-MSC) isolated in Example 1, and then the culture was treated with complete media Five days after the replacement, the conditioned medium was collected and treated with cord blood-derived mononuclear cells (MNC) and Jurkat cells separately cultured in Example 2 above. In addition, 5-azacytidine was treated with adult stem cells at a concentration as in Example 3, and Direct MLR and Indirect MLR were confirmed using cord blood-derived mononuclear cells.

As a result, it was confirmed that inhibition of inflammatory cells was increased only when 5-azacytidine was treated (FIGS. 1A and 1B). In addition, direct MLR and indirect MLR were confirmed using umbilical cord blood-derived MNC, and it was confirmed that MLR was inhibited by Indirect MLR in a dose-dependent manner by 5-azacytidine (C and D in FIG. 1).

Experimental Example  2: 5- Azacitidine  Analysis of gene promoter methylation by treatment

In order to analyze the change of gene promoter methylation by 5-azacytidine treatment, adult stem cells were treated with 2 μM 5-azacytidine for 24 hours, and then a promoter methylation array was prepared as in Example 8 (A to C in Fig. 2).

As a result, it was confirmed that the promoter of 7,734 genes among the examined promoters was demethylated after treatment with 5-azacytidine, and the promoter of 5,615 genes increased methylation after 5-azacytidine treatment (FIG. 2D).

In addition, the expression of IFN-γ / TNF-α increased the expression of IFN-γ / TNF-α and the expression of IFN-γ / TNF-α increased the expression of methylation by 5-azacytidine Of the total 150 genes, 30 genes were hypomethylated after treatment with 5-azacytidine (FIG. 2E).

Experimental Example  3: Inflammation control Related Parameters  analysis

Experimental Example  3-1: 5- Azacitidine  Inflammation regulation by treatment Of related factors  Expression analysis

Of the factors that increased secretion from adult stem cells by the IFN-γ / TNF-α treatment, among the factors that were hypomethylated by 5-azacytidine, It was confirmed in two kinds of adult stem cells (hUBC-MSC; 1272-3 and 01-2) whether expression was increased by 5-azacytidine treatment. Specifically, the degree of expression of each gene was measured by the real-time quantitative-PCR and Western blotting methods described in Examples 4 and 5 above.

As a result, the expression of COX2, PTGES, PTGES2, IL-6, IL-1β, ICAM and VCAM was significantly increased in both types of cells (FIG.

In addition, in order to examine whether pretreatment of 5-azacytidine affects the IFN-γ / TNF-α of adult stem cells, 5-azacytidine was pretreated for 24 hours and IFN-γ / TNF-α After 24 hours of treatment, the expression of the factors was checked.

As a result, when 5-azacytidine was pretreated compared with IFN-γ / TNF-α alone, the expression levels of PTGES and ICAM were significantly increased in both adult stem cells, In addition, it was confirmed that expression of COX2, LIF, IL-1β, and VCAM did not increase or change depending on the cells, and IL-6 tended to increase or decrease depending on the cells (FIGS.

In order to analyze the expression levels of COX2, PTGES, PGE2 and the like depending on the concentration of 5-azacytidine, the degree of expression of each factor was measured by the real-time quantitative-PCR and Western blotting methods described in Examples 4 and 5 Respectively.

As a result, it was confirmed that when 5-azacytidine was treated with adult stem cells at a concentration, COX2 and PTGES increased in a concentration-dependent manner (FIGS. 5A and 5B). In addition, it was confirmed that as the concentration of 5-azacytidine increases, the concentration of PGE2 also gradually increases in the stem cell culture fluid (FIG. 5C).

In the same manner as described above, IFN-y treatment of 5 cord blood-derived adult stem cells with 2 [mu] M 5-azacytidine for 24 hours resulted in an increase in COX2 (Fig. 4A). As shown in Fig.

Similarly, when two kinds of umbilical cord blood-derived adult stem cells were pretreated with 2 uM of 5-azacytidine for 24 hours and treated with IFN-? / TNF- ?, IDO (Fig. 4 (B)).

Experimental Example  3-3: 5- Azashitidine  by MLR and PGE2 Analysis of correlation between secretions

In order to confirm the relationship between the decrease in MLR by 5-azacytidine in Experimental Example 1 and the increase in the amount of PGE2 secretion in Experimental Example 3-1, RNA interference by siRNA was used as in Example 6 COX2 was inhibited, 5-azacytidine was treated, and co-cultured with monocytes derived from cord blood.

As a result, it was confirmed that the MLR decreased by 5-azacytidine increased again when COX2 was suppressed. This suggests that the increase in the inhibitory effect of 5-azacytidine on inflammatory cells of adult stem cells is closely related to the performance of PGE2 aggregation via COX2 (FIG. 5D).

Experimental Example  3-4: 5- Azashitidine  by COX2  And PTGES And the relationship between the increased expression of the promoter and the demethylation of the promoter

In order to confirm the relationship between the increase of expression of COX2 and PTGES by 5-azacytidine and the demethylation of the promoter, methylation changes of each gene promoter after 5-azacytidine treatment were carried out in the above Example 9 methyl-specific PCR (methyl-specific PCR).

As a result, it was confirmed that methylation of both the promoters of COX2 and PTGES was decreased (FIG. 5E).

Experimental Example  4: Move Related Parameters  analysis

Experimental Example  4-1: 5- Azacitidine  Stem cell migration by treatment Of related factors  Expression analysis

CXCR2, CXCR4, and CXCR6, which are involved in the migration of adult stem cells, are not specifically increased by IFN-γ / TNF-α but by the treatment of 5-azacytidine with a specific hypomethylation ) Was confirmed through the promoter array in Example 3, and whether or not the expression of these factors was actually increased by treatment with 5-azacytidine was evaluated using two kinds of adult stem cells (hUBC-MSC; 1272-3 and 01-2). Specifically, the expression levels of the respective receptor genes were measured by the real-time quantitative-PCR and Western blotting methods described in Examples 4 and 5 above.

As a result, it was confirmed that expression of these three kinds of receptor genes was significantly increased by 5-azacytidine treatment (FIG. 3 D).

Furthermore, in order to confirm whether pretreatment of 5-azacytidine affects the expression level of CXCR after IFN-γ / TNF-α treatment, pretreatment of 5-azacytidine for 24 hours and IFN- γ / TNF- After 24 hours of treatment, the expression of CXCR was confirmed.

As a result, the expression levels of CXCR2 and CXCR4 were significantly increased in both types of adult stem cells when 5-azacytidine was pretreated compared with IFN-γ / TNF-α alone. However, the expression level of CXCR6 was increased or decreased depending on the cell type (E and F in FIG. 3).

As a result of analyzing the expression amounts of CXCR2 and CXCR4 according to the concentration of 5-azacytidine in the same manner, it was confirmed that CXCR2 and CXCR4 were increased in a concentration-dependent manner when 5-azacytidine was treated with adult stem cells at different concentrations (A and B in Fig. 6).

Experimental Example  4-2: 5- Azacitidine  Depending on the treatment Adult stem cells  Mobility analysis

In order to analyze the mobility of adult stem cells according to the treatment with 5-azacytidine, the mobility of the stem cells was observed through the mobility assay described in Example 10 above.

As a result, migration of 5-azacytidine-treated group to CXCL8 and SDF-1, which are ligands for CXCR2 and CXCR4, respectively, was increased compared with the untreated control group (C ).

In addition, migration of IFN-γ / TNF-α was increased in the 5-azacytidine-treated group compared to the untreated control group (FIG. 6D).

Experimental Example  4-3: 5- Azashitidine  by CXCR2  And CXCR4 And the relationship between the increased expression of the promoter and the demethylation of the promoter

In order to confirm the relationship between the increase of expression of CXCR2 and CXCR4 by 5-azacytidine and the demethylation of the promoter, methylation changes of each gene promoter after 5-azacytidine treatment were measured in the same manner as in Example 9 methyl-specific PCR (methyl-specific PCR).

As a result, it was confirmed that methylation of both promoters of CXCR2 and CXCR4 was decreased (FIG. 6E).

Experimental Example  5: Animal experiment

In this study, we investigated the effects of 5-azacytidine on adult stem cells and the effects of inflammatory and migration factors on DSS-induced colitis mouse model Treated adult adult stem cells and 5-azacytidine-treated adult stem cells.

Specifically, C57BL / 6J mice (male; 8-10 weeks old) were obtained from Jackson laboratory (Bar Harbor, ME). Mice were bred under specific pathogenic-free conditions in an experimental animal facility at Seoul National University. All experiments were carried out in accordance with the provisions of the Institute of Laboratory Animals Resources (SNU-100125-8, SNU-111223-1, and SNU-130130-2; Seoul National University). Acute colitis was induced in mice by the addition of% w / v dextran sulfate sodium (DSS, MP Biochemicals, Solon, OH, USA). 2 μM 5-azacytidine was added to the medium Suspension of cells (hUCB-MSCs) and washing with PCS to remove residual 5-azacytidine. After 1 day of DSS administration, resuspended in PBS (2 x 10 ⁶ cells / 200 μl) hCB-MSC was administered intraperitoneally, and the body weight was measured for 10 days after the administration, and the colon length was measured by sacrificing the mice on the day 10, and the colon sample was fixed with 4% paraformaldehyde , Frozen section compound was stored at -80 ° C (C) sections cut and stained with H & E in a thickness of 5 mm.

As a result, in the experimental group treated with 5-azacytidine, the weight loss was significantly inhibited as compared with the control group treated with 5-azacytidine (FIG. 7A), and the decrease in the length of the colon was also significantly (Fig. 7B and Fig. 7C). As a result of H & E staining, it was confirmed that infiltration of inflammatory cells was reduced in the experimental group treated with 5-azacytidine, and the morphology of the villi was maintained at a level similar to normal (FIG.

<110> Kang Stem Biotech CO., LTD. <120> Pharmaceutical composition comprising stem cells treated with DNA          methyltransferase or culture thereof for prevention and treatment          of immune diseases and inflammatory diseases <130> KPA140006-KR-P1 <150> KR 10-2014-0001974 <151> 2014-01-07 <160> 54 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX2 forward primer <400> 1 tgagcatcta cggtttgctg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX2 reverse primer <400> 2 tgcttgtctg gaacaactgc 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IDO forward primer <400> 3 actgtgtcct ggcaaactgg aag 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IDO reverse primer <400> 4 aagctgcgat ttccaccaat agag 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM forward primer <400> 5 tcatcactgt ggtagcagcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM reverse primer <400> 6 gataggttca gggaggcgtg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VCAM forward primer <400> 7 acgaatgagg ggaccacatc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VCAM reverse primer <400> 8 tccagagggc cactcaaatg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGES1 forward primer <400> 9 atgtgagtcc ctgtgatggc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGES1 reverse primer <400> 10 tgagccagag agaagactgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 forward primer <400> 11 gccatcctct ccctggaaat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 reverse primer <400> 12 cctactgtga cccgaaccat 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 forward primer <400> 13 ctccagaagc catcagacag g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 reverse primer <400> 14 ctgggttgca agggggaaat 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 forward primer <400> 15 ggcagaggag ttagccaaga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 reverse primer <400> 16 tagtgggcta agggcacaag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR6 forward primer <400> 17 gacactctgg ctggtttgga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR6 reverse primer <400> 18 tcatccccct tggtttcagc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIF forward primer <400> 19 ccacccatgt cacaacaacc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIF reverse primer <400> 20 ccccctgggc tgtgtaatag 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RPL13A forward primer <400> 21 catcgtggct aaacaggtac tg 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RPL13A reverse primer <400> 22 gcacgacctt gagggcagcc 20 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm1 methyl forward primer <400> 23 ttgttgtatt ttaatatggt agaagagc 28 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm1 methyl reverse primer <400> 24 atcataaaaa ctccatactc acgaa 25 <210> 25 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm1 unmethyl forward primer <400> 25 ttgttgtatt ttaatatggt agaagagtg 29 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm1 unmethyl reverse primer <400> 26 aatcataaaa actccatact cacaaa 26 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm2 methyl forward primer <400> 27 gtgggagttg aataatgaga ttatac 26 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm2 methyl reverse primer <400> 28 ttctaaaaca catatacaaa acgta 25 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm2 unmethyl forward primer <400> 29 gggagttgaa taatgagatt atatgg 26 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX2 pm2 unmethyl reverse primer <400> 30 ttctaaaaca catatacaaa acata 25 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm1 methyl forward primer <400> 31 tgtagttttt aaatttgtga tatcga 26 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm1 methyl reverse primer <400> 32 tcaaaaaact aaaacaaaaa aatcg 25 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm1 unmethyl forward primer <400> 33 tgtagttttt aaatttgtga tattga 26 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm1 unmethyl reverse primer <400> 34 aaaaaactaa aacaaaaaaa tcact 25 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm2 methyl forward primer <400> 35 tttttagttt cgaacgggat tc 22 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm2 methyl reverse primer <400> 36 cctctaacga ataaaccta ccgta 25 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm2 unmethyl forward primer <400> 37 ttttttagtt ttgaatggga tttg 24 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PTGES pm2 unmethyl reverse primer <400> 38 ccctctaaca aataaaacct accata 26 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm1 methyl forward primer <400> 39 tagttgtacg tgtttaagtt gtcga 25 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm1 methyl reverse primer <400> 40 cattaataat aataatcccc cgaa 24 <210> 41 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm1 unmethyl forward primer <400> 41 ttagttgtat gtgtttaagt tgttga 26 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm1 unmethyl reverse primer <400> 42 cattaataat aataatcccc caaa 24 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm2 methyl forward primer <400> 43 taattttggg tttttaaagg tagtc 25 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm2 methyl reverse primer <400> 44 taacctcaaa aattactttc tcgtc 25 <210> 45 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm2 unmethyl forward primer <400> 45 attttgggtt tttaaaggta gttgt 25 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PTGES2 pm2 unmethyl reverse primer <400> 46 ctaacctcaa aaattacttt ctcatc 26 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 pm methyl forward primer <400> 47 gtttttaatt ggttgatagg tgttc 25 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 pm methyl reverse primer <400> 48 ataacactca ctacaaaaat ccgta 25 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 pm unmethyl forward primer <400> 49 tgtttttaat tggttgatag gtgttt 26 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CXCR2 pm unmethyl reverse primer <400> 50 ataacactca ctacaaaaat ccata 25 <210> 51 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 pm methyl forward primer <400> 51 tgagtagagg ataagttttg gtacga 26 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 pm methyl reverse primer <400> 52 accaatctaa aatcgcttta aacg 24 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 pm unmethyl forward primer <400> 53 tgagtagagg ataagttttg gtatga 26 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 pm unmethyl reverse primer <400> 54 accaatctaa aatcacttta aacatc 26

Claims (16)

Prevention of autoimmune diseases, graft rejection, arthritis, graft-versus-host disease, bacterial infection, sepsis or inflammation including stem cells or cultures thereof cultured by adding 5-azacytidine to stem cells; A pharmaceutical composition for therapeutic use.
delete The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises stem cells or culture thereof cultured by additionally adding IFN-?, TNF-? Or both to 5-azacytidine.
The pharmaceutical composition according to claim 1, wherein the stem cell is a human adult stem cell, a human pluripotent stem cell, an induced pluripotent stem cell, an animal embryonic stem cell, or an adult adult stem cell.
The pharmaceutical composition according to claim 4, wherein the adult stem cell is a mesenchymal stem cell, a mesenchymal stromal cell derived from a human tissue, a mesenchymal stem cell derived from a human tissue, a multipotential stem cell or an amniotic epithelial cell .
[Claim 5] The method according to claim 5, wherein the adult stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose derived mesenchymal stem cells, Wherein the stem cell is a mesenchymal stem cell selected from the group consisting of stem cells, skin-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells.
delete The method of claim 1, wherein the autoimmune disease is selected from the group consisting of Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, malignant anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism, , Scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, , Systemic scleroderma, asthma, and ulcerative colitis.
The method of claim 1, wherein the 5-azacytidine is selected from the group consisting of COX-2 (Cyclooxygenase-2), PTGES (Prostaglandin E synthase), IDO (Indoleamine 2,3-dioxygenase), IL- (Interleukin-1?), Leukemia inhibitory factor (LIF), Intercellular adhesion molecule, VCAM, CXCR2, 6). &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
10. The method of claim 9, wherein the 5-azacytidine is selected from the group consisting of COX-2 (Cyclooxygenase-2), PTGES (Prostaglandin E synthase), IDO (Indoleamine 2,3-dioxygenase), IL- (Interleukin-1?), Leukemia inhibitory factor (LIF), Intercellular adhesion molecule, VCAM, CXCR2, 6). &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
A method for inhibiting an immune response or inflammatory response of a subject other than a human, comprising the step of administering to a subject a stem cell or culture thereof cultured by adding 5-azacytidine to the stem cell.
A method for producing an immunosuppressant or an anti-inflammatory agent, which comprises culturing stem cells by adding 5-azacytidine.
(COX-2), PTGES (Prostaglandin E synthase), IDO (Indoleamine 2,3-dioxygenase), and IL-6 Interleukin-6, Interleukin-1?, Leukemia inhibitory factor (LIF), Intercellular adhesion molecule, VCAM, CXCR2, ) And CXCR6 (CXC Chemokine Receptor 6).
Transplants for the treatment of autoimmune diseases, including stem cells and 5-azacytidine, graft rejection, arthritis, graft versus host disease, bacterial infection, sepsis or inflammation.
A complex for the treatment of autoimmune diseases, including stem cells and 5-azacytidine, graft rejection, arthritis, graft versus host disease, bacterial infection, sepsis or inflammation.
delete

Images