KR101766341B1 - Pharmaceutical composition comprising stem cells treated with granules of mast cells 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 the prevention or treatment of immune diseases and inflammatory diseases including stem cells or cultures thereof cultured by adding mast cell granules to stem cells.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating immune diseases or inflammatory diseases, which comprises stem cells treated with mast cell granules or cultures 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 mast cell granules 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 (immune tolerance) which controls immune suppression and immunity which promotes immunity. These two immune functions are balanced to form immunological homeostasis . However, if for some reason the balance is broken and the immune response is greater than that of immune tolerance, immune and inflammatory diseases are caused.

Immunosuppression for the prevention or treatment of an immunological 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 steroids not only promote atherosclerosis but also cause hypertension, gastric ulcer, diabetes, growth inhibition, 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.

Meanwhile, mast cells are involved in the inflammatory reaction. When the IgE antibody is activated and cross-linked by other external antigens or allergens, various mast cell pathways cause obesity And releases inflammation-inducing substances such as histamine, β-hexosaminidase and leukotriene, which are trapped in the cell granules, into the plasma membrane. These released inflammation-inducing substances act to further activate inflammation by activating inflammation-inducing receptors and cytokines.

In particular, the release of these substances from mast cells is closely related to allergic diseases, and allergic diseases are generally associated with IgE antibodies genetically / immunologically, and the acquired factors, such as environmental and psychological factors, It is known to be sensitized and sensitized by exposure. These immunologic factors are now the target of treatment, and immunologically it will be possible to treat allergic diseases by controlling the release of these allergenic substances into the extracellular membranes. However, it has not been known that when these granules in the mast cells are extracted and treated with stem cells, the expression of inflammatory regulators and the like secreted from stem cells is rather increased, thereby preventing or treating inflammatory diseases or immune diseases.

Accordingly, the present inventors have completed the present invention by confirming that the expression of inflammatory regulatory factors secreted from stem cells is remarkably increased when cultured by adding the granules of mast cells to the stem cells.

It is an object of the present invention to provide a method for the treatment and prophylactic treatment of immune diseases or inflammatory diseases, including stem cells or cultures thereof, which are cultured by adding mast cells granules to stem cells without any adverse effect that can replace conventional immunosuppressants and inflammation- Or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method of preventing or treating an immunological disease or an inflammatory disease comprising the step of administering the composition to a subject other than a human.

It is 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.

Another object of the present invention is to provide a method for producing an immunosuppressant or an anti-inflammatory agent comprising the step of culturing stem cells with mast cell granules.

Another object of the present invention is to provide a method for mass production of COX-2 (Cyclooxygenase-2) protein comprising culturing stem cells in a medium supplemented with mast cell granules.

It is another object of the present invention to provide grafts and complexes comprising stem cells and mast cell granules.

It is still another object of the present invention to provide a stem cell or a culture thereof cultured by adding mast cell granules.

In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an immune disease or inflammatory disease comprising stem cells or cultures thereof obtained by culturing stem cells with mast cell granules.

In the present invention, when granules of mast cell granules, specifically mast cells derived from cord blood, are extracted and treated with stem cells, the expression of inflammatory cell inhibitory factors (COX-2, PGE ₂ ) secreted from the stem cells is increased , Thus confirming the regulation of immunity and inflammation. In addition, as a result of administering the stem cells to an animal model of an inflammatory disease and an immune disease, it was confirmed that there is an effect of improving not only gross lesion but also histopathological lesion. That is, the present inventors confirmed that stem cells treated with mast cell granules and cultures thereof can be used as a cell therapy agent for immunomodulation and inflammation control, and the stem cells cultured by adding mast cell granules to stem cells or stem cells The present invention can be used as a pharmaceutical composition for the prevention or treatment of an immunological disease or an inflammatory disease including a culture.

In the present invention, the term "mast cell" is widely distributed in vertebral connective tissues and the like, and histamine, serotonin, heparin, beta-hexosaminidase (β -hexosaminidase) and leukotriene in the form of granules. When cell collapse releases granules in cells, hypersensitivity (inflammation) occurs in tissues against them. These cells are known to act on blood clotting inhibition, permeability of blood vessels, and blood pressure.

The mast cell is mainly known as a bone marrow-derived cell, but may be a mast cell derived from cord blood. In the examples of the present invention, mast cells isolated from cord blood were cultured in a medium containing SCF (stem cell factor) and IL-6 to obtain mast cells.

In the present invention, the mast cell granule is also referred to as a mast cell extract, and includes not only the substance contained in the mast cell but also the substance existing in the granule, that is, the substance which can be released while the granule is broken. Thus, the mast cell granules themselves can be used in an optimal combination to exhibit an excellent effect. Therefore, it is more advantageous to achieve the object of the present invention than to use various combinations of the inflammation inducing substances contained in the mast cells Can be used.

In the present invention, in the "addition and cultivation" of mast cell granules, for example, the above substance may be added to a culture medium of stem cells and cultured. Preferably, 10 ² to 10 ⁵ mast cell granules may be contained per 1 mL of the medium, and the cells may be cultured at a concentration within the above range for 0.1 to 200 hours, more preferably for 1 to 72 hours. In addition, it may be cultured by adding mast cell granules, and then culturing by replacing the medium.

In the present invention, treatment of mast cell granules or culturing with addition thereof belongs to the scope of the present invention to treating inflammation-inducing substances contained in the granules.

In the present invention, "stem cells" may be cells having the ability to differentiate into various tissues, i.e., undifferentiated cells.

In the present invention, the stem cells may be adult stem cells, pluripotent stem cells, pluripotent stem cells or embryonic stem cells.

Multipotent stem cells are pluripotent stem cells derived from various adult cells such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), and fat (or adipose tissue 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.

On the other hand, the adult stem cells may be derived from tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta. In addition, the adult stem cells may be mesenchymal stromal cells or multipotential stem cells, preferably human umbilical cord blood stem cell stem cells (hUCB -MSC). ≪ / RTI > The method of obtaining stem cells in each tissue 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.

For example, DMEM (Dulbecco's modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) may be used as a culture medium for culturing the stem cells. Can be used.

The 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, it was confirmed that the expression of COX-2 and the secretion amount of PGE2 were increased in stem cells cultured by adding mast cell granules. Since COX-2 is known as an enzyme that plays a key role in the synthesis of PGE2, it is apparent that the amount of PGE2 synthesized increases as COX-2 expression level is increased. In addition, PGE2 has been reported to suppress the secretion of cytokines such as inflammatory cytokines such as interleukin-1 [beta] and TNF- [alpha].

In another embodiment of the present invention, it has been confirmed that stem cells cultured with the addition of mast cell granules inhibit the proliferation of immune cells. Excessive immune cell proliferation leads to excessive inflammatory and immune responses, thereby suppressing abnormal inflammatory and immune responses by inhibiting proliferation of the cells.

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 means a disease in which inflammation is the main lesion, and includes, but is not limited to, edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, , Pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, acne spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendonitis, , Nephritis, sjogren's syndrome or multiple sclerosis.

The term "autoimmune disease" in the present invention means a disease which is a problem when a specific immune response occurs. Preferably, the autoimmune disease, graft rejection, graft versus host disease, Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto thyroiditis, anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperlipidemia, scleroderma, Behcet's disease, inflammatory bowel disease, And can be selected from the group consisting of scleroderma, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic scleroderma, asthma or ulcerative colitis have.

The autoimmune disease may be Crohn's disease, erythema disease, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, malignant disease, autoimmune diseases or inflammatory diseases. The autoimmune diseases may be autoimmune diseases, graft rejection, graft- versus host disease, arthritis, bacterial infection, sepsis or inflammation. Anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperlipidemia, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, - Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma or ulcerative colitis, and the like.

In the present invention, "prevention" means any action that inhibits or delays the onset of an immunological disease or inflammatory disease upon administration of the composition, and "treatment" means that the administration of the composition improves the symptoms of the immune or inflammatory disease, Means any act that

In addition, the stem cell or culture thereof according to the present invention is preferably used in an amount of 1.0 × 10 ⁵ to 1.0 × 10 ⁹ , preferably 1.0 × 10 ⁶ to 1.0 × 10 ⁸ , more preferably 1.0 × 10 ⁸ , ⁷ cells.

The stem cells or cultures thereof according to the present invention may 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 another aspect, the present invention provides a method of preventing or treating an immune disease or an inflammatory disease, comprising the step of administering the composition to a subject other than a human.

The term "administering" of the present invention means introducing a given substance into an individual in a suitable manner.

The term "individual" of the present invention means all animals including mice, mice, livestock and the like, including humans who have developed or are capable of developing an immunological disease or inflammatory disease. As a specific example, it may be a mammal including a human.

In addition, the stem cells or cultures thereof according to the present invention can be formulated into a unit dosage form suitable for administration to a patient in the body according to a conventional method in the pharmaceutical field, and the preparation can be administered once or several times Lt; RTI ID = 0.0 > administration. ≪ / RTI > 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 stem cells, cultures or pharmaceutical preparations according to the present invention thus prepared may be used in combination with other stem cells used for transplantation and other uses or a mixture thereof with such stem cells And preferably can be directly implanted or implanted in a disease site of a patient in need of treatment, or can be implanted or injected directly into the abdominal cavity, but the present invention is not limited thereto. 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 embodiment, the present invention provides a method for inhibiting the immune response or controlling inflammation by administering a stem cell treated with the mast cell granule according to the present invention and a culture thereof, The present invention provides a method for inhibiting an immune reaction or an inflammatory reaction of a subject comprising administering a stem cell or culture thereof to a subject.

In the present invention, the term "subject " means mammals including cows, dogs, pigs, chickens, sheep, horses, and humans, but is not limited thereto. In addition, preferably, the stem cell cultured with the addition of the mast cell granule or the culture thereof 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 be characterized by 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 culturing stem cells with mast cell granules.

In the present invention, the term "immunosuppressive agent" as used herein refers to a stem cell obtained by culturing stem cells with mast cell granules added thereto, or a preparation containing the same, wherein the immune response is inhibited, .

As described above, the term "anti-inflammatory agent" means a preparation capable of treating an inflammatory disease by suppressing inflammation with a stem cell obtained by culturing stem cells with mast cell granules added thereto or a culture thereof do.

In another embodiment, the present invention provides a method for producing stem cells comprising the step of culturing stem cells in a medium supplemented with mast cell granules, wherein the expression of COX-2 (Cyclooxygenase- 2) or PGE ₂ (Prostaglandin E2) protein.

According to the embodiment of the present invention, adult stem cells treated with mast cell granules significantly increase expression of COX-2 and synthesis amount of PGE ₂ , which are known as inflammation regulators (Experimental Examples 2 and 3). This suggests that the stem cells of the present invention can be used as a therapeutic agent for the prevention or treatment of inflammatory diseases by increasing the synthesis amount of PGE ₂ inhibiting the secretion of interleukin-1? And TNF-?, Which are inflammation-inducing cytokines do.

According to another embodiment of the present invention, it was confirmed that adult stem cells treated with mast cell granules and mast cell granules inhibit the proliferation of immune cells (Experimental Example 4). This suggests that the stem cells of the present invention can be used as immunosuppressants for the prevention or treatment of immune diseases or inflammatory diseases by inhibiting the proliferation of immune cells.

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 present invention provides an implant comprising stem cells and mast cell granules.

"Implant " as used herein refers to a material that can be implanted in a human or animal, isolating the damaged site from the outside or containing implanted cells or secreted therapeutic material. Such grafts may include a biocompatible synthetic polymer as a support for tissue engineering and a variety of materials used in the art such as natural materials. Since the graft of the present invention contains stem cells and mast cell granules, there is an advantage that the graft does not cause an immune rejection reaction or an inflammation reaction. 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 an implant having stem cells removed after culturing stem cells by adding mast cell granules on a support.

In another aspect, the present invention provides a method for producing an implant comprising culturing stem cells on a support by adding mast cell granules.

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 stem cells and mast cell granules. The complex may be used as a cell therapy agent.

In another aspect, the present invention provides a stem cell or a culture thereof cultured by adding mast cell granules. The culture may exhibit an effect of preventing or treating an immunological disease or an inflammatory disease, and inflammatory factors such as COX-2 and PGE ₂ may be present.

The stem cells or cultures thereof according to the present invention can be used for the prevention or treatment of immune diseases and inflammatory diseases. In addition, the stem cells according to the present invention can produce an inflammatory regulator or a cell migration-related factor with a simple, economical and chemical process and an improved yield. The stem cell or culture thereof according to the present invention is a cell therapy agent which can be used economically and has no side effect, replacing existing immunosuppressive agents and inflammation inhibitors known to have side effects, and can be used for immunization such as Clonus disease, rheumatoid arthritis, Can be used for the prevention or treatment of diseases and inflammatory diseases.

FIG. 1 is a graph showing the degree of disease activity (weight loss, diarrhea, blood cell count, disease activity index, and the like) of an inflammatory bowel disease after administering stem cells treated with stem cells and mast cell granules to an animal model of inflammatory bowel disease. (MSC), and the MC-MSC was induced in the mast cells of the umbilical cord blood-derived stem cells (SCCs). Group)
FIG. 2 is a graph showing the results of visual observation of lesions after administration of stem cells treated with stem cells and mast cell granules in an atopic dermatitis animal model ((-): negative control group / Df: atopic dermatitis Induced positive control / Fibroblast: induced fibroblast-subcutaneously-administered group / MSC: induced cord blood stem cell-treated group / MC-MSC: induced mast cell-treated cord blood stem cell-
FIG. 3 is an image showing histopathological lesions observed after administration of stem cells treated with stem cells and mast cell granules in an atopic dermatitis animal model. MSC: Implanted cord blood stem cells / MC-MSC: induced mast cell preconditioning Umbilical cord blood stem cells (MSCs) were injected into the umbilical cord blood stem cells Group)
4 is an image and a graph showing an increase in the expression level of COX-2 due to pretreatment of mast cell granules. ((-): negative control / CM (culture medium): cell culture / CC MNC (mononuclear cell): Monocyte / Mast Cell: Mast cell
FIG. 5 is an image and a graph showing an increase in the expression level of COX-2 due to pre-treatment of mast cell granules in various types of umbilical cord blood-derived stem cells.
FIG. 6 is a graph showing an increase in the amount of PGE ₂ secretion in the stem cell treated with mast cell granules (MSC: cord blood stem cell treatment group without mast cell granulation, MC-MSC: cord blood stem treated with mast cell granule Cell treatment group)
FIG. 7 is a graph showing the ability of stem cells treated with mast cell granules to inhibit the proliferation of immune cells ((-): Negative control group / ConA: Concanavalin A treated group, MSC: Umbilical cord blood stem cells treated with cord blood stem cells , MC-MSC: cord blood stem cells treated with mast cell granules)

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.

Manufacturing example  1. Manufacture of animal models

An inflammatory bowel disease animal model was prepared by ingesting Dextran sulfate sodium (DSS, 3%) in the negative water to induce inflammatory bowel disease.

An atopic dermatitis animal model was prepared by applying ointment derived from house dust mite six times and inducing it.

Example  1. Cord blood From mononuclear cells  Extraction of granules in mast cells

Mononuclear cells isolated from cord blood were cultured in medium containing SCF (stem cell factor) and IL-6 for 6 to 8 weeks. When the proportion of mast cells in the mononuclear cells reached 90% or more, the cells were rapidly frozen and thawed Was repeated 3 times to extract granules in mast cells.

Experimental Example  1. Treated granules in mast cells Intermediate lobe  Stem cell treatment and symptom change analysis

The mesenchymal stem cells (about 1 x 10 ⁴ ) extracted from the above Example 1 were treated for 48 hours in 8 mL medium containing cord blood derived mesenchymal stem cells, washed 5 times, The inflammatory bowel disease and atopic dermatitis of Preparation Example 1 were intraperitoneally and subcutaneously administered to two animal models, respectively.

As a result of observing the symptomatic changes of the two animal models after the stem cell administration, it was found that when the umbilical cord blood derived mesenchymal stem cells were administered to the animal model of the inflammatory bowel disease, the disease activity (loss of body weight, diarrhea, And the index score). In addition, it was confirmed that the effect of improving the disease activity was significantly improved when the umbilical cord blood-derived mesenchymal stem cells that had been pre-cultured in the mast cell granules extracted in Example 1 were administered (FIG. 1).

In addition, in the atopic dermatitis animal model, the improvement effect was also observed when the umbilical cord blood-derived mesenchymal stem cells were administered, not only when the visual lesion was observed (FIG. 2), but when the histopathological lesion was observed (FIG. 3) Similarly, the improvement effect was better than that in the case of mesenchymal stem cells derived from umbilical cord blood derived from the mast cell granules extracted in Example 1 above.

Experimental Example  2. Analysis of COX-2 expression levels

PGE ₂ secreted by cord blood derived stem cells plays an important role in the therapeutic effect of inflammatory bowel disease or atopic dermatitis. The expression level of COX-2, an enzyme that plays a key role in the synthesis of PGE ₂ , was measured in order to confirm the change in the amount of PGE ₂ secreted by the pre-treatment of mast cell granules.

As a result, the amount of COX-2 expressed in stem cells was significantly increased during the treatment of mast cell granules, and this phenomenon was not observed when monocyte culture medium or extract was treated. On the other hand, it was confirmed that the amount of COX-2 expression was only slightly increased when mast cell cultures were treated (Fig. 4).

In addition, it was confirmed that COX-2 expression was increased in various types of cord blood-derived stem cells due to the preconditioning of the granules in the mast cells (FIG. 5).

Experimental Example  3. PGE ₂  Analysis of secretion level

In Experimental Example 2, the expression level of COX-2, an enzyme that plays a key role in the synthesis of PGE ₂ , one of the key immunoregulatory factors, was found to increase, and the level of PGE ₂ secretion was measured.

Specifically, after the mast cell granules were treated, the PGE ₂ secretion amount of the stem cells was measured in the culture medium of the stem cells using commercially available PGE ₂ measurement kit (R & D systems).

As a result, it was confirmed that the stem cell treated with the mast cell granule significantly increased the secretion amount of PGE ₂ as compared with the stem cell not treated with the granule, which is a result of the increase of COX-2 expression (Fig. 6).

Experimental Example  4. Analysis of the degree of immune cell proliferation

Since excessive proliferation of immune cells is a factor that exacerbates immune diseases or inflammatory diseases, the inhibitory effect on the proliferation of immune cells of stem cells treated with mast cell granules was analyzed.

Specifically, immune cells were treated with Concanavalin A to induce proliferation, and co-cultured with the stem cells treated with the mast cell granules or the stem cells not treated with the granules. Subsequently, the degree of inhibition of proliferation of immune cells was measured by BrdU incorporation assay.

As a result, it was confirmed that a large number of immune cells proliferated when only Concanavalin A was treated. On the other hand, it was confirmed that the stem cells treated with the mast cell granules inhibited the proliferation of the immune cells significantly compared with the stem cells not treated with the above granules. Although the cells were treated with Concanavalin A, Inhibiting the proliferation of immune cells (Fig. 7).

From the above results, it was confirmed that the stem cells treated with the mast cell granules of the present invention can be used as immunosuppressants.

Claims (17)

A pharmaceutical composition for preventing or treating an immunological disease or inflammatory disease comprising stem cells or cultures thereof cultured by adding a mast cell extract to stem cells.
The pharmaceutical composition according to claim 1, wherein the mast cell is derived from cord blood.
The method of claim 1, wherein the mast cell extract comprises histamine, serotonin, heparin, beta-hexosaminidase, and leukotriene. Composition.
The pharmaceutical composition according to claim 1, wherein the stem cell cultured by adding the mast cell extract increases the expression level of COX-2 (Cyclooxygenase-2) in stem cells.
The pharmaceutical composition according to claim 1, wherein the stem cells are adult stem cells, pluripotent stem cells, induced pluripotent stem cells or embryonic stem cells.
6. The pharmaceutical composition according to claim 5, wherein the adult stem cells are derived from a tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta.
The pharmaceutical composition according to claim 5, wherein the adult stem cells are mesenchymal stem cells, mesenchymal stromal cells or multipotential stem cells.
The pharmaceutical composition according to claim 1, wherein the immunological disease or inflammatory disease is an autoimmune disease, graft rejection, arthritis, graft versus host disease, bacterial infection, sepsis or inflammation.
9. The method of claim 8, 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, , 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. ≪ RTI ID = 0.0 > 11. < / RTI >
9. A method for preventing or treating an immune or inflammatory disease, comprising administering the composition of any one of claims 1 to 9 to a subject other than a human.
A method for inhibiting an immune response or an inflammatory response of a subject other than a human, comprising the step of administering to the subject a stem cell or culture thereof cultured by adding a mast cell extract to the stem cell.
A method for producing an immunosuppressant or an anti-inflammatory agent, comprising culturing stem cells by adding a mast cell extract.
In the media supplemented with mast cell extract comprising the step of culturing the stem cells, COX-2 (Cyclooxygenase-2 ) , or PGE ₂ (Prostaglandin E2) method of mass production of proteins.
14. The method according to any one of claims 11 to 13, characterized in that the mast cell is derived from cord blood.
The pharmaceutical composition according to claim 1, wherein the mast cell extract is obtained by freezing and thawing mast cells. delete delete

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