KR101512171B1 - A composition comprising stem cell for preventing or treating of immune or inflammatory disease - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of immune diseases or inflammatory diseases containing stem cells or cultures thereof and a process for producing the composition.
The stem cell of the present invention or a culture thereof and the stem cell cultured by adding the NOD2 agonist can be effectively used for the prevention or treatment of an immunological disease or an inflammatory disease including atopic dermatitis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing or treating an immunological disease or inflammatory disease containing stem cells as an active ingredient,

The present invention relates to a composition for the prevention or treatment of immune diseases or inflammatory diseases containing stem cells or cultures thereof and a process for producing the composition.

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 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 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 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.

Atopic dermatitis refers to recurrent, chronic allergic eczema with symptoms such as severe itching and dry skin. Atopy has high IgE expression in blood and has the same characteristics as eosinophilia. Recently, atopic dermatitis is estimated to occur in about 10 to 20% of the total population, but there is no clear cure that can be cured. Especially, it is mostly diagnosed at the young age of 5 years or less. It is diagnosed within 24 months. The prevalence of atopic dermatitis has increased in the last 10 years in the National Epidemiologic Survey conducted by the Korean Society of Pediatric Allergy Respiratory Society. Early diagnosis and management of atopic dermatitis, which is the starting point of allergy progression, is very important to prevent progression of adult allergies since 50 to 75% of children with atopic dermatitis have allergic diseases progressing to asthma and rhinitis.

Atopic dermatitis is caused by many factors such as activation of T lymphocytes, abnormalities of cytokine system, decrease of cell mediated immunity, immunologic abnormality such as increase of IgE, physiological factors and skin biochemical defects. To treat such atopic dermatitis, treatment with a drug such as steroids is required as well as moisturizing of the dried skin. When symptoms are mild as a therapeutic agent for atopic dermatitis, moisturizers, topical steroids, antihistamines, antibiotics, and local immune response modifiers are used. In severe atopic dermatitis, systemic steroids and immunosuppressants are used. However, long-term use has side effects. If the drug is discontinued, there is a high possibility of recurrence of the lesion. Therefore, safe and effective treatment is also required for long-term use. Patients with atopic dermatitis are more likely to have an intolerant gang disorder, social and school indications, depression, anorexia, anxiety, sleep disturbance, severe mental, physical and social impairment, and develop asthma and allergic rhinitis Atopic dermatitis is a major disease that causes not only skin but also immunological diseases in the body, but currently used atopic dermatitis remedy is a remedy that alleviates symptoms. Therefore, development of a more fundamental and epoch-making therapeutic agent is urgent.

Therefore, the present inventors have demonstrated immunological characteristics of negative hematopoietic stem cell markers or immunoreactivity-related markers, thereby separating stem cells expressing ZNF281, which is less likely to be rejected at the time of transplantation, to be effective in the treatment of atopic dermatitis And that the stem cells obtained by culturing the stem cells with the addition of the NOD2 agonist exhibit more excellent therapeutic effect of atopic dermatitis, the stem cells of the present invention are also identified as NOD2-expressing stem cells, Thereby completing the invention.

It is an object of the present invention to provide a composition for preventing or treating immunological diseases or inflammatory diseases containing isolated stem cells or cultures thereof.

Another object of the present invention is to provide a method for producing a composition for preventing or treating an immunological disease or an inflammatory disease containing stem cells or a culture thereof.

In one aspect for accomplishing the above object, the present invention provides a composition for preventing or treating an immune disease or inflammatory disease containing stem cells or a culture thereof.

The term "stem cell" of the present invention means a cell having an excellent proliferative potential and capable of differentiating into various tissues of the body.

The term "culture product" of the present invention means a culture medium containing growth factors, cytokines, chemokines and the like secreted from cells other than the cells from the cell culture solution.

In the present invention, the term "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 administration of the composition improves the symptoms of the immune or inflammatory disease Or to be benefited.

The stem cells according to the present invention are characterized by expressing ZNF281. The term "ZNF281" of the present invention is a protein encoded by the ZNF281 gene in human, also referred to as zinc finger protein 281. [ It is a protein involved in electronic regulation and inhibits the transcription of many genes, including gastrin and ornithin decarboxylase. Binds to the G-rich box of the enhancer region of the gene and belongs to the krueppel C2H2-type zinc finger protein family.

Since the stem cell expressing ZNF281, which is an active ingredient of the composition of the present invention, is a cell in which HLA-DR (class II) is not expressed, which is the most important cause of rejection in tissue or organ transplantation, For the prevention or treatment of cancer.

Preferably, the stem cells of the present invention are characterized by expressing NOD2. The term "NOD2" of the present invention refers to the NOD domain of the CARD (caspase-recruitment domain) involved in protein-protein interaction, the NOD domain in the middle, and the LRR domain in the C- One of the NOD family proteins consisting of the domain is a protein having two CARD domains at the N-terminus. In addition, there is a NOD1 protein with a single CARD domain at the N-terminus, which together is called NLR (NOD like receptor), which is involved in the in vivo immune response with TLR (toll like receptor). It is a protein distributed in bone marrow-derived macrophages, neutrophils, dendritic cells, and small intestinal parenchyma cells, and its distribution is expressed in a limited region rather than NOD1. Among the agonists (a.k.a. ligands) recognized by NOD2, the most widely known is MDP (muramyl dipeptide) which is common to PGN components of Gram-negative bacteria and Gram-positive bacteria.

In addition, the stem cells of the present invention have at least one of the following characteristics:

(a) exhibits positive immunological properties for the transcription factor c-myc.

(b) The extracellular matrix is coated on the bottom and proliferates in a linear or spherical cell population between 5 and 30 days after adherence.

(c) a cumulative population doubling level (CPDL) of 30 to 45.

(d) Immunological properties of the negative for CD14, CD31, CD34, CD45 and HLA-DR

I get it.

(e) Differentiation into mesodermal, endodermal, and ectodermal cells.

IL-6, IL-8, IL-10, IL-12p40, IL-12, At least one cytokine or chemokine selected from the group consisting of IL-13, IL-16, IP-10, Leptin, MCP-2, MIG, MIP-3a, b-NGFm sTNFRI and PFGF-bb is secreted.

The expression of Oct-4, Sox-2, Rex-1, c-myc and ZNF281 in the stem cells of the present invention means that the undifferentiated state is maintained.

In addition, the stem cells of the present invention show a CPDL (cumulative population doubling level) of 30 to 45, indicating that the stem cells have excellent proliferative power.

In addition, the stem cells of the present invention show the immunological characteristics of negative for CD14, CD31, CD34, CD45 and HLA-DR, which are known as hematopoietic stem cell markers or immunoreaction-related markers. As described above, the stem cells of the present invention lack hematopoietic and immune rejection-related markers and can minimize angiogenesis and rejection upon transplantation, and thus can be used as cells useful for allogenic transplantation.

The stem cells according to the present invention are characterized by being human adult stem cells, human pluripotent stem cells, induced pluripotent stem cells, animal embryonic stem cells, or animal adult stem cells.

Preferably, the stem cells may be adult stem cells. The term "adult stem cells" of the present invention can be mesenchymal stem cells, mesenchymal stem cells derived from human tissues, mesenchymal stem cells derived from human tissues such as fats and bone marrow, multipotential stem cells or amniotic epithelial cells. Adult stem cells are generally noted because they can avoid unlimited sources and ethical problems faced by researchers compared to embryonic stem cells. Furthermore, stem cells isolated from umbilical cord blood have greater advantages than other adult stem cells because, unlike bone marrow or adipose tissue, donors are not likely to suffer additional harm.

More preferably, the stem cell of the present invention is a mesenchymal stem cell, umbilical cord blood-derived mesenchymal stem cell, bone marrow-derived mesenchymal stem cell, adipose-derived mesenchymal stem cell, muscle-derived mesenchymal stem cell, , Skin-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells.

The term "cord blood" of the present invention refers to blood collected from an umbilical vein connecting the placenta of a mammal and a fetus. The umbilical cord blood can be easily collected from the umbilical vein of the donor at birth. More specifically, in the case of a normal vaginal delivery, the uterus can be collected from the umbilical vein which has been exposed to the uterus while the placenta still remains in the uterus. In the case of a cesarean section, the placenta is also drawn from the umbilical vein in a state of being exposed outside the uterus after delivery of the baby.

Since stem cells expressing ZNF281, which is an active ingredient of the composition of the present invention, are cells that do not express HLA-DR (class II), the most important cause of rejection in tissue or organ transplantation, Or rejection, and thus can be used as well as autologous or homologous. Preferably, it may be isolated from cord blood of a mammal including a human. Most preferably, it can be a cord blood-derived stem cell disclosed in Korean Patent Application No. 10-2009-0023821.

In the present invention, the immunological disease or inflammatory disease is characterized by autoimmune disease, graft rejection, arthritis, graft versus host disease, bacterial infection, sepsis or inflammation. The autoimmune diseases include autoimmune diseases such as Crohn's disease, erythema disease, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperlipemia, scleroderma, Inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic scleroderma, Ulcerative colitis, < / RTI > and ulcerative colitis.

Among the immunological diseases or inflammatory diseases of the present invention, "atopic dermatitis" is a representative skin disease occurring in a person having atopic allergy. Often, it is a chronic skin disease called as fever, and dry skin and itching are the main symptoms. It has immunologic characteristics and it is accompanied by other allergic diseases such as urticaria, metal allergy, asthma or allergic rhinitis, and it tends to have a family history. Symptoms usually appear at 2 to 6 months of age, especially at 1 year of age and 85% at age 5. It is usually known to be a short-lived disease at a young age, but 50% of the patients disappear within two months, 25% appear until adolescence, and the remaining 25% continue to disappear even as adults. Unbearable itching can distract attention, interfere with learning, cause sexual degradation, and can lead to damage or emotional hurt by other children's teasing or bullying.

The composition of the present invention may contain 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cells, preferably 1.0 × 10 ⁶ to 1.0 × 10 ⁸ cells, more preferably 1.0 × 10 ⁷ cells per ml .

The compositions of the present invention can be used without being frozen or frozen for future use. When it is required to be frozen, a standard cryopreservative (for example, DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, Sorbitol, i-inositol, D-lactose, choline chloride, Epilife ( ^R ) cell freezing medium (Cascade Biologics)) may be added to the pre-frozen 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 prepared by mixing with the injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the injection solution. The injection bag may be made of polyvinyl chloride or polyethylene and may be made of a material selected from the group consisting of Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo An injection bag can be illustrated.

The pharmaceutical preparation may further comprise one or more conventional pharmaceutically inert carriers and diluents in addition to the active ingredient. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the stem cells and the recipient thereof. Diluents include, but are not limited to, saline, aqueous buffer, solvents and / or dispersion media. In addition, preservatives, analgesics, solubilizing agents or stabilizers may be added in the case of injections, and bases, excipients, lubricants or preservatives in the case of topical preparations.

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.

The present invention relates to a method for producing the composition, comprising the steps of: isolating stem cells; And culturing the isolated stem cells. The present invention also provides a method for preparing a composition for preventing or treating an immunological disease or an inflammatory disease.

The step of isolating the stem cells of the present invention can be carried out by any separation method known in the art. For example, there are density gradient fractionation, immunoselection, and differential adhesion separation.

According to a specific embodiment of the present invention, stem cells expressing ZNF281 from cord blood were isolated and cultured. Methods for isolating and culturing stem cells from the umbilical cord blood can be used in any of the methods previously used, including the method disclosed in Korean Patent Application No. 10-2009-0023821.

The step of culturing the stem cells may further include adding NOD2 agonist.

The term "agonist " of the present invention generally refers to a chemical substance that positively stimulates a receptor and is also called an agonist. That is, as opposed to antagonists and antagonists, agonists play a positive role, instead of interfering with the normal ligand or acting in the opposite way. In the present invention, the term " ligand ", which generally means a chemical substance binding to a receptor, can be used in parallel.

The term "NOD2 agonist" of the present invention means without limitation the substance capable of binding to the NOD2 receptor to activate NOD2, for example, MDP (muramyl dipeptide).

In the present invention, "adding and culturing" an agonist may be performed, for example, by adding an agonist to a culture solution of a stem cell of the present invention. Preferably, the agonist may be cultured by adding the agonist at a concentration of 1 to 100 μg / ml for 0.1 to 200 hours, more preferably for 1 to 72 hours. Alternatively, the agonist may be added and cultured, and then the medium may be replaced and cultured.

Preferably, human stem cells are treated with MDP at a concentration of 1 to 100 μg / ml for 0.1 to 200 hours. When treating MDP for less than 0.1 hour, the NOD2 pathway may not be fully activated, and since treating MDP for 200 hours or more is not economically advantageous, more preferably 0.1 to 200 hours, Can be cultured by adding MDP for 1 to 72 hours, most preferably for 24 hours.

For example, DMEM (Dulbecco ' s modified Eagle medium), McCoys 5A medium, Eagle ' s basal medium, CMRL < (R) > L-15 medium, RPMI 1640 medium or Keratinocyte-SFM (Keratinocyte serum free medium), and most preferably Dmedia (Gibco) medium, ) Can be used.

The stem cell medium may be supplemented with an additive. In general, the concentration of the essential amino acids, such as glutamine, and non-essential amino acids, such as neutral buffer (e.g., phosphate and / or high concentration bicarbonate) and protein nutrients (e.g., fetal bovine serum, horse or human serum, serum substitute or albumin, Essential amino acids). 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). Antibiotics and antifungal agents to prevent microbial contamination may also be added.

In another aspect, the present invention provides a method for preventing or treating an immune disease or an inflammatory disease, comprising the step of administering to a subject suffering from an immunological disease or inflammatory disease comprising atopic dermatitis, A method for treating an immunological disease or an inflammatory disease.

The term "individual" in the present invention means a mammal including, but not limited to, cattle, dogs, pigs, chickens, sheep, horses, and humans.

Stem cells expressing ZNF281 isolated from human adult stem cells of the present invention were found to be effective for the prevention or treatment of atopic dermatitis similarly to the case of oral administration of prednisolone currently used, and MDP, an agonist of NOD2, The stem cells expressing ZNF281 of the present invention also showed NOD2 expression, indicating that the stem cells treated with the cultured stem cells showed a superior therapeutic effect. Accordingly, the stem cells expressing ZNF281 of the present invention, the culture thereof, and the stem cells cultured by adding the NOD2 agonist can be effectively used for the prevention or treatment of immune diseases or inflammatory diseases including atopic dermatitis.

1 is a diagram showing an experimental schedule of an animal experimental model. The time at which the atopy induced ointment was treated on one straight line and the time when the stem cells of the present invention were treated were simultaneously shown.
FIG. 2 is a view showing a site where a stem cell of the present invention is injected. And injected intrasubcutaneous injection into the dorsal and auricular area, which is the site of atopic dermatitis.
FIG. 3 is a graph showing the results of observation of gross lesions after administration of stem cells in a model of atopic dermatitis. A is a view showing a visual lesion score for each test group evaluated at the time of autopsy (Day 30), and B is a chart showing a result of a gross lesion observation according to time.
FIG. 4 is a graph showing histological changes after administration of stem cells in a mouse model of atopic dermatitis. A shows the frequency of inflammatory cells with H & E staining, and B shows the thickness of the epithelial layer.
FIG. 5 is a graph showing deaggregation of mast cells after administration of stem cells in a model of atopic dermatitis mice. A is a result of staining with toluidine blue and confirmed by confocal microscopy, and B is a figure obtained by quantifying degranulation of mast cells.
FIG. 6 shows degranulation of mast cells in skin tissue of atopic dermatitis-induced mice injected with stem cells. The skin tissue of each group was pulverized to extract the protein and the amounts of tryptase found in the degranulation of mast cells were compared.
FIG. 7 is a graph showing the concentration of immunoglobulin E (IgE) in serum after administration of stem cells in a model of atopic dermatitis.
FIG. 8 is a diagram showing the distribution of the stem cells injected in vivo in the atopic dermatitis-induced mouse into the skin tissue.

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 to these examples.

Example  1: Experimental animal

To evaluate the efficacy of umbilical cord blood derived mesenchymal stem cells against atopic dermatitis, experiments were carried out using NC / Nga atopic dermatitis mice. Seven-week-old male BALB / cAnNCrjBgi-nu mice were purchased from the central laboratory animals, and general symptoms were observed in the laboratory for about one week and only healthy animals were used in the experiment. 39 males of 8 weeks of age were randomly divided into groups. This experiment was conducted at Seoul National University College of Veterinary Medicine, Seoul National University, which was set at a temperature of 22 ± 3 ° C, a relative humidity of 50 ± 10%, a ventilation frequency of 10 to 12 times / hr, an illumination time of 12 hours (07:00 to 19:00) This was done on an MI lathe equipped with a HEPA filter in the laboratory. During the refinement and experiment period, they were kept in a polycarbonate MI cage (26 × 42 × 18 cm, manufactured by Myungjin Machinery) at a density of 7 grains / box. In the breeding box, the tag containing the experiment number, the animal number, and the drug dose was attached. During the frying period, feeds were fed free of high pressure steam sterilized animal feed (Purina) and free water was used for drinking low - pressure steam sterilized water.

Example  2: Establishment of an animal model of atopic dermatitis

The animals used for the experiment were 7 weeks old mice as NC mice (NC / Nga mice) expressing atopic dermatitis, and they were used for the experiment at 8 weeks of age after a week of purifying period. In order to induce atopic dermatitis, the dorsal part of the NC / Nga mouse and the upper part of the pinna were epilated as much as possible on the first induction day, and the hair was completely removed using a hair removal cream. After removal of the hair removal cream, 100 mg of atopic ointment ointment was uniformly applied to the dorsal side of the pinna using a flat rod. From the second induction date, the newly developed hairs from the back of the NC / Nga mouse to the upper part of the ear canal were removed with a maximum of the epilator, and then 4% SDS (sodium dodecyl sulfate) aqueous solution 150 μl) was sprayed, dried with a cool wind of the hair dryer, completely dried for about 2 to 3 hours, and 100 mg of atopic ointment ointment was uniformly applied to the dorsal and auricular portion using a flat rod. The atopic dermatitis mouse model was established by applying atopy - induced ointment twice a week for 3 weeks in total 6 times. The atopy induction ointment used in this experiment was applied to mice with atopic dermatitis (NC / Nga mouse) as an ointment containing American house dust mite extract (Dermatophagoides farina; Df) 1999, 120 Suppl. 1: 70-75; Vestergaard, C. et al., Mol. Med. Today, 2000, 6 ( 5: 209-210; Matsuda, H. et al., Int. Immunol., 2997, 9 (3): 461-466).

Example  3: hUCB - USC ( human umbilical cord blood - universal stem cell ), Culture and proliferation

Umbilical blood used to isolate hUCB-USC was obtained from Allcord, a donation cord blood bank from Boramae Hospital. Specifically, the method of isolating hUCB-USC from cord blood is as follows. First, red blood cells were removed using HetaSep ^™ (Stem Cell Technologies INC, Vancouver, BC), and mononuclear cells were separated using a Ficoll gradient. The isolated mononuclear cells were cultured in the specific culture medium described in Korean Patent Application No. 10-2009-0023821, and the stem cells expressing ZNF281 were isolated and proliferated by continuous subculture.

Example  4: hUCB - USC Transplantation

The hUCB-USC expressing ZNF281 isolated and propagated in Example 3 was injected intrasubcutaneous injection into the dorsal and auricular portion of atopic dermatitis. The experimental groups were constructed as shown in Table 1 below. In addition, hUCBUSC was applied to NC / Nga mice once a week, and then injected four times a day (Fig. 1). In the experimental group, hUCB-USC treated with hUCB-USC expressing ZNF281 cultured for 24 hours with 10 μg / ml MDP (muramyl dipeptide) added to the culture solution of hUCB-USC of the present invention 1 day before cell administration As a group, the MDP is a substance represented by the following chemical formula 1, which is a peptidoglycan which is a component of a gram-negative and gram-positive bacteria, thereby increasing the expression amount of the NOD-2 receptor of USC and ultimately controlling the immune regulation It is an additive to maximize ability.

The negative control group (-) consisted of mice (NC / Nga mice) that did not induce atopic dermatitis and the atopic dermatitis control group (Df) induced atopic dermatitis using atopy induced ointment (Df) Mice. In addition, the fibroblast control group (Fb) was a group consisting of mice that induced atopic dermatitis using atopic induction ointment and treated with the same amount of fibroblasts at the same site as the cell administration site in hUCB-USC group expressing ZNF281, and MDP Pre-treatment The hUCB-USC group (MDP) was administered with hUCB-USC expressing ZNF281 cultured with MDP in the culture medium 24 hours before the cell recovery at the site of administration of hUCB-USC by inducing atopic dermatitis using atopic induction ointment (PDS) is a group consisting of mice that induce atopic dermatitis using atopic induction ointment and oral administration of prednisolone, an immunosuppressant once a day for 3 weeks.

Example  5: Gross lesion  evaluation

After injection of hUCB-USC expressing ZNF281 in atopic dermatitis-induced mouse model, the lesion was checked with the naked eye to determine the remedy effect. The severity of the disease was assessed according to the degree of dryness, excoriation, erythema and edema according to the severity of symptoms from 0 to 3 (0, none; 1, mild; 2 , moderate; 3, severe). From the beginning of the experiment, the mean value of each experimental group on the 4th, 11th, 18th, 24th and 30th day was compared and analyzed.

The experimental results are shown in Fig. Atopic evaluation of atopic dermatitis in each experimental group (Day 30) was significantly higher in the atopic dermatitis-induced control group than in the negative control group, and this increase was significantly decreased in the hUCB-USC treated group expressing ZNF281. This was comparable to that of oral administration of prednisolone, a commercially available immunosuppressant, and was found to be superior to MDP group cultured with NOD2 agonist MDP for one day, whereas fibroblast The experimental group had no effect on atopic dermatitis relief (Fig. 3A). This showed a similar pattern throughout the experiment as well as during the autopsy (Fig. 3B). These results indicate that the hUCB-USC expressing ZNF281 of the present invention exhibits a therapeutic effect on atopic dermatitis as well as a superior therapeutic effect in the MDP group, Was also expressed.

Example  6: Tissue Inspection

30 days after the start of the experiment (Day 30), the experimental animals were euthanized, and atopic dermatitis lesion tissue was secured at the time of necropsy, and tissues were fixed in 4% paraformaldehyde and embedded in paraffin to obtain H & E (hematoxylin and eosin) Stained with blue (toluidine blue) and analyzed using confocal microscopy system.

The results of the experiment are shown in FIG. This study was conducted to investigate the difference between the control group and the hUCB-USC group expressing ZNF281 in the pathogenesis of atopic dermatitis. When compared with the general H & E staining, the expression of inflammatory cells in the group treated with ZNF281- (Fig. 4A). It was also found that the thickness of the epithelial layer was reduced to a level similar to that of the immunosuppressant PDS group in the hUCB-USC administration group expressing ZNF281 compared to the atopic dermatitis control group (Fig. 4B).

Example  7: mast cell Degranulation  inspection

It is known that atopic dermatitis is induced by IgE, one of the immunoglobulins, by inducing degranulation of mast cells. Therefore, the degree of degranulation of mast cells was compared between the control group and the hUCB-USC group expressing ZNF281 at the onset of atopic dermatitis. For this purpose, skin tissue was embedded in paraffin and then confirmed by confocal microscopy through toluidine blue staining.

As a result, it was confirmed that the degranulation of mast cells was significantly reduced in the hUBC-USC-treated group expressing ZNF281 compared with the atopic dermatitis-induced control group (Fig. 5). In addition, the expression level of tryptase found in the degranulation of mast cells after the crushing of the skin tissue of each experimental group was significantly reduced in the hUCB-USC treated group expressing ZNF281 (Fig. 6).

Example  8: Serum IgE  Concentration assessment

Thirty days after the start of the experiment (Day 30), the experimental animals were euthanized and serum samples collected at the time of autopsy were analyzed for serum immunoglobulin E (BD) using the Opt EIA Mouse Set (BD Bioscience, Mississauga, Canada) (IgE) levels were compared.

As shown in FIG. 7, the serum IgE level increased by atopic dermatitis induced by hUCB-USC, which expresses ZNF281, was significantly lower than that of the atopic dermatitis-induced control group, which was similar to that of the immunosuppressant prednisolone- Respectively. On the other hand, it was confirmed that the MDP pretreatment hUCB-USC group, which is a NOD2 agonist, decreased to a level lower than that of hUCB-USC group or prednisolone group.

Example  9: Cell Tracking Test

In order to confirm the distribution of the transplanted cells in the animal body, CFDA-SE (Vybrant CFDA SE Cell Tracer Kit, Invitrogen) was labeled with hUCB-USC expressing ZNF281 and then administered to mice and autopsied The skin tissue was observed. The skin tissue was collected and fixed with 4% paraformaldehyde, and then frozen sections were prepared and processed. By observing the green wavelength of CFDA-SE using a confocal microscope system, the in vivo distribution of hUCB-USC expressing transplanted ZNF281 The location was traced and analyzed.

As shown in Fig. 8, it was confirmed that hUCB-USC expressing transplanted ZNF281 was observed under the skin of atopic dermatitis lesion. In other words, it was confirmed that hUCB-USC expressing ZNF281 injected into an animal model is directly distributed in the disease site and is effective in relieving atopic dermatitis.

<Statistical processing of data>

One-way ANOVA was performed for the statistical analysis of the data from the experiment. The significance was determined at the p = 0.05 level. When the significance was confirmed, Dunnett's t-test was performed. Significance was tested (p <0.05).

Claims (12)

An immunological disease or inflammatory disease selected from the group consisting of atopy, graft rejection, graft-versus-host disease, and dermatitis, wherein the stem cell expressing ZNF281 (zinc finger protein 281) and NOD2 (nucleotide-binding oligomerization domain protein 2) &Lt; / RTI &gt;
delete delete The method according to claim 1,
Wherein the stem cell has at least one of the following characteristics:
(a) exhibits positive immunological properties for the transcription factor c-myc (cellular myelocytomatosis oncogene);
(b) the extracellular matrix is coated on the bottom and proliferates in a linear or spherical cell population between 5 and 30 days after attachment;
(c) show a cumulative population doubling level of 30 to 45;
(d) immunological characterization of negative for CD (cluster of differentiation) 14, CD31, CD34, CD45 and human leukocyte antigen-DR (HLA-DR);
(e) differentiable into mesoderm, endoderm and ectodermal cells; or
(f) TIMP-2 (TIMP metallopeptidase inhibitor 2), TGF-beta (transforming growth factor beta), RANTES (regulated on activation, normal T cell expressed and secreted), CINC-3 (cytokine-induced neutrophil chemoattractant 3) IL-8, IL-10, IL-12p40, IL-12, IL-10, IL-12, IL-13, IL-16, IP-10 (interferon gamma-induced protein 10), Leptin, MCP-2 (monocyte chemoattractant protein 2), MIG (monokine induced by gamma interferon) at least one cytokine or chemokine selected from the group consisting of alpha-beta, beta-nerve growth factor (b-NGF), soluble tumor necrosis factor receptor (sTNFRI) and platelet- Secretion.
The method according to claim 1,
Wherein the stem cells are human adult stem cells, human pluripotent stem cells, induced pluripotent stem cells, animal embryonic stem cells, or animal adult stem cells.
6. The method of claim 5,
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.
6. The method of claim 5,
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, muscle derived mesenchymal stem cells, A mesenchymal stem cell, a mesenchymal stem cell, an amniotic membrane-derived mesenchymal stem cell, and a placenta-derived mesenchymal stem cell.
delete delete An immune disease selected from the group consisting of autoimmune diseases, graft rejection, graft versus host disease, and dermatitis, which contain stem cells expressing ZNF281 (zinc finger protein 281) and NOD2 (nucleotide-binding oligomerization domain protein 2) A method of preparing a composition for the prevention or treatment of an inflammatory disease,
Isolating stem cells expressing ZNF281 and NOD2; And
And culturing the isolated stem cells.
11. The method of claim 10,
Wherein the step of culturing the stem cells further comprises adding NOD2 agonist and culturing.
12. The method of claim 11,
Wherein the concentration of the agonist of NOD2 is 1 to 100 占 퐂 / ml with respect to the medium, and the incubation time is 0.1 to 200 hours.

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