WO2018038575A1 - Pharmaceutical composition for treating rheumatoid arthritis, comprising mesenchymal stem cell-derived secretory proteome, and therapeutic method using same - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating an autoimmune disease using mesenchymal stem cell-derived secretory proteome. A pharmaceutical composition according to the present invention increases the expression of IL-10, which is a representative immunomodulatory cytokine, and reduces the expression of inflammatory cytokines such as TNF- α, IL-6 and IL-12 (p70), and thus can effectively prevent or treat various autoimmune diseases including rheumatoid arthritis.

Description

Pharmaceutical composition for treating rheumatoid arthritis comprising mesenchymal stem cell-derived secretory protein and method of treatment using same

The present invention relates to a composition for preventing, improving or treating autoimmune diseases using mesenchymal stem cell-derived secretory proteins.

Rheumatoid arthritis is the most common inflammatory arthritis in adults, and its cause is not yet fully understood, but it is understood as an inflammatory response of the synovial membrane of the joint due to an autoimmune mechanism. Although antirheumatic drugs and biological agents used to treat rheumatoid arthritis have been shown to improve arthritis through inflammation control, they are still insufficient to induce remission, the ultimate therapeutic target. In many patients, the therapeutic effect is incomplete, side effects associated with the therapeutic agent are impeding the treatment, and when the biological agent is used for a long time, antibodies to the drug are generated, thereby reducing the therapeutic effect.

Mesenchymal stem cells are undifferentiated adult stem cells that exist between differentiated cells of tissues or organs, and can be isolated from various tissues in the human body such as bone marrow, fat, and muscle, and self-renewing self-renewing. Have self-renewal In addition, since it can be easily proliferated in vitro and can be differentiated into various tissue cells such as adipocytes, bone cells, chondrocytes, and myocytes, existing stem cell researches have been concentrated on regeneration studies using differentiation functions.

The immunomodulatory function of mesenchymal stem cells, which has been recently revealed by various studies, protects hematopoietic stem cells from damage caused by immune responses, and acts at each stage of the immune response, resulting in an immunomodulatory effect. And anti-inflammatory responses. This immunomodulatory function occurs through interaction with various immune cells such as natural killer cells (NK cells), dendritic cells, macrophages (macrophage), T cells and B cells. Although the anti-inflammatory effects of mesenchymal stem cells are described as various growth factors secreted from stem cells and paracrine, where damaged tissues are repaired by proteins, studies on the treatment of inflammatory diseases using the adjacent secretory effects have been It is inadequate.

On the other hand, the secreted protein can be manufactured and synthesized in a large amount from other (homologous) cell lines that have completed both characterization, contamination analysis, and quality control for clinical use, and thus, unlike cell therapeutics for cell replacement, It is a biologic that can easily overcome shortages or medical problems.

One object of the present invention is to provide a composition that can effectively prevent, ameliorate and treat autoimmune diseases by using a secretory protein derived from the culture medium of mesenchymal stem cells that do not have ethical problems and do not have immunogenicity.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, a mesenchymal stem cell-derived secretory protein (secretome), comprising a pharmaceutical composition for the prevention or treatment of autoimmune diseases.

In the present invention, the "mesenchymal stem cell" refers to an undifferentiated cell having a multipotency derived from an adult human cell of a mammal, including a human, for example, bone marrow, blood And various adult cells such as brain, skin, fat (ie, adipose tissue or adipocytes), umbilical cord blood, umbilical cord Barton's jelly and the like.

In addition, in the present invention, the "secretome" (secretome) means the sum of the protein components among the components secreted from the mesenchymal stem cells. Secretory proteins refer to components that are released into the extracellular environment by a cell after transcription, translation, and post-translational modification of the gene in the cell. Representative expression markers of secreted proteins correspond to growth factors such as EGF and VEGF and extracellular matrix proteins such as collagen and fibronectin.

In the present invention, the secreted protein may be isolated from the culture solution obtained by culturing the mesenchymal stem cells.

The method of culturing the mesenchymal stem cells in the present invention may be by a two-dimensional culture or a three-dimensional culture method.

In the present invention, the two-dimensional culture of the mesenchymal stem cells, the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium for 24 to 96 hours and then carried out in a serum-free medium for 24 to 72 hours Can be.

Here, the composition of the mesenchymal stem cell culture medium is not particularly limited but may be serum medium. For example, Dulbecco's modified Eagle's medium (DMEM) or RPMI containing 5-15 wt% Fetal bovine serum (FBS) and 0.05-0.2 mM mercaptoethanol. -1640 medium, or the like, or serum-free medium such as StemPro medium, MSCGro medium, MesenCult medium, or NutriStem medium, but is not limited thereto, and any medium that can be used for culturing mesenchymal stem cells in the art is not limited thereto. Can be used.

In addition, the serum-free medium may be Dulbecco's modified Eagle's medium (DMEM) that excludes phenol red and antibiotics, but is not limited thereto. It can be used for the culture of mesenchymal stem cells, including the available medium, and any medium in which fetal bovine serum is excluded can be used without limitation.

In the present invention, depending on the culture conditions of the mesenchymal stem cells, the secretion amount and secretion of the secreted protein may be different, the normal culture is carried out under the normal oxygen partial pressure (20% by volume level of oxygen). However, the environment in the body is low oxygen partial pressure, by providing such an environment in vitro to cultivate stem cells when the growth and differentiation and neovascularization ability of the cells, thereby increasing the therapeutic effect of stem cells. Therefore, in the present invention, the secreted protein is not only obtained by culturing mesenchymal stem cells under normal oxygen culture conditions (20% by volume O ₂ ), but also by culturing mesenchymal stem cells under hypoxic conditions (0.5-1% by volume O _2). It may be obtained by culturing under).

In the present invention, as the secretory protein, centrifugation of the culture solution obtained by the two-dimensional culture at 500 to 1,500 xg, the supernatant is recovered, and the concentrate of the polymer component obtained is used to suppress the expression of inflammatory cytokines. Expression of anti-inflammatory cytokines is preferred because it can be activated to further suppress the immune response.

In one embodiment of the present invention, the concentrate of the polymer component may include filtering the supernatant from the supernatant obtained by centrifugation with a 0.1 to 0.3 μM filter, preferably a 0.2 μM filter; And filtering the molecules up to 3 kDa.

Here, the method of filtering the molecules below 3 kDa, it may be performed by diafiltration using a tangential flow filtration (TFF) device. In addition, in the present invention, the supernatant may be concentrated at 0 to 5 ° C. while diluting the supernatant with injection water using a peristatic tubing pump.

In addition, in another embodiment of the present invention, the concentrate of the polymer component may be obtained by reacting the supernatant obtained by centrifugation with an alcohol polar solvent and concentrating the active ingredient.

Here, the alcohol polar solvent may be used alone or two or more of a lower alcohol having 1 to 6 carbon atoms, a dilution solution of the alcohol, for example, 95% or 90% alcohol aqueous solution, or acetone to be isopropyl alcohol by a reducing agent. Can be.

In addition, the alcohol solution in the present invention means a dilution solution of alcohol, for example, may include 95% ethanol, 90% ethanol and the like.

In the present invention, the alcohol polar solvent is preferably mixed in an amount of 2 to 5 times the weight ratio with respect to the supernatant, since only the active ingredient of the polymer concentrate in the supernatant can be effectively concentrated.

In addition, in the present invention, the reaction between the supernatant and the polar polar solvent is preferably performed at -30 to 0 ° C for 5 to 500 minutes.

According to one example of the present invention, 100% alcohol may be added to the supernatant obtained by centrifugation of the culture solution obtained by the two-dimensional culture, and left for 5 to 500 minutes at -30 to 0 ° C. Thereafter, after centrifugation, the precipitate may be centrifuged again by adding 90% alcohol to the precipitate. Alternatively, the precipitate obtained after centrifugation can be suspended by adding sterile water and frozen.

In the present invention, if necessary, further comprising the step of freeze drying the polymer concentrate obtained as described above for 6 to 10 hours. In the present invention, the freeze-dried polymer concentrate of the secreted protein can be obtained in powder form.

In addition, the method of three-dimensional culturing the mesenchymal stem cells in the present invention, after culturing the mesenchymal stem cells in the medium for mesenchymal stem cell culture for 24 to 96 hours and suspended in serum-free medium for 24 to 72 hours While incubated.

Here, the composition of the mesenchymal stem cell culture medium is not particularly limited but may be serum medium. For example, it may be Dulbecco's modified Eagle's medium (DMEM) containing 5 to 15% by weight of fetal bovine serum (FBS) and 0.05 to 0.2 mM of mercaptoethanol. However, the present invention is not limited thereto, and any medium that can be used for culturing mesenchymal stem cells in the art may be used without limitation.

However, in the present invention, prior to floating and culturing the mesenchymal stem cells in the serum-free medium during the three-dimensional culture, it is possible to process recombinant trypsin without trypsin or animal-derived components. In the present invention, the "recombinant trypsin" may be a recombinant trypsin without an animal-derived component, for example, may be a recombinant trypsin produced in corn. Recombinant trypsin without the animal-derived component is commercially available and may be, for example, TrypLETM Select (GIBCO Invitrogen), TrypLETM Express (GIBCO Invitrogen), TrypZeanTM (Sigma Aldrich) or Recombinant Trypsin SolutionTM (Biological Industries). There is no restriction in particular.

The serum-free medium in the present invention may be Dulbecco's modified Eagle's medium (DMEM), but is not limited thereto and may be used for culturing mesenchymal stem cells in the art. Any media that has been excluded can be used without limitation.

In the present invention, when the mesenchymal stem cells are suspended and cultured in a serum-free medium during the three-dimensional culture, the inflammatory cytokine expression is more than that in the case of stationary culture by rotating at a rotational speed of 30 to 90 rpm using a spinner flask. Is inhibited and the expression of anti-inflammatory cytokines can be activated to further suppress the immune response.

In the present invention, the secreted protein is centrifuged at 500 to 1,500 xg of the culture medium obtained by the three-dimensional culture to recover the supernatant, and then the concentration of the polymer component obtained is used to suppress the expression of inflammatory cytokines. Expression of anti-inflammatory cytokines is preferred because it can be activated to further suppress the immune response.

In one embodiment of the present invention, the concentrate of the polymer component may include filtering the supernatant from the supernatant obtained by centrifugation with a 0.1 to 0.3 μM filter, preferably a 0.2 μM filter; And filtering the molecules up to 3 kDa.

Here, the method of filtering the molecules below 3 kDa, it may be performed by diafiltration using a tangential flow filtration (TFF) device. In addition, in the present invention, the supernatant may be concentrated at 0 to 5 ° C. while diluting the supernatant with injection water using a peristatic tubing pump.

In addition, in another embodiment of the present invention, the concentrate of the polymer component may be obtained by reacting the supernatant obtained by centrifugation with an alcohol polar solvent and concentrating the active ingredient.

Here, the alcohol polar solvent may be used alone or two or more of a lower alcohol having 1 to 6 carbon atoms, a dilution solution of the alcohol, for example, 95% or 90% alcohol aqueous solution, or acetone to be isopropyl alcohol by a reducing agent. Can be.

In addition, the alcohol solution in the present invention means a dilution solution of alcohol, for example, may include 95% ethanol, 90% ethanol and the like.

In the present invention, the alcohol polar solvent is preferably mixed in an amount of 2 to 5 times the weight ratio with respect to the supernatant, since only the active ingredient of the polymer concentrate in the supernatant can be effectively concentrated.

In addition, in the present invention, the reaction between the supernatant and the polar polar solvent is preferably performed at -30 to 0 ° C for 5 to 500 minutes.

According to one example of the present invention, 100% alcohol may be added to the supernatant obtained by centrifugation of the culture solution obtained by the two-dimensional culture, and left for 5 to 500 minutes at -30 to 0 ° C. Thereafter, after centrifugation, the precipitate may be centrifuged again by adding 90% alcohol to the precipitate. Alternatively, the precipitate obtained after centrifugation can be suspended by adding sterile water and frozen.

In the present invention, if necessary, further comprising the step of freeze drying the polymer concentrate obtained as described above for 6 to 10 hours. In the present invention, the freeze-dried polymer concentrate of the secreted protein can be obtained in powder form.

Mesenchymal stem cell-derived secretory proteins obtained as described above in the present invention can effectively prevent, improve or treat autoimmune diseases.

In the present invention said autoimmune disease is a non-malignant disease or disorder that occurs and is directed against an individual's own tissue.

One of the most important traits of all normal individuals is that they do not deleteriously react with the antigenic substances that make up self, while non-self antigens can be recognized and reacted to eliminate them. Biological nonresponsiveness to autoantigens is called immunologic unresponsiveness or tolerance. However, when an abnormality occurs in inducing or maintaining self-tolerance, an immune response occurs to autoantigens, and as a result, an attack occurs on the tissues. The disease caused by this process is called an autoimmune disease.

The autoimmune disease is an inflammatory disease in which antibodies are produced against its own organ tissues or components thereof, and may refer to diseases causing chronic systemic inflammation in many tissues and organs.

In one embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, inflammatory spondyloarthritis, adult stature disease, macrophage activity syndrome, systemic sclerosis, multiple myositis, dermatitis, vasculitis, mixed connective diseases, Sjogren's syndrome, Ulcerative colitis, Crohn's disease, allergic asthma, allergic rhinitis, atopic dermatitis, gallbladder, lacrimitis, conjunctivitis, psoriasis, vulgaris ulcer, Parkinson's disease, muscular dystrophy, myasthenia gravis, multiple sclerosis, Alzheimer's disease, stroke At least one selected from the group consisting of atherosclerosis, vascular restenosis, type I diabetes, type II diabetes, diabetic retinopathy, and chronic thyroiditis, but preferably may be rheumatoid arthritis.

In the present invention, the "prevention" means a reduction in the extent of the development of the pathological cells of the animal or damage or loss of cells. Prevention can be complete or partial. In this case, it may mean that the occurrence or abnormal immune action of pathological cells in the subject is reduced compared to the case where the composition for preventing and treating the autoimmune disease is not used.

In the present invention, the "treatment" refers to all actions that are clinically involved in altering the natural process of the subject or cell to be treated, and can be performed during or during clinical pathology. The desired therapeutic effect may prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, slow the progression of the disease, or Mitigating or temporarily alleviating the condition or improving the prognosis. In other words, the treatment may be interpreted as encompassing all behaviors in which the symptoms of the autoimmune disease are improved or cured by the composition.

In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical compositions of the present invention are not limited to these, but can be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc., and in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.

Examples of suitable carriers, excipients, and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , Sublingual or rectal. Oral or parenteral release is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention is dependent on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, extent of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg per day. / kg or 0.001-50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

According to another embodiment of the present invention, a mesenchymal stem cell-derived secretory protein (secretome), comprising a food composition for preventing or improving autoimmune diseases.

Detailed description of mesenchymal stem cells, secreted proteins and autoimmune diseases in the present invention is duplicated as described in the pharmaceutical composition, the following description is omitted to avoid excessive complexity of the description.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads and the like. Since the food composition of the present invention is composed of mesenchymal stem cell-derived secretory proteins with little toxicity and side effects, it can be used with confidence even for long-term use for prophylactic purposes.

When the secretion protein of the present invention or a concentrate of the polymer component comprising the same is included in the food composition, the amount may be added at a ratio of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in the form of a drink, there is no particular limitation other than the containing the food composition in the ratio indicated, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).

Other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.

These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

The composition according to the present invention increases the expression of IL-10, a representative immunomodulatory cytokine, and decreases the expression of inflammatory cytokines such as TNF-α, IL-6, IL-12 (p70), thereby reducing the expression of rheumatoid arthritis. It can effectively prevent or treat autoimmune diseases.

The effects of the present invention are not limited to the above-described effects, but should be understood to include all the effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of evaluating arthritis severity after treatment with MTX.

2 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the change in the foot thickness of the mouse after the MTX treatment.

3 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And after treatment with MTX, the mouse's foot picture and H & E stained picture of the foot tissue are shown.

4A to 4C are secreted proteins isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of quantitatively scoring the extent of inflammatory cell infiltration, synovial hyperplasia and bone erosion after MTX treatment.

FIG. 5 shows indocyanine green in tissues of mouse foot tissue after treatment with secreted protein and MTX isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention. Optical imaging photographs taken of fluorescence are shown.

6a to 6e are secreted proteins isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in the expression level of inflammation-related cytokines (TNF-α, IL-12 (p70), IL-6, IL-10, IL-1β) after treatment with MTX.

Figures 7a and 7b is a collagen-induced arthritis mouse model in one embodiment of the present invention TGF after treating the secreted protein derived from human adipose-derived mesenchymal stem cells and the secreted protein isolated and concentrated from the two-dimensional culture solution TGF The change in the expression level of -β1 and galectin-1 is shown graphically.

Figure 7c and 7d is a collagen-induced arthritis mouse model in the embodiment of the present invention treated with a three-dimensional rotational culture-derived secretion protein derived from human adipose-derived mesenchymal stem cells and three-dimensional stationary culture-derived secreted protein TGF-β1 and The graph shows the change in the expression level of galectin-1.

8A and 8B are secreted proteins isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention, respectively; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of analyzing the number of total splenocytes and the expression of CD4 + T cells in splenocytes after treatment with MTX.

9A and 9B are secreted proteins isolated and concentrated from two-dimensional cultures of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in CD4 ⁺ IFN-γ ⁺ T cells and CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells in the spleen after MTX treatment.

Figure 10a and Figure 10b is a secreted protein isolated and concentrated from the two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention, respectively; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in dendritic cells and intraperitoneal inflammatory (M1) and anti-inflammatory (M2) macrophages in the spleen after treatment with MTX.

11 is a biological process (biological process), the relationship between a total of 310 cytokines confirmed the expression in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional culture medium and three-dimensional rotation culture medium in one embodiment of the present invention, The results are analyzed by molecular function and cellular component.

12A, 12B and 12C show biological processes, molecular functions and cytokines of cytokines expressed in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional culture medium and three-dimensional spin culture medium in one embodiment of the present invention, respectively. The results show a summary of the functions that are highly relevant by cellular composition.

FIG. 13 shows cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotating cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures in one embodiment of the present invention; Cytokines are expressed as fold changes by the top 20.

14A to 14C are cytokines whose expression is increased or decreased in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures in one embodiment of the present invention. Among the cytokines having an effective meaning in the protein network (protein network) analysis and quantitative analysis results are shown.

According to one embodiment of the present invention, a mesenchymal stem cell-derived secretory protein (secretome), comprising a pharmaceutical composition for the prevention or treatment of autoimmune diseases.

According to another embodiment of the present invention, a mesenchymal stem cell-derived secretory protein (secretome), comprising a food composition for preventing or improving autoimmune diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Reagents and Chemical products

Dulbecco modified Eagle's medium-low glucose (DMEM), Fetal Bovine Serum (FBS), penicillin / streptomycin, 2-mercaptoethanol (× 1000), trypsin / EDTA (0.05%) and trypan blue (0.4%) It was purchased from Rosen.

From human fat cells Mesenchyme  Stem Cells Secured

Bone marrow-derived human mesenchymal stem cells were cultured at an early passage from the Yonsei University Cell Therapy Center that complies with the Korean Food and Drug Administration's standards (GMP). Medium stem cell culture medium (10% FBS, DMEM low glucose with 0.1 mM mercaptoethanol added) was added to a culture dish of clinically approved human mesenchymal stem cells of KFDA, and cultured for 72 to 86 hours. Medium was exchanged every 2-3 days, cell cultures were passaged with 70-85% confluency and the fifth passage was used for the study.

Mesenchyme  Stem Cell Culture

1. Two-dimensional Culture

The mesenchymal stem cells were cultured in 80% confluence, washed 4 times or more with PBS buffer to remove protein components such as fetal bovine serum, and then serum-free medium (DMEM-low glucose) excluding antibiotics and fetal bovine serum. After culturing for 48 hours, the culture was recovered.

2. Three-dimensional Spinner Rotational Culture

The mesenchymal stem cells were cultured at 80% confluence and then recovered by trypsin treatment, washed twice with PBS buffer to remove serum components, and then suspended in serum-free medium, followed by 60 spinner flasks. Incubated for 48 hours while rotating at rpm to recover the culture.

3. 3D Political Culture

In order to compare the effect with the three-dimensional spinner rotation culture method, the mesenchymal stem cells were cultured with 80% confluence, then trypsin-treated and recovered, washed twice with PBS buffer to remove serum components, and then serum-free medium. After the suspension was suspended in the culture for 48 hours, the culture solution was recovered.

Secreted from the culture Proteomic  Separation and Concentration

The mesenchymal stem cell culture cultured in large quantities by the two-dimensional and three-dimensional culture method was centrifuged at 1000 x g once to remove the cell residues first. Afterwards, diafiltration was performed using a tangential flow filtration (TFF) capsule (PALL, minimate TFF capsule), which secondly filters large particles such as cell debris with a 0.2 μM filter and filters molecules of 3 kDa or less. In the process of filtration with a diafiltration system, the culture solution was continuously concentrated at 4 ° C. with a dilution continuously with a water for injection (saline solution or Ringer injection) using a peristatic tubing pump. The concentration of the protein in the concentrated culture was confirmed by refractometer measurement and Bradford reagent, and stored at -80 ° C until the experiment.

However, to compare the concentration effect, the two-dimensional culture solution that did not undergo the above filtration and concentration process was also stored at −80 ° C. until the experiment (secretory protein derived from the two-dimensional culture solution).

Production of Collagen-Induced Arthritis Animal Model and Validation of Its Treatment Effect

Six to seven week old DBA / 1 mice were used to make collagen-induced arthritis animal models. The induction of arthritis was performed by mixing quantified bovine type II collagen with an equal amount of complete Freud's adjuvant, followed by intradermal injection of the tail starting portion of the mouse by 100 μg. immunization). Two weeks later, 50 μg of bovine-derived type II collagen and an incomplete Freud's adjuvant were mixed to further inject intradermal injection into the tail of the mouse (2nd boosting). In order to confirm the therapeutic effect, when the severity of arthritis was 6-8 points, the obtained secretory protein was started to be administered and treated for 5 weeks. Negative control (group not causing arthritis) and positive control group (untreated group without arthritis but not treated) three times a week were intraperitoneally injected with saline, and treated groups were methotrexate ( MTX, 35 mg / kg), secreted protein derived from two-dimensional culture (200 μg / mouse), secreted protein isolated and concentrated from two-dimensional culture (200 μg / mouse), secreted protein isolated and concentrated from three-dimensional rotary culture ( 200 μg / mouse) and secreted protein (200 μg / mouse) separated and concentrated from three-dimensional stationary culture were intraperitoneally injected and the severity of arthritis was measured.

The severity of arthritis was measured by visual observation of foot redness, swelling, and deformity twice a week, and the severity of arthritis at each time period was quantified according to the following scores. Indicated. In addition, the paw thickness was measured using a caliper twice a week, and the change of the mouse paw thickness according to each treatment is shown in FIG. 2.

0: is based on the negative control and is normal

1: Mild inflammation (redness, edema) in the center of the foot or toes, ankles, and knee joints

2: Severe arthritis in several areas

3: A condition in which severe inflammation involving the entire foot is observed

4: Inflammation results in stiffness or loss of joint movement

As shown in Figure 1, when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotational culture, arthritis severity was significantly reduced compared to the non-treated, as a treatment for arthritis The therapeutic effect was equivalent to that of a large amount of MTX used.

In addition, as shown in Figure 2, when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotational culture, the thickness of the arthritis-induced mouse foot was significantly reduced compared to the non-treated In addition, it showed the same level of therapeutic effect as a significant amount of MTX, which was previously used for treating arthritis.

Biopsy

After the articular tissue of each of the treated collagen-induced mouse arthritis models was fixed with formalin, it was fixed with paraffin by demineralization in 4% formic acid. After sectioning, hematoxylin-eosin (H & E) staining was performed. Stained tissue slides were analyzed for infiltration of inflammatory cells, hyperproliferation of synovial cells and cartilage destruction. Figure 3 shows the picture of the mouse's foot and the stained tissue according to each treatment. In addition, in the results of staining slides of mouse foot tissues according to each treatment, the degree of inflammatory cell infiltration, synovial hyperplasia and bone erosion was quantitatively scored. 4a to 4c.

As shown in FIG. 3, when treated with 2D and 3D rotating cultures, the secreted proteins (2D, 3D) separated and concentrated, the thickness of the feet of arthritis-induced mice was significantly reduced compared to the case without treatment. Equivalent to MTX, which was used as a treatment for arthritis,

As shown in Figures 4a to 4c, when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotary culture, infiltration of the inflammatory cells, synovial proliferation and bone compared to the non-treated The degree of erosion was remarkably decreased, and the treatment effect was equivalent to that of a large amount of MTX, which was previously used as an arthritis treatment agent.

optics Imaging (Optic imaging)

After 3 minutes after injecting 645 uM of indocyanine green (ICG) into the tail vein of the collagen-induced mouse arthritis model treated with the secreted protein isolated and concentrated in the two-dimensional culture, the eXplore Optix System ( Advanced Research Technologies Inc., Montreal, Canada) was used to measure the fluorescence remaining in the joint cavity, and the results are shown in FIG. 5.

As shown in FIG. 5, when treated with secreted secretory proteins isolated and concentrated in a two-dimensional culture, indocyanine green fluorescence was significantly reduced in mouse foot tissue compared with untreated secreted proteins. It can be seen that the fluorescence level was equivalent to that of MTX administration.

secretion Protein  Measurement of Inflammation-Related Cytokines After Administration

Inflammation-related cytokines (TNF-α, IL-12 (p70), IL-6, IL-10, by collecting serum from collagen-induced mouse arthritis model treated with two-dimensional culture medium and three-dimensional culture-derived secreted protein as described above) IL-1β) and galectin-1 were measured by ELISA, and the results are shown in FIGS. 6A to 6E and 7A to 7D.

As shown in FIGS. 6A to 6C and 6E, when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotation culture, TNF- inflammatory cytokine compared to the non-treated The expression levels of α, IL-12 (p70), IL-6 and IL-1β were significantly decreased, and the reduction effect was superior to that of MTX. In addition, as shown in FIG. 6D, IL-10, an anti-inflammatory cytokine, was treated with secreted proteins (2D and 3D) separated and concentrated in a two-dimensional culture and a three-dimensional rotary culture, and there was no treatment. It can be seen that the expression level is significantly increased compared with the case of MTX treatment.

In addition, as shown in FIGS. 7A and 7B, when the secreted protein (2D (enriched)) separated and concentrated from the two-dimensional culture solution is treated, the secreted protein derived from the two-dimensional culture solution (2D (non-concentrated)) is simply treated. It can be seen that the expression levels of anti-inflammatory cytokines TGF-β1 and galectin-1 were increased more than one case.

In addition, as shown in FIGS. 7C and 7D, when the secreted protein (3D (rotation)) separated and concentrated from the three-dimensional rotational culture solution is treated, the secreted protein (3D (politics) separated and concentrated from the three-dimensional stationary culture solution It was found that the expression levels of anti-inflammatory cytokines TGF-β1 and galectin-1 were increased more than those treated with)).

Through this, the two-dimensional culture of mesenchymal stem cells after the secretion protein obtained through the separation and concentration process according to the present invention can be seen that the anti-inflammatory effect is superior to the secreted protein obtained after the two-dimensional culture, and also the mesenchymal stem cells It can be seen that the secretory protein obtained by three-dimensional rotational culture is significantly superior to the anti-inflammatory effect than the secreted protein obtained by tertiary stationary culture.

Flow cytometry  (flow cytometry )

Splenocytes were isolated from lymphatic vessels and spleen of collagen-induced mouse arthritis model treated with 2D and 3D culture-derived secretory proteins as described above, and then, total flow was measured using a flow cytometer (FacsVerse, BD Pharmigen, USA). The number of splenocytes was measured, analyzed for the expression level of CD4, and the results were analyzed using FlowJo, and the results are shown in FIG. 8. In addition, after analyzing the distribution of Tregs cells by flow cytometry (FacsVerse, BD Pharmigen, USA) using CD4 + CD25 + FoxP3 +, a marker of regulatory T cells (Tregs), which play an important role in immune regulation in the spleen cells, FlowJo The results are analyzed using and shown in FIG. 9. In addition, the distribution of Th1 cells was analyzed using CD4 + IFN-γ +, and the maturation and activity of dendritic cells and intraperitoneal macrophage in the spleen that regulate innate immunity were analyzed by CD86, MCH, and CD206. After analyzing the expression level, the results are analyzed using FlowJo and shown in FIG. 10.

As shown in Figure 8a, when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotating culture, it can be seen that the number of spleen cells significantly reduced compared to the case without treatment, As shown in Figure 8b, it can be seen that the fraction of CD4 + T cells was reduced even when treated with MTX.

As shown in FIG. 9A, CD4 + IFN-γ + T cells showing Th1 cells when treated with secreted proteins (2D, 3D) isolated and concentrated in two-dimensional and three-dimensional rotary cultures were not treated. It can be seen that the fraction of was significantly reduced, as shown in Figure 9b, the fraction of CD4 + CD25 + FoxP3 + T cells showing regulatory T cells did not show a significant difference with each treatment.

As shown in Figure 10a, it can be seen that the expression level of CD86 and MHC, which is a marker of the activity of dendritic cells in the spleen, does not differ significantly between treatment groups. On the other hand, as shown in Figure 10b, in the case of the CD86, which is a marker of inflammatory macrophages (M1) intraperitoneal of the mouse, treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotating culture solution The expression level was significantly decreased compared with the case of no treatment or MTX treatment.In the case of CD206, an indicator of anti-inflammatory macrophage (M2), the secreted protein separated and concentrated in two-dimensional and three-dimensional rotary cultures It was found that the amount of expression increased significantly compared to the case of no treatment or the case of MTX treatment.

Cytokine Profiling by Antibody Assay Cytokine  profiling by antibody array)

To analyze the proteins produced in mesenchymal stem cell-derived secreted proteins, Fullmoon BioSystems Antibody Array (SCK100) was used. Specifically, 60 μl of the secreted protein separated and concentrated from the two-dimensional culture medium and the three-dimensional culture solution was adjusted to a total volume of 75 μl using a labeling buffer, and then cultured by biotin labeling. Blocking. Detection consisted of Cy-3 streptoavidin and the expression of 310 cytokines in the shaded state was measured.

FIG. 11 shows the relationship between a total of 310 cytokines expressed in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional cultures and three-dimensional rotational cultures. Biological process, molecular function And analysis results with cellular components.

12 shows the results of functional enrichment analysis of cytokines expressed in secretory proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional culture medium and three-dimensional rotary culture medium. More specifically, FIGS. 12A, 12B, and 12C are results of the 310 proteins identified using the DAVID program, which summarize functions related to biological processes, molecular functions, and cellular configurations.

In addition, Table 1 below shows 55 cytokines among 85 cytokines whose expression is increased in secreted proteins isolated and concentrated from three-dimensional rotational cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. .

List of increased cytokine	Fold change (3D secretome / 2D secretome)
Plasminogen activator inhibitor-1, PAI-1	3.615
Ferritin	2.160
Insulin-like growth factor-binding protein 7, IGF-BP7	1.898
Osteoprotegerin, OPG	1.644
CTGFL / WISP-2	1.636
Beta-2-Microglobulin	1.517
Macrophage colony-stimulating factor, M-CSF	1.497
Tissue inhibitors of metalloproteinases-1, TIMP-1	1.441
Galectin-1	1.408
C-Kit	1.329
Insulin-like growth factor-binding protein 3, IGF-BP3	1.288
Matrix metalloproteinases 2, MMP-2	1.272
Matrix metalloproteinases 10, MMP-10	1.200
Galectin-3	1.187
Catenin-alpha	1.139
Transforming growth factor-β2, TGF-β2	1.108
Catenin-gamma	1.098
S 100A10 / P11	1.089
Trefoil factor 2, TFF-2	1.086
soluble tumor necrosis factor-receptor II, sTNF-receptor II	1.078
Transforming growth factor beta Receptor Ⅲ, TGF-β Receptor Ⅲ	1.064
Transforming growth factor beta Receptor II, TGF-β Receptor II	1.062
Vascular endothelial growth factor receptor 1 (Flt-1)	1.056
Stem cell growth factor (SCGF) -beta	1.055
Vascular endothelial growth factor B (VEGFB)	1.053
Cancer Antigen 15-3 (CA15-3)	1.052
Serine / threonine protein kinase 2 (AKT2)	1.045
B-cell lymphoma / leukemia 10 (BCL-10)	1.044
Soluble Tumor necrosis factor-related apoptosis-inducing ligand (s-TRAIL) Receptor-2	1.043
Platelet-derived growth factor receptor (PDGFR) alpha	1.041
Vitamin K-dependent protein Z (PROZ)	1.038
Platelet-derived growth factor subunit (PDGFB)	1.037
Interleukin (IL) -1 beta	1.035
Tyrosine-protein kinase (LYN)	0.135
Myostatin (GDF-8)	1.034
B-cell receptor (Scd22)	1.032
CD14	1.031
IGF1 receptor (IGF 1R)	1.031
Tyrosine-protein kinase receptor (TYRO3)	1.029
Mucosae-associated epithelial chemokine (MEC)	1.027
Activated leukocyte cell adhesion molecule (ALCAM)	1.027
Vascular endothelial growth factor receptor 2 (KDR (VEGFR2))	1.025
Histone-lysine N-methyltransferase (MLL)	1.023
FGFR1 Oncogene Partner	1.020
Ovarian carcinoma antigen (CA125)	1.020
Androgen receptor	1.019
Tissue inhibitor of metalloproteinases 3 (TIMP3)	1.019
Stromal cell-derived factor (SDF) -1 alpha	1.018
Glycoprotein hormones alpha chain (alpha hCG)	1.018
sTRAIL / APO2L	1.018
Intercellular adhesion molecule (ICAM) -1	1.016
Visfatin	1.015
Monocyte chemoattractant protein (MCP) -2	1.015
Interleukin-13 receptor subunit alpha-1 (CA19-9)	1.015
Integrin alpha-5 (ITGA-5)	1.014

In addition, Table 2 shows 55 cytokines among 224 cytokines whose expression is reduced in secreted proteins isolated and concentrated from three-dimensional rotational cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. .

List of decreased cytokine	Fold change (3D secretome / 2D secretome)
Fibroblast growth factor 5 (FGF-5)	0.585
Follistatin	0.774
Fibroblast growth factor (FGF) -acidic	0.776
Interleukin (IL) -1RA	0.783
Adipolean Variant	0.792
4-1BB Receptor	0.802
sCD40 Ligand	0.812
Insulin-like growth factor (IGF) -I	0.815
Granulocyte-macrophage colony-stimulating factor (GM-CSF)	0.816
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF)	0.819
Granulocyte colony-stimulating factor (G-CSF)	0.822
Insulin-like growth factor (IGF) -II	0.822
Soluble receptor activator of NF-kappa B (sRANK Receptor)	0.822
Fibroblast growth factor (FGF) -basic	0.822
Adiponectin	0.824
Interleukin (IL) -6	0.826
Eotaxin	0.827
Interleukin (IL) -4	0.827
sFas Ligand / Apo1L	0.829
Epidermal growth factor receptor (EGFR)	0.830
Tumor necrosis factor (TNF) -beta	0.834
Vascular cell adhesion protein (VCAM) -1	0.838
Platelet-derived growth factor subunit B (PDGF-BB)	0.838
RANTES	0.839
Interleukin (IL) -7	0.839
Tumor necrosis factor ligand superfamily member 9 (4-1BBL)	0.840
Vascular endothelial growth factor (VEGF)	0.845
Activation-inducible TNF-related ligand (AITRIL)	0.849
Tumor necrosis factor (TNF) -alpha	0.850
Interleukin-2 receptor subunit (sIL-2R) alpha	0.850
A proliferation-inducing ligand (APRIL)	0.857
Fms-related tyrosine kinase 3 (Flt3) -Ligand	0.858
Exodus-2	0.863
sRANKL	0.864
B cell-attracting chemokine (BCA) -1	0.866
Eotaxin-2	0.868
Artemin	0.869
Beta-defensin 104 (BD-4)	0.870
C-X-C motif chemokine (CXCL) 16	0.871
Eotaxin-3	0.872
Fibroblast growth factor (FGF) -10	0.873
Beta-defensin 4A (BD-2)	0.874
Fibroblast growth factor (FGF) -16	0.875
Beta-defensin 103 (BD-3)	0.875
Beta-defensin 1 (BD-1)	0.877
B cell activating factor (BAFF)	0.878
Platelet-derived growth factor subunit A (PDGF-AA)	0.881
Connective tissue growth factor (CTGF)	0.883
Interleukin (IL) -8	0.883
Bone morphogenetic protein (BMP) -7 / OP-1	0.886
Endostatin	0.887
Fibroblast growth factor (FGF) -17	0.889
Ciliary neurotrophic factor (CNTF)	0.889
C-C motif chemokine 27 (CTACK)	0.890
Interleukin (IL) -10	0.893

In addition, FIG. 13 is different from the secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional culture, compared to the cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotational culture, and the reduced cytokines. The result is a fold change of 20 units. Cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotating cultures compared to secreted proteins isolated and concentrated in two-dimensional cultures include Plasminogen activator inhibitor-1 (PAI-1), Ferritin, Insulin-like growth factor-binding protein 7, IGF-BP7, Osteoprotegerin (OPG), CTGFL / WISP-2, Beta-2-micro Globulin (Beta-2-Microglobulin), Macrophage colony-stimulating factor (M-CSF), Tissue inhibitors of metalloproteinases-1 (TIMP-1), galectin -1 (Galectin-1), C-Kit, Insulin-like growth factor-binding protein 3 (IGF-BP3), Matrix metalloproteinases 2, MMP- 2), MMP-10, galectin-3, catenin-alpha, TG F-β2, Catnin-gamma, S 100A10 / P11, TFF-2, sTNF-receptor II. On the other hand, cytokines with reduced expression in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in two-dimensional cultures had fibroblast growth factor 5 (FGF-5) and follistatin. (Follistatin), fibroblast growth factor (FGF) -acidic, Interleukin (IL) -1RA, Adipolean Variant, 4-1BB receptor (4-1BB Receptor), sCD40 Ligand, IGF-I, GM-CSF, EG-VEGF, G-CSF, IGF-II, sRANK Receptor, FGF-basic, Adiponectin, IL-6, Eotaxin, IL-4, sFas Ligand / Apo1L, EGFR.

14A to 14C show cytokines having an effective meaning among cytokines whose expression is increased or decreased in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures Protein network analysis and quantitative analysis results. (A) is a signaling network of cytokines with an increased expression of at least 1.25-fold in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. Schematic diagram showing. Figure 16 (b) is a schematic diagram showing a signal network of cytokines reduced expression less than 0.8-fold in the secreted protein isolated and concentrated from the three-dimensional rotational culture compared to the secreted protein isolated and concentrated in the mesenchymal stem cell-derived two-dimensional culture . Figure 16 (c) is a cytokine that is increased by 1.25 times or more, or 0.8 or less times reduced expression in secreted proteins isolated and concentrated from the three-dimensional rotation culture medium compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional culture The result of quantitative analysis of Cain Cytokines with a fold increase of 1.25-fold or more in three-dimensional rotational culture-derived secreted proteins compared to two-dimensional cultured-derived secreted proteins include plasminogen activator inhibitor-1 (PAI-1) and ferritin. Insulin-like growth factor-binding protein 7, IGF-BP7, Osteoprotegerin (OPG), CTGFL / WISP-2, Beta-2-microglobulin (Beta- 2-Microglobulin), Macrophage colony-stimulating factor (M-CSF), Tissue inhibitors of metalloproteinases-1 (TIMP-1), and galectin-1 (Galectin) -1), C-Kit, (insulin-like growth factor-binding protein 3, IGF-BP3) and matrix metalloproteinases 2 (MMP-2) Three-dimensional rotational culture compared to secreted proteins derived from two-dimensional culture Cytokines with reduced fold values of 0.8-fold or less in derived secreted proteins include fibroblast growth factor 5 (FGF-5), follistatin, and fibroblast growth factor (FGF)- acidic), Interleukin (IL) -1RA) and Adipolean Variant.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.

The present invention uses mesenchymal stem cell-derived secretory proteins, rheumatoid arthritis, inflammatory spondyloarthritis, adult type S. disease, macrophage activity syndrome, systemic sclerosis, multiple myositis, dermatitis, vasculitis, mixed connective diseases, Sjogren's syndrome, Ulcerative colitis, Crohn's disease, allergic asthma, allergic rhinitis, atopic dermatitis, gallbladder, lacrimitis, conjunctivitis, psoriasis, vulgaris ulcer, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, Alzheimer's disease, stroke, The present invention relates to a composition for preventing, ameliorating or treating various autoimmune diseases such as atherosclerosis, vascular restenosis, type I diabetes, type II diabetes, diabetic retinopathy, and chronic thyroiditis.

Claims (22)

1. A pharmaceutical composition for preventing or treating autoimmune diseases, including mesenchymal stem cell-derived secretory proteins.
2. The method of claim 1,

   The secreted protein is isolated from the culture solution obtained by culturing the mesenchymal stem cells, pharmaceutical composition.
3. The method of claim 2,

   The culturing of the mesenchymal stem cells is by two-dimensional culture or three-dimensional culture, pharmaceutical composition.
4. The method of claim 3,

   The two-dimensional culture of the mesenchymal stem cells is carried out by culturing the mesenchymal stem cells in mesenchymal stem cell culture medium for 24 to 96 hours and then incubated in serum-free medium for 24 to 72 hours. .
5. The method of claim 4, wherein

   The mesenchymal stem cell culture medium is Dulbecco's modified Eagle's medium containing 5 to 15% by weight of fetal bovine serum (FBS) and 0.05 to 0.2 mM mercaptoethanol. , DMEM), RPMI-1640 medium, StemPro medium, MSCGro medium, MesenCult medium and NutriStem medium, any one selected from the group consisting of.
6. The method of claim 3,

   The secreted protein is a pharmaceutical composition obtained by recovering the supernatant by centrifuging the culture medium obtained by culturing the mesenchymal stem cells in two or three dimensions at 500 to 1,500 xg.
7. The method of claim 6,

   The concentrate is filtered by the supernatant with a 0.1 to 0.3 μM filter; And filtering the molecules up to 3 kDa.
8. The method of claim 7, wherein

   The filtration of the molecules below 3 kDa is carried out by diafiltration using a tangential flow filtration (TFF) device.
9. The method of claim 6,

   Wherein the concentrate is obtained by reacting the recovered supernatant with an alcohol polar solvent.
10. The method of claim 9,

    The reaction of the supernatant and the polar polar solvent is carried out for 5 to 500 minutes at -30 to 0 ℃, pharmaceutical composition.
11. The method of claim 9,

    The alcohol polar solvent is mixed in an amount of 2 to 5 times by weight of the supernatant, pharmaceutical composition.
12. The method of claim 6,

    Wherein said concentrate is lyophilized.
13. The method of claim 3,

    The three-dimensional culture of the mesenchymal stem cells is performed by culturing the mesenchymal stem cells in mesenchymal stem cell culture medium for 24 to 96 hours and then floating in serum-free medium and culturing for 24 to 72 hours. Pharmaceutical composition.
14. The method of claim 13,

    A pharmaceutical composition for treating recombinant trypsin without trypsin or animal-derived components prior to suspending and culturing the mesenchymal stem cells in serum-free medium in the three-dimensional culture.
15. The method of claim 13,

    The serum-free medium is phenol red (Dulbecco's modified Eagle's medium, DMEM) excluding antibiotics, pharmaceutical composition.
16. The method of claim 13,

    When the three-dimensional culture is carried out by rotating at a rotational speed of 30 to 90 rpm using a spinner flask, pharmaceutical composition.
17. The method of claim 1,

    The autoimmune diseases include rheumatoid arthritis, inflammatory spondyloarthritis, adult Stil disease, macrophage activation syndrome, systemic sclerosis, multiple myositis, dermatitis, vasculitis, mixed connective diseases, Sjogren's syndrome, ulcerative colitis, Crohn's disease, allergic disease Asthma, allergic rhinitis, atopic dermatitis, gallbladder, lacrimitis, conjunctivitis, psoriasis, vulgaris swelling, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I A pharmaceutical composition, selected from the group consisting of diabetes mellitus, type II diabetes, diabetic retinopathy and chronic thyroiditis.
18. The method of claim 17,

    The autoimmune disease is rheumatoid arthritis, pharmaceutical composition.
19. Mesenchymal stem cell-derived secretory proteins (secretome), comprising a food composition for the prevention or improvement of autoimmune diseases.
20. The method of claim 19,

    The secreted protein is a food composition that is isolated from the culture obtained by culturing the mesenchymal stem cells.
21. The method of claim 19,

    The culture of the mesenchymal stem cells are by two-dimensional culture or three-dimensional culture, food composition.
22. A method for preventing or treating autoimmune diseases, comprising administering the pharmaceutical composition of any one of claims 1 to 18 to a subject in need thereof for preventing or treating autoimmune diseases.

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