WO2019190218A1 - Composition, comprising tonsil-derived mesenchymal stem cell-controlling medium, for prevention or treatment of hepatic disease - Google Patents
Composition, comprising tonsil-derived mesenchymal stem cell-controlling medium, for prevention or treatment of hepatic disease Download PDFInfo
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- WO2019190218A1 WO2019190218A1 PCT/KR2019/003626 KR2019003626W WO2019190218A1 WO 2019190218 A1 WO2019190218 A1 WO 2019190218A1 KR 2019003626 W KR2019003626 W KR 2019003626W WO 2019190218 A1 WO2019190218 A1 WO 2019190218A1
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- Prior art keywords
- liver disease
- mesenchymal stem
- tonsil
- stem cells
- derived mesenchymal
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
Definitions
- the present invention relates to a composition for the prevention, improvement or treatment of liver disease, comprising as an active ingredient the medium of tonsil-derived mesenchymal stem cells.
- the present invention relates to a food composition for preventing or improving liver disease, including an adjusting medium of tonsil derived mesenchymal stem cells.
- the liver plays various roles in metabolism of lipids, detoxification, bile excretion, storage of various nutrients, hematopoietic and blood coagulation, and regulation of circulating blood volume, and is one of the essential organs for life support.
- the function of the liver is to manage energy metabolism, so that all the nutrients absorbed from food are metabolized into substances that can produce energy in the liver and are supplied or stored throughout the body.
- the liver has the function of synthesizing, storing, and distributing about 2,000 kinds of enzymes, albumin, and serum proteins of coagulation factors, bile acids, phospholipids, and cholesterol.
- the function of detoxifying drugs, alcohol, toxic substances, etc. the function of excretion of various metabolites into the duodenum, and the immune function plays an important role in maintaining life.
- hepatitis which causes inflammation of the liver, accounts for the majority of liver diseases, and according to aspects, it can be divided into acute hepatitis and chronic infection, depending on the cause, viral hepatitis, alcoholic hepatitis, and drug hepatitis.
- liver diseases caused by such abnormalities include fatty liver, hepatitis, cirrhosis, liver cancer, and the like.
- the mechanism of the progression of liver disease is not fully understood, but it is known that the development of advanced liver disease such as fatty liver and cirrhosis is accompanied by secondary cell damage after primary fatty liver development.
- liver disease since liver disease is not detected in the early stage of the disease and is found to be progressed considerably, it is at the top of the cause of death both domestically and globally.
- Stem cells are cells that can differentiate into various cells constituting biological tissues, which collectively refer to undifferentiated cells of differentiation stages that can be obtained from embryonic, fetal and adult tissues.
- stem cell cultures have an advantageous advantage to be developed as a therapeutic agent, and related research and development is being conducted.
- mesenchymal stem cells In order to proceed with the stem cell-related research as described above, smooth supply and demand of stem cells is required.
- some stem cells are difficult to use because they have a great limitation in obtaining cells.
- mesenchymal stem cells derived from cord blood and adipose tissue must be obtained using invasive methods.
- the most non-invasive method of obtaining cells is mesenchymal stem cells through bone marrow harvesting, but bone marrow harvesting requires anesthesia and causes pain, which limits its use.
- a cell acquisition method using peripheral blood is required to separate patient-specific stem cells, but the peripheral blood alone is too small to separate mesenchymal stem cells from adults, and the separation method is not economical.
- the growth is not as smooth as the amount that can be used for cell therapy, so there is a need for an alternative to increase the practicality.
- the adult stem cells obtained from the elderly patients can be naturally separated from the low-age patients because they are significantly lowered in cell capacity and the secretion of various factors and the ability of stem cells to move compared to cells obtained from the low age. Need to get cells from tissue.
- the cells thus obtained can be easily secured in an quantitative manner for experiments, and it is necessary to maintain the differentiation capacity during passage of cells.
- stem cells differ in their properties.
- the present invention provides a pharmaceutical composition for preventing or treating liver disease, which comprises as an active ingredient the mediating medium of tonsil derived mesenchymal stem cells.
- the present invention provides a food composition for preventing or improving liver disease, including an adjusting medium of tonsil derived mesenchymal stem cells.
- the present invention provides a method for treating liver disease comprising administering a modulating medium of tonsil derived mesenchymal stem cells to a subject in need thereof.
- the present invention provides the use of a media for the adjustment of tonsil-derived mesenchymal stem cells in the manufacture of a medicament for the treatment of liver disease.
- the present invention provides a composition comprising a control medium of tonsil derived mesenchymal stem cells for use in the treatment of liver disease.
- the media of the tonsil-derived mesenchymal stem cells may be any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin.
- the present invention was completed by confirming that it is effective in the treatment of liver disease by having a high content of the medium in the control medium and having a different composition of the control medium and other profiles of other derived mesenchymal stem cells.
- the amygdala-derived mesenchymal stem cells are tissues which are located at the inside of the neck and the back of the nose to primarily protect the body from substances invading from the outside and act as lymphatic epithelial immune tissues. It refers to an undifferentiated stem cell having the ability to differentiate into two or more new cells while having a self-replicating ability derived from tonsils.
- tonsil-derived mesenchymal stem cell control medium means a culture medium or supernatant from which tonsil-derived mesenchymal stem cells are removed from a culture medium obtained by culturing tonsil-derived mesenchymal stem cells, Mesenchymal stem cell culture supernatant "," tonsil derived mesenchymal stem cell condition culture solution “or” tonsil derived mesenchymal stem cell culture medium ".
- prevention refers to any action that inhibits or delays the onset of liver disease by administration of a media for the adjustment of tonsil derived mesenchymal stem cells according to the present invention.
- treatment refers to any action that improves or advantageously changes the symptoms of the disease by administration of a control medium of tonsil derived mesenchymal stem cells according to the present invention.
- Immprovement in the present invention means any action that improves the bad state of liver disease by administering or ingesting the composition of the present invention to a subject.
- liver disease refers to a problem with one or more of the various functions performed by the liver, which means that normal metabolism cannot be performed.
- the most common hepatitis is hepatitis, which is divided into acute hepatitis and chronic hepatitis. In general, acute hepatitis is easy to treat and is benign. Acute hepatitis includes viral hepatitis, alcoholic hepatitis, and addictive hepatitis.
- Liver disease in the present invention can be included without limitation as long as it can be treated using a modulator medium of tonsil derived mesenchymal stem cells, for example, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, liver ischemia, alcoholic liver disease, hepatic At least one selected from the group consisting of lethargy, liver atrophy and liver cancer.
- the control medium of the tonsil derived mesenchymal stem cells was used for the inhibition and treatment of liver disease.
- the liver disease is cirrhosis.
- the present invention provides a pharmaceutical composition for preventing or treating liver disease, which comprises as an active ingredient the mediating medium of tonsil derived mesenchymal stem cells.
- the control medium of tonsil-derived mesenchymal stem cells has a high content of any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin, as compared to the control medium of other derived mesenchymal stem cells.
- the media of the tonsil-derived mesenchymal stem cells according to the present invention may be at least one level selected from the group consisting of IL-1Ra, proMMP-1, and preprostromelysin, for example, to exhibit a therapeutic effect.
- IL-1Ra IL-1Ra
- proMMP-1 proMMP-1
- preprostromelysin for example
- control medium of tonsil-derived mesenchymal stem cells is easy to acquire tonsils, which are tissues of origin, compared to other stem-derived mesenchymal stem cells, and can be recycled after initial operation. Since the yield is very high, a control medium to be injected can be easily obtained, and the amount of injectable can be freely controlled in comparison with other stem cell control medium, which has a big advantage.
- the control medium of tonsil-derived mesenchymal stem cells includes a control medium prepared by separating, culturing and special manipulation from an individual, and is used as a medicine used for the purpose of treatment, diagnosis and prevention, and for liver disease. Can be used.
- Liver disease in the present invention is as mentioned above.
- tonsil-derived mesenchymal stem cells are extractable from tonsils according to methods known in the art. For example, it can be extracted from the tonsil according to the method described in Korean Patent No. 10-1508413.
- the amygdala-derived mesenchymal stem cell culture may be obtained by culturing tonsil-derived mesenchymal stem cells and removing the cells, and then obtained the culture solution, the culture supernatant, or a concentrate thereof, or a freeze-dried product thereof.
- the amygdala-derived mesenchymal stem cell culture may be obtained by passage to the amygdala-derived mesenchymal stem cells in serum medium, followed by culturing in serum-free medium.
- Tonsil-derived mesenchymal stem cells can be conventionally cultured using a stem cell culture medium. Tonsil-derived mesenchymal stem cell cultures are obtained by subcultured tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum medium and then passaged in serum-free medium, and are used as is or filtered using centrifugation or a filter. The supernatant obtained after removing the stem cells and macromolecules can be used. In addition, the supernatant obtained can be used as it is or as a concentrate obtained by concentration.
- the amygdala-derived mesenchymal stem cell culture may be a control medium obtained by passage of the amygdala-derived mesenchymal stem cells continuously in a serum-free medium without adding a serum.
- the culture in the serum-free medium it may be used as it is, or the supernatant obtained after removing the stem cells and macromolecules by centrifugation or filtration using a filter can be used.
- the supernatant obtained can be used as it is or as a concentrate obtained by concentration.
- the culture medium used to obtain the control medium according to the present invention is, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, alanine, aminoacetic acid, proline, serine, N-acetyl Amino acids such as -L-cysteine and the like; Na +, K +, Mg 2+ , Cl -, acetate -, maleate - electrolytes, such as and the like; And vitamins such as retinol palmitate, ergocalciferol, tocopherol acetate, ascorbic acid, thiamine chloride, sodium riboflavin phosphate, nicotinic acid, pyridoxine chloride, dexpanthenol and the like.
- the serum medium is a medium suitable for maintaining and storing the same cell type as tonsil-derived mesenchymal stem cells, and may be DMEM medium supplemented with serum.
- the serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight relative to the total weight of the serum medium.
- FBS fetal bovine serum
- antibiotics, antifungal agents, mycoplasma inhibitors, and the like may be included, which may be 1% by weight relative to the total weight of the medium.
- Antibiotics include antibiotics commonly used in cell cultures such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include gentamicin, ciprofloxacin, tyrosine, etc. It is not limited to this.
- culturing in serum-free medium may be performed after removing the culture solution in the serum medium and washing the cells with phosphate buffer.
- the serum medium may be DMEM medium supplemented with 5-12% fetal bovine serum and 1% antibiotic.
- the serum-free medium may be DMEM medium containing no serum.
- the amygdala-derived mesenchymal stem cell culture is chopped tonsil tissue isolated from the amygdala, the tonsil tissue is attached to the bottom of the culture plate, 10% fetal bovine serum (optional) and 1% Culturing in a 37 ° C. and 5% CO 2 incubator in DMEM medium supplemented with penicillin-streptomycin, separating and passaging the cells when the density of the cells reaches 80% saturation After culturing the cells for 3 to 4 days, washing the cells with phosphate buffer, culturing the washed cells in a DMEM medium containing no serum and antibiotics, and incubating the cells at 37 ° C. and 5% CO 2. Can be obtained.
- the amygdala-derived mesenchymal stem cell culture may be obtained by centrifuging or filtering the amygdala-derived mesenchymal stem cell culture to remove the cells.
- compositions of the present invention may be formulated in a variety of formulations, including solutions, suspensions, emulsions, lotions, ointments, lyophilizers, etc., in accordance with conventional methods.
- the pharmaceutical composition of the present invention may be formulated and administered in a unit dosage form of a pharmaceutical formulation suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, wherein the formulation is effectively administered by one or several administrations. Contains amount. Suitable formulations for this purpose are parenteral, injectables, injectables and the like.
- the pharmaceutical composition for preventing or treating liver disease may include a pharmaceutically acceptable conventional inert carrier and diluent.
- Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical compositions of the invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, and the like. Binders such as, alginate, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, polysorbates, cetyl alcohol, and glycerol Active agents include, but are not limited to.
- the pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the modulating medium of tonsil-derived mesenchymal stem cells and the beneficiaries to be administered it.
- Diluents include, but are not limited to, saline, aqueous buffers, solvents and / or dispersion media.
- a preservative, a painless agent, a solubilizer, or a stabilizer may be further included, and in the case of a topical administration agent, a base, an excipient, a lubricant, or a preservative may be further included.
- the compositions of the present invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) can be added to the cell population before freezing.
- standard cryopreservatives eg DMSO, glycerol, Epilife cell freezing medium (Cascade Bi
- the administration may be both a non-surgical administration using a catheter and surgical administration methods such as injection after dissection of the disease site.
- the dosage may vary depending on the concentration, but may be administered 10 ⁇ l / kg to 1 ml / kg body weight once or divided into several times.
- the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It does not limit the scope of the present invention in terms of aspects.
- the present invention also provides a food composition for preventing or ameliorating liver disease, including an adjusting medium of tonsil-derived mesenchymal stem cells.
- the present invention can be generally used as a commonly used food.
- the food composition of the present invention can be used as a dietary supplement.
- health functional food means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the law on health functional foods, and “functionality” refers to the structure and function of the human body. It means the ingestion for the purpose of obtaining a useful effect for health use such as nutrient control or physiological action.
- the food composition of the present invention may include a conventional food additive, and the suitability as the "food additive" is applicable according to the General Regulations and General Test Law of the Food Additives approved by the Ministry of Food and Drug Safety unless otherwise specified. Judge according to the standards and standards for the item.
- Food Additive Revolution examples include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar pigment preparation, are mentioned.
- the food composition of the present invention may comprise 0.01 to 95% by weight, preferably 5 to 90% by weight of the media of the tonsil-derived mesenchymal stem cells based on the total weight of the composition.
- the food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and / or improving liver disease.
- hard capsules of the health functional food in the form of capsules can be prepared by filling a mixture with additives such as control medium, and excipients of tonsil-derived mesenchymal stem cells according to the present invention in a conventional hard capsules
- Soft capsules may be prepared by filling a mixture with an additive such as a food composition and an excipient according to the present invention in a capsule base such as gelatin.
- the soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
- excipients binders, disintegrants, glidants, copulation agents, flavoring agents, etc. are described in the literature known in the art and include those having the same or similar functions (Korean Pharmacopoeia, Munseongsa, Korea Pharmacy University Council, 5th edition, p33-48, 1989).
- the present invention provides a method for treating liver disease comprising administering a modulating medium of tonsil derived mesenchymal stem cells to a subject in need thereof.
- the subject refers to an animal, and may typically be a mammal that may exhibit a beneficial effect with treatment with a modulating medium of tonsil derived mesenchymal stem cells of the invention.
- Preferred examples of such subjects may include primates, such as humans.
- such subjects may include all subjects with or at risk of having symptoms of liver disease.
- the present invention also provides the use of a media for the adjustment of tonsil derived mesenchymal stem cells in the manufacture of a medicament for the treatment of liver disease.
- the present invention also provides a composition comprising a control medium of tonsil derived mesenchymal stem cells for use in the treatment of liver disease.
- the present invention also provides the use of a modulated medium of tonsil derived mesenchymal stem cells for the treatment of liver disease.
- the pharmaceutical composition for preventing or treating liver disease of the present invention has a different profile of immunomodulatory factors and hepatic fibrosis inhibitors in the media, compared to other media for the regulation of other mesenchymal stem cells, thereby preventing and treating liver diseases. Has an excellent effect.
- Figure 1 shows the schedule of the test for confirming the therapeutic effect of the control medium (T-MSC CM) of tonsil-derived mesenchymal stem cells in liver disease model.
- Figure 2 shows the results confirmed by H & E staining and Sirius staining the recovery effect of the control medium treatment of amygdala-derived mesenchymal stem cells in CCl 4 pretreatment damaged liver tissue.
- Figure 3 shows the results confirmed the recovery of fibrosis according to the media treatment of tonsil-derived mesenchymal stem cells in liver tissue damaged by CCl 4 pretreatment.
- Figure 4 shows the expression of collagen type 1 alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) according to control medium treatment of tonsil-derived mesenchymal stem cells in CCl 4 pretreated liver tissue. The result of confirming the change is shown.
- Tonsil-derived mesenchymal stem cells were extracted from patients undergoing tonsillectomy in Otorhinolaryngology-Head and Neck Surgery at Lee Dae Mok Dong Hospital Passed by ethics committee review: ECT 11-53-02. It was prepared as Korean Patent No. 10-1508413, tonsil-derived mesenchymal stem cells (7-8 passages) were cultured under DMEM supplemented with 10% FBS, 1% penicillin / streptomycin.
- amygdala-derived mesenchymal stem cells were cultured to 80% confluency, after which they were exchanged with fresh culture media without FBS and antibiotics. The cells were incubated an additional 48 hours and the conditioned medium was recovered. The conditioned medium was concentrated 20-fold by centrifugation at 5000 g for 60 minutes using an Amicon Ultra centrifugal filter (3 kDa cut-off).
- Liver cirrhosis was induced using carbon tetrachloride (CCl 4 ), and the experimental schedule is shown in FIG. 1.
- mice Seven-week-old C57BL / 6 mice were randomly divided into untreated and carbon tetrachloride treated groups.
- the single dose of carbon tetrachloride was 10 ⁇ l / g, and the carbon tetrachloride was diluted to 10% in mineral oil and administered intraperitoneally twice a week for 3 weeks.
- the experiments and procedures were performed with the approval of the Ewha Womens University Animal Ethics Committee, and all experiments were performed under relevant remedies and guidelines.
- liver tissue paraffin sections were prepared and stained with hematoxylin and eosin (H & E). Fibrosis of liver tissue was confirmed by Sirius Red staining, and the collagen index was calculated by quantifying the degree of staining of collagen fibers by pixel densities per unit area.
- liver tissue damaged by CCl 4 pretreatment was recovered by tonsilocyte-mediated media (T-CM) treatment.
- T-CM tonsilocyte-mediated media
- liver fibrosis was confirmed by administration of toxin-mediated mesenchymal stem cells (T-CM) (*** p ⁇ 0.001).
- liver mRNAs were extracted and synthesized into cDNA, followed by real-time PCR. Expression of collagen type 1, alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) was confirmed.
- the sequence of the used primer is as follows.
- the gene expression analysis revealed that the expression of increased cirrhosis marker genes (Col1a1, Tgfb1, Timp1) in the CCl4 pretreatment group was statistically significantly reduced by the control medium of tonsil-derived mesenchymal stem cells. (* P ⁇ 0.05, ** p ⁇ 0.01).
- control medium of tonsil-derived mesenchymal stem cells according to the present invention has an excellent effect on the prevention and treatment of liver disease.
Abstract
The present invention relates to a composition comprising a tonsil-derived mesenchymal stem cell-controlling medium as an effective ingredient for prevention, alleviation, or treatment of hepatic diseases. The composition for prevention, alleviation, or treatment of hepatic diseases of the present invention exhibits different profiles for immunoregulatory elements and hepatic fibrosis inhibiting elements in the controlling medium in contrast with controlling media of mesenchymal stem cells derived from other tissues and, as a result, has excellent effects on the prevention, alleviation, and treatment of hepatic diseases.
Description
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 유효성분으로 포함하는 간질환 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prevention, improvement or treatment of liver disease, comprising as an active ingredient the medium of tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for preventing or improving liver disease, including an adjusting medium of tonsil derived mesenchymal stem cells.
간은 우리 몸에서 지질 등의 대사 작용, 해독, 담즙의 배설, 각종 영양소의 저장, 조혈이나 혈액 응고, 및 순환 혈액량의 조절 등 다양한 역할을 담당하고 있으며, 생명 유지를 위한 필수적인 장기 중 하나이다. 간의 기능을 보다 구체적으로 살펴보면, 우선, 에너지 대사를 관리하는 기능이 있어 음식물에서 흡수된 모든 영양소들이 간에서 에너지를 생산할 수 있는 물질로 대사되어 전신에 공급되거나 저장된다. 둘째, 간은 약 2,000 여종의 효소, 알부민, 응고 인자들의 혈청 단백질, 담즙산, 인지질, 콜레스테롤 등의 지방 등을 합성하고 저장하며 분배하는 기능이 있다. 셋째, 해독 및 분해 기능으로서 약물, 술, 독성물질 등을 해독시키는 기능, 각종 대사산물을 십이지장으로 배설하는 기능 및 면역 기능이 있어 생명 유지에 중요한 역할을 하고 있다.The liver plays various roles in metabolism of lipids, detoxification, bile excretion, storage of various nutrients, hematopoietic and blood coagulation, and regulation of circulating blood volume, and is one of the essential organs for life support. In more detail, the function of the liver is to manage energy metabolism, so that all the nutrients absorbed from food are metabolized into substances that can produce energy in the liver and are supplied or stored throughout the body. Second, the liver has the function of synthesizing, storing, and distributing about 2,000 kinds of enzymes, albumin, and serum proteins of coagulation factors, bile acids, phospholipids, and cholesterol. Third, as a detoxification and decomposition function, the function of detoxifying drugs, alcohol, toxic substances, etc., the function of excretion of various metabolites into the duodenum, and the immune function plays an important role in maintaining life.
일반적으로 간에 염증이 생기는 간염이 간질환의 대부분을 차지하며, 양상에 따라 급성 간염과 만성 감염, 원인에 따라 바이러스성 간염, 알코올성 간염, 약물성 간염 등으로 나눌 수 있다. 또한, 이런 이상으로 유발되는 간질환에는 지방간, 간염, 간경변증, 간암 등이 있다. 간질환의 진행 과정의 기전은 완전히 밝혀지지는 않았으나 일차적으로 지방간이 발생된 후 이차적인 세포 손상이 수반되어 지방간 및 간경화 등의 진행성 간질환으로 발전하는 것으로 알려져 있다. 또한, 간질환은 초기에 자각 증상이 없어 상당히 진행되어서야 발견되기 때문에, 국내뿐만 아니라 세계적으로 사망 원인의 수위를 차지하고 있다.In general, hepatitis, which causes inflammation of the liver, accounts for the majority of liver diseases, and according to aspects, it can be divided into acute hepatitis and chronic infection, depending on the cause, viral hepatitis, alcoholic hepatitis, and drug hepatitis. In addition, liver diseases caused by such abnormalities include fatty liver, hepatitis, cirrhosis, liver cancer, and the like. The mechanism of the progression of liver disease is not fully understood, but it is known that the development of advanced liver disease such as fatty liver and cirrhosis is accompanied by secondary cell damage after primary fatty liver development. In addition, since liver disease is not detected in the early stage of the disease and is found to be progressed considerably, it is at the top of the cause of death both domestically and globally.
한편, 줄기세포(stem cell)는 생물 조직을 구성하는 다양한 세포들로 분화할 수 있는 세포로서 배아, 태아 및 성체의 각 조직에서 얻을 수 있는 분화되기 전 단계의 미분화 세포들을 총칭한다. Stem cells, on the other hand, are cells that can differentiate into various cells constituting biological tissues, which collectively refer to undifferentiated cells of differentiation stages that can be obtained from embryonic, fetal and adult tissues.
치료 방법으로서 최근 줄기세포 및 조직공학을 이용한 이식치료법이 개발되고 있다. 하지만, 이러한 이식 치료 기법으로는 치료효과가 뛰어나고 인체의 면역반응을 일으키지 않는 안정한 세포 치료제를 개발하기 어렵다는 문제가 있다. 그 동안 여러 가지 줄기세포를 이용하여 세포 치료제로써 활용하기 위한 시도들이 진행되고 있지만, 면역 조절 능력에 대한 확인은 아직 부분적으로만 진행되고 있다. 또한, 최근에는 줄기세포를 직접 손상된 조직에 이식하는 방법 이외에 줄기세포의 배양액에 포함된 매개성 물질의 탁월한 측분비 효과가 생체내 또는 생체외 실험에서 입증되고 있다. 줄기세포는 인체 내에서 일생 동안 세포의 성장과 재생에 관련된 다양한 성장인자와 사이토카인 등을 분비하고, 줄기세포 배양액에는 이러한 단백질 성분들이 함유되어 있으며, 세포이식 시의 부작용인 면역거부반응을 회피할 수 있으므로, 줄기세포 배양액은 치료제로 개발하기에 유리한 장점을 가지기 때문에 관련 연구개발이 진행되고 있다. Recently, transplantation therapy using stem cells and tissue engineering has been developed as a treatment method. However, there is a problem that it is difficult to develop a stable cell therapy which is excellent in the therapeutic effect and does not cause an immune response of the human body by such transplantation treatment technique. Attempts have been made to utilize various stem cells as cell therapeutics, but the identification of immune regulation is only partially progressed. Recently, in addition to the method of directly transplanting stem cells into damaged tissues, excellent side secretion effects of mediators contained in the culture of stem cells have been demonstrated in vivo or ex vivo. Stem cells secrete various growth factors and cytokines related to cell growth and regeneration in the human body throughout life, and stem cell cultures contain these protein components and avoid immune rejection, a side effect of cell transplantation. As such, stem cell cultures have an advantageous advantage to be developed as a therapeutic agent, and related research and development is being conducted.
상기와 같은 줄기세포 관련 연구를 진행하기 위해서는 줄기세포의 원활한 수급이 필요하다. 그러나, 중간엽 줄기세포 중에서도 몇몇 줄기세포는 그 이용에 있어서 세포를 얻는데 큰 제한이 있기 때문에 그 이용이 어렵다. 예를 들어, 제대혈, 지방조직으로부터 유래한 중간엽 줄기세포는 침습적인 방법을 이용해 얻어야만 한다. 가장 비침습적인 방법을 이용해 얻을 수 있는 세포는 골수채취를 통한 중간엽 줄기세포이지만, 골수채취는 마취가 필요하고 고통을 유발하여, 그 이용에 제한이 있다. 이에 대한 대안으로 환자 맞춤형 줄기세포를 분리하기 위해 말초혈액을 이용한 세포획득법 등이 요구되고 있으나 말초혈액만으로는 성인에서 분리할 수 있는 중간엽 줄기세포의 수가 너무 적고 분리방법이 경제적이지 못하며, 분리해낸다고 해도 세포치료에 사용가능한 양만큼 증식이 원활하지 않은 경우가 대부분이기에 좀 더 실용성을 높일 수 있는 대체 방안이 필요하다. 또한, 고령인 환자에서 얻는 성체 줄기세포는 낮은 연령에게서 얻는 세포들에 비해 세포능이 현저히 떨어지며 각종 인자들의 분비 및 줄기세포의 이동 능력 등이 떨어지기 때문에, 저연령의 환자로부터 자연스럽게 분리될 수 있거나 버려지는 조직으로부터 세포를 얻을 필요성이 있다. 또한, 이렇게 얻어진 세포는 실험에 용이한 양적 확보가 가능하고 세포의 계대 배양 시에 분화능이 잘 유지될 필요성이 있다. In order to proceed with the stem cell-related research as described above, smooth supply and demand of stem cells is required. However, among the mesenchymal stem cells, some stem cells are difficult to use because they have a great limitation in obtaining cells. For example, mesenchymal stem cells derived from cord blood and adipose tissue must be obtained using invasive methods. The most non-invasive method of obtaining cells is mesenchymal stem cells through bone marrow harvesting, but bone marrow harvesting requires anesthesia and causes pain, which limits its use. As an alternative, a cell acquisition method using peripheral blood is required to separate patient-specific stem cells, but the peripheral blood alone is too small to separate mesenchymal stem cells from adults, and the separation method is not economical. Even if it does, the growth is not as smooth as the amount that can be used for cell therapy, so there is a need for an alternative to increase the practicality. In addition, the adult stem cells obtained from the elderly patients can be naturally separated from the low-age patients because they are significantly lowered in cell capacity and the secretion of various factors and the ability of stem cells to move compared to cells obtained from the low age. Need to get cells from tissue. In addition, the cells thus obtained can be easily secured in an quantitative manner for experiments, and it is necessary to maintain the differentiation capacity during passage of cells.
또한, 줄기세포들은 그 유래에 따라 그 특성이 상이하다. In addition, stem cells differ in their properties.
따라서 특정 질환에 대한 치료를 위해 질환에 따라 치료에 적합한 특성을 가지는 줄기세포를 선택하고 이의 특성을 이용하여 특정 질환에 대한 치료 방법을 개발할 필요가 있다.Therefore, it is necessary to select a stem cell having a characteristic suitable for treatment according to the disease for the treatment of a specific disease and to develop a treatment method for the specific disease using the characteristics thereof.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 유효 성분으로 포함하는 간질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver disease, which comprises as an active ingredient the mediating medium of tonsil derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or improving liver disease, including an adjusting medium of tonsil derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 치료학적 유효량으로 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 간질환의 치료방법을 제공한다.The present invention provides a method for treating liver disease comprising administering a modulating medium of tonsil derived mesenchymal stem cells to a subject in need thereof.
본 발명은 간질환의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다. The present invention provides the use of a media for the adjustment of tonsil-derived mesenchymal stem cells in the manufacture of a medicament for the treatment of liver disease.
본 발명은 간질환의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물을 제공한다.The present invention provides a composition comprising a control medium of tonsil derived mesenchymal stem cells for use in the treatment of liver disease.
본 발명자들은 간질환을 예방 또는 치료할 수 있는 적합한 종류의 줄기세포를 확인하던 중, 편도 유래 중간엽 줄기세포의 조정배지가 IL-1Ra, proMMP-1 및 preprostromelysin로 이루어진 군으로부터 선택되는 어느 하나 이상을 높은 함량으로 조정 배지 내 함유하고 타 유래 중간엽 줄기세포의 조정배지와 다른 프로파일의 함량으로 그 조성을 달리 가짐으로써 간질환의 치료에 효과적임을 확인하여 본 발명을 완성하였다. While the present inventors have identified suitable types of stem cells capable of preventing or treating liver disease, the media of the tonsil-derived mesenchymal stem cells may be any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin. The present invention was completed by confirming that it is effective in the treatment of liver disease by having a high content of the medium in the control medium and having a different composition of the control medium and other profiles of other derived mesenchymal stem cells.
본 발명에 있어서, 편도 유래 중간엽 줄기세포란 목의 안쪽과 코의 뒷부분에 위치하여 외부에서 침입하는 세균 등의 물질로부터 일차적으로 우리 몸을 방어함과 동시에 림프상피 면역조직으로 작용을 수행하는 조직인 편도에서 유래된 자기 복제 능력을 가지면서 두 개 이상의 새로운 세포로 분화하는 능력을 가진 미분화된 줄기세포를 의미한다. In the present invention, the amygdala-derived mesenchymal stem cells are tissues which are located at the inside of the neck and the back of the nose to primarily protect the body from substances invading from the outside and act as lymphatic epithelial immune tissues. It refers to an undifferentiated stem cell having the ability to differentiate into two or more new cells while having a self-replicating ability derived from tonsils.
본 발명에 있어서, "편도 유래 중간엽 줄기세포 조정 배지"는 편도 유래 중간엽 줄기세포를 배양하여 수득된 배양액으로부터 편도 유래 중간엽 줄기세포가 제거된 배양액 또는 상층액을 의미하며, "편도 유래 중간엽 줄기세포 배양 상층액", "편도 유래 중간엽 줄기세포 조건 배양액" 또는 "편도 유래 중간엽 줄기세포 배양 배지"와 호환적으로 사용될 수 있다.In the present invention, "tonsil-derived mesenchymal stem cell control medium" means a culture medium or supernatant from which tonsil-derived mesenchymal stem cells are removed from a culture medium obtained by culturing tonsil-derived mesenchymal stem cells, Mesenchymal stem cell culture supernatant "," tonsil derived mesenchymal stem cell condition culture solution "or" tonsil derived mesenchymal stem cell culture medium ".
본 발명에서 용어 “예방”이란 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지의 투여로 간 질환의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지의 투여로 상기 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명에서 "개선"이란 본 발명의 조성물을 개체에 투여하거나 섭취시켜 간질환의 나쁜 상태를 좋게 하는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of liver disease by administration of a media for the adjustment of tonsil derived mesenchymal stem cells according to the present invention. In the present invention, the term "treatment" refers to any action that improves or advantageously changes the symptoms of the disease by administration of a control medium of tonsil derived mesenchymal stem cells according to the present invention. "Improvement" in the present invention means any action that improves the bad state of liver disease by administering or ingesting the composition of the present invention to a subject.
본 발명에서 용어 “간 질환”이란 간이 수행하는 여러 가지 기능 중 한 가지 이상의 기능에 문제가 생겨 정상적으로 대사를 수행할 수 없는 것을 말한다. 간 질환에서 가장 많이 나타나는 것은 간염으로 급성 간염과 만성 간염으로 나뉘며, 일반적으로는 급성 간염이 치료하기 쉽고 양성에 속한다. 급성 간염은 원인 별로 바이러스성 간염, 알코올성 간염, 중독성 간염 등이 있다. 본 발명에서 간 질환은 편도 유래 중간엽 줄기세포의 조정 배지를 이용하여 치료될 수 있는 것이라면 제한 없이 포함될 수 있으나, 그 예로 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 본 발명에서는 편도 유래 중간엽 줄기세포의 조정 배지를 사용하여 간질환의 억제 및 치료효과를 확인하였다. 바람직하게 상기 간질환은 간경변이다. In the present invention, the term "liver disease" refers to a problem with one or more of the various functions performed by the liver, which means that normal metabolism cannot be performed. The most common hepatitis is hepatitis, which is divided into acute hepatitis and chronic hepatitis. In general, acute hepatitis is easy to treat and is benign. Acute hepatitis includes viral hepatitis, alcoholic hepatitis, and addictive hepatitis. Liver disease in the present invention can be included without limitation as long as it can be treated using a modulator medium of tonsil derived mesenchymal stem cells, for example, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, liver ischemia, alcoholic liver disease, hepatic At least one selected from the group consisting of lethargy, liver atrophy and liver cancer. In the present invention, the control medium of the tonsil derived mesenchymal stem cells was used for the inhibition and treatment of liver disease. Preferably the liver disease is cirrhosis.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 유효 성분으로 포함하는 간질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver disease, which comprises as an active ingredient the mediating medium of tonsil derived mesenchymal stem cells.
본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지는 타 유래 중간엽 줄기세포의 조정 배지와 대비하여 IL-1Ra, proMMP-1 및 preprostromelysin로 이루어진 군으로부터 선택되는 어느 하나 이상의 함량이 높으며, 조정 배지 내 면역 조절 인자, 간섬유화 억제 인자들의 상이한 프로파일 등을 가짐으로써 간질환의 예방 및 치료에 우수한 효과를 가진다. 특히 간의 섬유화 억제에 우수한 효과를 나타낸다.The control medium of tonsil-derived mesenchymal stem cells according to the present invention has a high content of any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin, as compared to the control medium of other derived mesenchymal stem cells. By having different profiles of immune regulation factors, hepatic fibrosis inhibitors, etc., it has an excellent effect on the prevention and treatment of liver disease. In particular, it shows an excellent effect on inhibiting liver fibrosis.
즉, 본원 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지는 IL-1Ra, proMMP-1 및 preprostromelysin로 이루어진 군으로부터 선택되는 어느 하나 이상을 일정 수준 이상, 예를 들어 치료학적으로 효과를 나타낼 수 있는 일정 수준 이상으로, 함유함으로써 간질환의 예방 및 치료에 우수한 효과를 가진다. That is, the media of the tonsil-derived mesenchymal stem cells according to the present invention may be at least one level selected from the group consisting of IL-1Ra, proMMP-1, and preprostromelysin, for example, to exhibit a therapeutic effect. By more than a certain level, by containing it has an excellent effect in the prevention and treatment of liver disease.
또한, 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지는 편도 유래 중간엽 줄기세포가 타 유래 줄기세포와 대비하여 기원 조직인 편도의 획득이 용이하며 수술 후 버려지는 편도 조직의 재활용이 가능하고 초기 수득율이 매우 높다는 점에서 주입해야 하는 조정 배지를 용이하게 얻을 수 있고 주입 가능한 양을 타유래 줄기세포 조정배지와 대비하여 자유롭게 조절할 수 있어 큰 장점을 가진다.In addition, the control medium of tonsil-derived mesenchymal stem cells according to the present invention is easy to acquire tonsils, which are tissues of origin, compared to other stem-derived mesenchymal stem cells, and can be recycled after initial operation. Since the yield is very high, a control medium to be injected can be easily obtained, and the amount of injectable can be freely controlled in comparison with other stem cell control medium, which has a big advantage.
본 발명에 있어서, 편도 유래 중간엽 줄기세포의 조정배지는 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 조정 배지를 포함하며, 치료, 진단 및 예방의 목적으로 사용되는 의약품으로 사용되어 간질환에 사용될 수 있다. In the present invention, the control medium of tonsil-derived mesenchymal stem cells includes a control medium prepared by separating, culturing and special manipulation from an individual, and is used as a medicine used for the purpose of treatment, diagnosis and prevention, and for liver disease. Can be used.
본 발명에 있어서 간질환은 상기 언급된 바와 같다. Liver disease in the present invention is as mentioned above.
본 발명에 있어서, 편도 유래 중간엽 줄기세포는 당업계에 알려진 방법에 따라 편도로부터 추출 가능하다. 예컨대, 대한민국특허 제10-1508413호 등에 기재된 방법에 따라 편도로부터 추출가능하다. In the present invention, tonsil-derived mesenchymal stem cells are extractable from tonsils according to methods known in the art. For example, it can be extracted from the tonsil according to the method described in Korean Patent No. 10-1508413.
편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기세포를 배양하고 세포를 제거한 후, 수득된 배양액, 배양 상층액 또는 이의 농축물이거나 이의 동결건조물일 수 있다.The amygdala-derived mesenchymal stem cell culture may be obtained by culturing tonsil-derived mesenchymal stem cells and removing the cells, and then obtained the culture solution, the culture supernatant, or a concentrate thereof, or a freeze-dried product thereof.
본 발명에 있어서, 편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기 세포를 혈청 배지에서 계대 배양한 후, 무혈청 배지에서 배양하여 수득될 수 있다.In the present invention, the amygdala-derived mesenchymal stem cell culture may be obtained by passage to the amygdala-derived mesenchymal stem cells in serum medium, followed by culturing in serum-free medium.
편도 유래 중간엽 줄기세포는 줄기세포 배양용 배지를 사용하여 통상적으로 배양될 수 있다. 편도 유래 중간엽 줄기세포 배양액은 편도 조직으로부터 수득된 편도 유래 중간엽 줄기세포를 혈청 배지에서 계대배양한 후, 무혈청 배지에서 계대 배양하여 수득되고, 이를 그대로 사용하거나 또는 원심분리나 필터를 이용한 여과에 의해 줄기세포 및 거대분자를 제거한 후에 수득된 상층액을 사용할 수 있다. 또한, 수득된 상층액은 그대로 사용하거나 또는 농축하여 수득된 농축물로 사용할 수 있다.Tonsil-derived mesenchymal stem cells can be conventionally cultured using a stem cell culture medium. Tonsil-derived mesenchymal stem cell cultures are obtained by subcultured tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum medium and then passaged in serum-free medium, and are used as is or filtered using centrifugation or a filter. The supernatant obtained after removing the stem cells and macromolecules can be used. In addition, the supernatant obtained can be used as it is or as a concentrate obtained by concentration.
본 발명에 있어서, 편도 유래 중간엽 줄기세포 배양액은 혈청이 첨가되는 단계가 없이 편도 유래 중간엽 줄기 세포를 지속적으로 무혈청 배지에서 계대 배양하면서 수득되는 조정 배지일 수 있다. In the present invention, the amygdala-derived mesenchymal stem cell culture may be a control medium obtained by passage of the amygdala-derived mesenchymal stem cells continuously in a serum-free medium without adding a serum.
위 무혈청 배지에서의 배양에 따라 이를 그대로 사용하거나 또는 원심분리나 필터를 이용한 여과에 의해 줄기세포 및 거대분자를 제거한 후에 수득된 상층액을 사용할 수 있다. 또한, 수득된 상층액은 그대로 사용하거나 또는 농축하여 수득된 농축물로 사용할 수 있다.Depending on the culture in the serum-free medium, it may be used as it is, or the supernatant obtained after removing the stem cells and macromolecules by centrifugation or filtration using a filter can be used. In addition, the supernatant obtained can be used as it is or as a concentrate obtained by concentration.
편도 유래 중간엽 줄기세포의 배양을 위한 배양용 배지 및 배양 조건은 본 발명이 속하는 기술분야에서 잘 알려져 있으며, 통상의 지식을 가진 자가 적절하게 선택하거나 변형하여 이용할 수 있다.Culture medium and culture conditions for the culture of tonsil-derived mesenchymal stem cells are well known in the art to which the present invention pertains, and those skilled in the art can appropriately select or modify them.
상기 본 발명에 따른 조정 배지를 얻기 위해 사용되는 배양 배지는 예를 들어 이소류신, 류신, 리신, 메티오닌, 페닐알라닌, 트레오닌, 트립토판, 발린, 아르기닌, 히스티딘, 알라닌, 아미노아세트산, 프롤린, 세린, N-아세틸-L-시스테인 등과 같은 아미노산류; Na
+, K
+, Mg
2+, Cl
-, 아세테이트
-, 말레이트
- 등과 같은 전해질류; 및 레티놀 팔미테이트, 에르고칼시페롤, 토코페롤 아세테이트, 아스코브산, 염화티아민,인산리보플라빈나트륨, 니코틴산, 염화피리독신, 덱스판테놀 등과 같은 비타민류 등을 포함할 수 있다.The culture medium used to obtain the control medium according to the present invention is, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, alanine, aminoacetic acid, proline, serine, N-acetyl Amino acids such as -L-cysteine and the like; Na +, K +, Mg 2+ , Cl -, acetate -, maleate - electrolytes, such as and the like; And vitamins such as retinol palmitate, ergocalciferol, tocopherol acetate, ascorbic acid, thiamine chloride, sodium riboflavin phosphate, nicotinic acid, pyridoxine chloride, dexpanthenol and the like.
상기 혈청 배지는 편도 유래 중간엽 줄기세포와 동일한 세포형을 유지하고 보관하는데 적합한 배지로서, 혈청이 보충된 DMEM 배지일 수 있다. 혈청은 우태아혈청(FBS)일 수 있으나, 이에 한정되지 않으며, 혈청 배지의 총 중량에 대해 1 내지 10 중량%일 수 있다. 필요에 따라, 항생제, 항진균제 및 마이코플라스마 억제제 등을 포함할 수 있으며, 이들은 배지의 총 중량에 대해 1 중량%일 수 있다. 항생제는 페니실린-스트렙토마이신 등 세포 배양에서 통상적으로 사용되는 항생제를 포함하고, 항진균제는 암포테리신-B(fungizone) 등을 포함하며, 마이코플라스마 억제제는 겐타마이신, 시프로플록사신, 타일로신 등을 포함하나, 이에 한정되지 않는다.The serum medium is a medium suitable for maintaining and storing the same cell type as tonsil-derived mesenchymal stem cells, and may be DMEM medium supplemented with serum. The serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight relative to the total weight of the serum medium. If desired, antibiotics, antifungal agents, mycoplasma inhibitors, and the like may be included, which may be 1% by weight relative to the total weight of the medium. Antibiotics include antibiotics commonly used in cell cultures such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include gentamicin, ciprofloxacin, tyrosine, etc. It is not limited to this.
상기 혈청을 포함하는 배지에서 배양 후, 무혈청 배지 중에서 배양은 혈청 배지에서의 배양액을 제거하고, 세포를 인산염 완충용액으로 세척한 후에 수행될 수 있다.After culturing in the serum-containing medium, culturing in serum-free medium may be performed after removing the culture solution in the serum medium and washing the cells with phosphate buffer.
바람직하게, 상기 혈청 배지는 5 내지 12% 우태아혈청 및 1%의 항생제가 보충된 DMEM 배지일 수 있다.Preferably, the serum medium may be DMEM medium supplemented with 5-12% fetal bovine serum and 1% antibiotic.
바람직하게, 상기 무혈청 배지는 혈청을 포함하지 않는 DMEM 배지일 수 있다.Preferably, the serum-free medium may be DMEM medium containing no serum.
본 발명의 일 실시양태에 따르면, 편도 유래 중간엽 줄기세포 배양액은 편도로부터 단리된 편도 조직을 잘게 자른 후, 편도 조직을 배양 플레이트의 바닥에 부착시키고, 10% 우태혈청(선택적으로) 및 1% 페니실린-스트렙토마이신이 보충된 DMEM 배지 중에서 37℃ 및 5% CO
2 상태의 배양기에서 배양하는 단계, 세포의 밀도가 80%의 포화상태 (confluence)에 도달했을 때, 세포를 분리하고 계대 배양시키는 단계, 세포를 3 내지 4일간 배양한 후, 인산염 완충용액으로 세척하는 단계, 세척된 세포를 혈청 및 항생제를 포함하지 않는 DMEM 배지에서, 37℃ 및 5% CO
2 상태의 배양기에서 배양하는 단계에 의해 수득될 수 있다.According to one embodiment of the invention, the amygdala-derived mesenchymal stem cell culture is chopped tonsil tissue isolated from the amygdala, the tonsil tissue is attached to the bottom of the culture plate, 10% fetal bovine serum (optional) and 1% Culturing in a 37 ° C. and 5% CO 2 incubator in DMEM medium supplemented with penicillin-streptomycin, separating and passaging the cells when the density of the cells reaches 80% saturation After culturing the cells for 3 to 4 days, washing the cells with phosphate buffer, culturing the washed cells in a DMEM medium containing no serum and antibiotics, and incubating the cells at 37 ° C. and 5% CO 2. Can be obtained.
본 발명의 일실시양태에 따르면, 편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기세포 배양액을 원심분리나 여과시켜 세포를 제거하는 단계에 의해 수득될 수 있다.According to one embodiment of the present invention, the amygdala-derived mesenchymal stem cell culture may be obtained by centrifuging or filtering the amygdala-derived mesenchymal stem cell culture to remove the cells.
본 발명의 약학 조성물은 통상의 방법에 따라 액제, 현탁액, 에멀젼, 로션, 연고, 동결건조제 등 다양한 제형으로 제제화될 수 있다. The pharmaceutical compositions of the present invention may be formulated in a variety of formulations, including solutions, suspensions, emulsions, lotions, ointments, lyophilizers, etc., in accordance with conventional methods.
본 발명의 약학 조성물은 약학적 분야의 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 약학적 제제로 제형화시켜 투여할 수 있으며, 상기 제제는 1회 또는 수회 투여에 의해 효과적인 투여량을 포함한다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제, 주입제 등이 바람직하다. 또한, 상기 간질환 예방 또는 치료용 약학 조성물은 약학적으로 허용가능한 통상의 불활성 담체 및 희석제를 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 약학적으로 허용가능한 담체 및 희석제는 전분, 당, 및 만니톨과 같은 부형제, 칼슘 포스페이트 등과 같은 충전제 및 증량제, 카르복시메틸셀룰로오스, 히드록시프로필셀룰로오스 등과 같은 셀룰로오스 유도체, 젤라틴, 알긴산염, 및 폴리비닐 피롤리돈 등과 같은 결합제, 활석, 스테아린산 칼슘, 수소화 피마자유 및 폴리에틸렌 글리콜과 같은 윤활제, 포비돈, 크로스포비돈과 같은 붕해제, 폴리소르베이트, 세틸알코올, 및 글리세롤 등과 같은 계면활성제를 포함하나, 이에 한정되지 않는다. 상기 약학적으로 허용되는 담체 및 희석제는 편도 유래 중간엽 줄기세포의 조정 배지 및 이를 투여받을 수혜자에 대해 생물학적 및 생리학적으로 친화적인 것일 수 있다. 희석제로는 이에 한정되지 않으나, 염수, 수용성 완충액, 용매 및/또는 분산제(dispersion media)를 들 수 있다. 이외에도, 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소투여용 제제의 경우에는 기제(base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다. 본 발명의 조성물은 동결되지 않은 채 사용되거나 차후 사용을 위해 동결될 수 있다. 동결되어야 할 경우, 표준 냉동보존제 (예를 들어 DMSO, 글리세롤, 에피라이프 (Epilife) 세포 동결 배지 (Cascade Biologics))가 동결 전 세포 집단에 첨가될 수 있다.The pharmaceutical composition of the present invention may be formulated and administered in a unit dosage form of a pharmaceutical formulation suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, wherein the formulation is effectively administered by one or several administrations. Contains amount. Suitable formulations for this purpose are parenteral, injectables, injectables and the like. In addition, the pharmaceutical composition for preventing or treating liver disease may include a pharmaceutically acceptable conventional inert carrier and diluent. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical compositions of the invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, and the like. Binders such as, alginate, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, polysorbates, cetyl alcohol, and glycerol Active agents include, but are not limited to. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the modulating medium of tonsil-derived mesenchymal stem cells and the beneficiaries to be administered it. Diluents include, but are not limited to, saline, aqueous buffers, solvents and / or dispersion media. In addition, for example, in the case of an injection, a preservative, a painless agent, a solubilizer, or a stabilizer may be further included, and in the case of a topical administration agent, a base, an excipient, a lubricant, or a preservative may be further included. The compositions of the present invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) can be added to the cell population before freezing.
또한, 당업계에서 통상적으로 사용하는 투여방법을 이용하여 투여될 수 있으며, 바람직하게는 치료가 필요한 환자의 질환 부위에 직접 투여가 가능하나 이에 한정되지는 않는다. 또한, 상기 투여는 카테터를 이용한 비외과적 투여 및 질환부위 절개 후 주입 등 외과적 투여방법 모두 가능하다. 투여량은 농축 정도에 따라 그 투여량이 달라질 수 있으나 10 μl/kg 내지 1 ml/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. In addition, it may be administered using a conventionally used method of administration in the art, and preferably, it is possible to directly administer to a disease part of a patient in need of treatment, but is not limited thereto. In addition, the administration may be both a non-surgical administration using a catheter and surgical administration methods such as injection after dissection of the disease site. The dosage may vary depending on the concentration, but may be administered 10 μl / kg to 1 ml / kg body weight once or divided into several times.
그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It does not limit the scope of the present invention in terms of aspects.
본 발명은 또한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for preventing or ameliorating liver disease, including an adjusting medium of tonsil-derived mesenchymal stem cells.
본 발명은 통상적으로 이용되는 식품으로써 일반적으로 사용될 수 있다. The present invention can be generally used as a commonly used food.
본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능식품"이라 함은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present invention can be used as a dietary supplement. The term "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the law on health functional foods, and "functionality" refers to the structure and function of the human body. It means the ingestion for the purpose of obtaining a useful effect for health use such as nutrient control or physiological action.
본 발명의 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 "식품 첨가물"로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. The food composition of the present invention may include a conventional food additive, and the suitability as the "food additive" is applicable according to the General Regulations and General Test Law of the Food Additives approved by the Ministry of Food and Drug Safety unless otherwise specified. Judge according to the standards and standards for the item.
상기 "식품 첨가물 공전"에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Examples of the items listed in the "Food Additive Revolution" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar pigment preparation, are mentioned.
본 발명의 식품 조성물은 조성물 총 중량에 대하여 편도 유래 중간엽 줄기세포의 조정배지를 0.01 내지 95 중량%, 바람직하게는 5 내지 90 중량%로 포함할 수 있다. The food composition of the present invention may comprise 0.01 to 95% by weight, preferably 5 to 90% by weight of the media of the tonsil-derived mesenchymal stem cells based on the total weight of the composition.
또한, 본 발명의 식품 조성물은 간질환의 예방 및/또는 개선을 목적으로, 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.In addition, the food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and / or improving liver disease.
예를 들어, 캡슐 형태의 건강기능식품 중 경질캡슐제는 통상의 경질캡슐에 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지, 및 부형제 등의 첨가제와의 혼합물을 충진하여 제조할 수 있으며, 연질캡슐제는 본 발명에 따른 식품 조성물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캡슐기제에 충진하여 제조할 수 있다. 상기 연질캡슐제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.For example, hard capsules of the health functional food in the form of capsules can be prepared by filling a mixture with additives such as control medium, and excipients of tonsil-derived mesenchymal stem cells according to the present invention in a conventional hard capsules, Soft capsules may be prepared by filling a mixture with an additive such as a food composition and an excipient according to the present invention in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 (대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989).The term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. are described in the literature known in the art and include those having the same or similar functions (Korean Pharmacopoeia, Munseongsa, Korea Pharmacy University Council, 5th edition, p33-48, 1989).
상기 식품의 종류에는 특별한 제한이 없으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the type of food, and includes all of the health functional foods in the ordinary sense.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 치료학적 유효량으로 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 간질환의 치료방법을 제공한다.The present invention provides a method for treating liver disease comprising administering a modulating medium of tonsil derived mesenchymal stem cells to a subject in need thereof.
본 발명에 "편도 유래 중간엽 줄기세포의 조정 배지", "간질환", "투여" 등의 용어는 상기에서 설명한 바와 동일하다.In the present invention, terms such as "modulating medium of tonsil-derived mesenchymal stem cells", "liver disease", and "administration" are the same as described above.
상기 대상체는 동물을 말하며, 전형적으로 본 발명의 편도 유래 중간엽 줄기세포의 조정 배지를 이용한 치료로 유익한 효과를 나타낼 수 있는 포유동물일 수 있다. 이러한 대상체의 바람직한 예로 인간과 같은 영장류가 포함될 수 있다. 또한 이와 같은 대상체들에는 간질환의 증상을 갖거나 이와 같은 증상을 가질 위험이 있는 대상체들이 모두 포함될 수 있다.The subject refers to an animal, and may typically be a mammal that may exhibit a beneficial effect with treatment with a modulating medium of tonsil derived mesenchymal stem cells of the invention. Preferred examples of such subjects may include primates, such as humans. In addition, such subjects may include all subjects with or at risk of having symptoms of liver disease.
본 발명은 또한 간질환의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다. The present invention also provides the use of a media for the adjustment of tonsil derived mesenchymal stem cells in the manufacture of a medicament for the treatment of liver disease.
본 발명은 또한 간질환의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물을 제공한다. The present invention also provides a composition comprising a control medium of tonsil derived mesenchymal stem cells for use in the treatment of liver disease.
본 발명은 또한 간질환의 치료를 위한 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다. The present invention also provides the use of a modulated medium of tonsil derived mesenchymal stem cells for the treatment of liver disease.
본 발명의 간질환 예방 또는 치료용 약학 조성물은 타유래 중간엽 줄기세포의 조정 배지와 대비하여, 조정 배지 내 면역 조절 인자, 간섬유화 억제 인자들의 상이한 프로파일을 가지고 이로 인하여 간질환의 예방 및 치료에 우수한 효과를 가진다. The pharmaceutical composition for preventing or treating liver disease of the present invention has a different profile of immunomodulatory factors and hepatic fibrosis inhibitors in the media, compared to other media for the regulation of other mesenchymal stem cells, thereby preventing and treating liver diseases. Has an excellent effect.
도 1은 간질환 모델에서 편도 유래 중간엽 줄기세포의 조정 배지(T-MSC CM)의 치료 효과 확인 실험의 스케쥴을 나타낸다. Figure 1 shows the schedule of the test for confirming the therapeutic effect of the control medium (T-MSC CM) of tonsil-derived mesenchymal stem cells in liver disease model.
도 2는 CCl
4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 회복 효과를 H&E 염색 및 Sirius 염색을 통해 확인한 결과를 나타낸다. Figure 2 shows the results confirmed by H & E staining and Sirius staining the recovery effect of the control medium treatment of amygdala-derived mesenchymal stem cells in CCl 4 pretreatment damaged liver tissue.
도 3은 CCl
4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 섬유화 회복 효과를 확인한 결과를 나타낸다. Figure 3 shows the results confirmed the recovery of fibrosis according to the media treatment of tonsil-derived mesenchymal stem cells in liver tissue damaged by CCl 4 pretreatment.
도 4는 CCl
4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 collagen type 1 alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1)과 metallopeptidase inhibitor 1 (Timp1)의 발현 변화를 확인한 결과를 나타낸다. Figure 4 shows the expression of collagen type 1 alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) according to control medium treatment of tonsil-derived mesenchymal stem cells in CCl 4 pretreated liver tissue. The result of confirming the change is shown.
본 발명의 이해를 돕기 위하여 실시예, 제조예를 제시한다. 하기의 실시예, 제조예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예, 제조예에 의해 본 발명의 내용이 한정되는 것은 아니다.Examples and preparation examples are provided to aid the understanding of the present invention. The following examples and preparations are merely provided to more easily understand the present invention, but the contents of the present invention are not limited by the examples and preparations.
<실시예 1> 편도 유래 중간엽 줄기세포의 조정 배지의 제조Example 1 Preparation of Modulating Media of Tonsil-Derived Mesenchymal Stem Cells
편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell, T-MSC) 는 이대목동병원 이비인후-두경부외과에서 편도적출술을 시행하는 환자로부터 적출된 편도조직(4-20세의 저연령층 조직, 임상윤리위원회 심의 통과: ECT 11-53-02)으로부터 얻었다. 이는 대한민국 등록 특허 제10-1508413호와 같이 제조되었으며, 편도 유래 중간엽 줄기세포 (7~8 passage)는 10% FBS, 1% 페니실린/스트렙토마이신이 보충된 DMEM 하에 배양되었다. Tonsil-derived mesenchymal stem cells (T-MSCs) were extracted from patients undergoing tonsillectomy in Otorhinolaryngology-Head and Neck Surgery at Lee Dae Mok Dong Hospital Passed by ethics committee review: ECT 11-53-02. It was prepared as Korean Patent No. 10-1508413, tonsil-derived mesenchymal stem cells (7-8 passages) were cultured under DMEM supplemented with 10% FBS, 1% penicillin / streptomycin.
조정 배지를 제조하기 위해 편도 유래 중간엽 줄기세포는 80 % confluency까지 배양되었고, 이에 도달한 후에 FBS와 항생제가 포함되지 않은 신선한 배양 배지로 교환되었다. 세포는 48 시간 추가로 배양되었고 조정 배지가 회수되었다. 조정 배지는 Amicon Ultra centrifugal filter(3 kDa cut-off)를 이용하여 60분 간 5000g로 원심분리에 의해 20배 농축되었다. To prepare conditioned media, amygdala-derived mesenchymal stem cells were cultured to 80% confluency, after which they were exchanged with fresh culture media without FBS and antibiotics. The cells were incubated an additional 48 hours and the conditioned medium was recovered. The conditioned medium was concentrated 20-fold by centrifugation at 5000 g for 60 minutes using an Amicon Ultra centrifugal filter (3 kDa cut-off).
<실시예 2> CCl4 간질환 모델에서 편도 유래 중간엽 줄기세포의 조정배지 투여에 의한 치료 효과 확인 Example 2 Confirmation of Therapeutic Effect by Modified Medium Administration of Amygdala-derived Mesenchymal Stem Cells in CCl4 Liver Disease Model
간경화는 사염화탄소(carbon tetrachloride, CCl
4)를 이용하여 유도하였으며, 실험 스케쥴은 도 1에 나타내었다. Liver cirrhosis was induced using carbon tetrachloride (CCl 4 ), and the experimental schedule is shown in FIG. 1.
7주령의 C57BL/6 마우스를 무작위로 미처리군과 사염화탄소 처리군으로 나누었다. 사염화탄소의 1회 투여양은 10 μl/g으로 사염화탄소를 미네랄오일에 10% 농도로 희석하여 일주일에 2차례 3주간에 걸쳐 복강내 투여하였다. 전처치를 마친 실험동물을 3그룹으로 나누어 사염화탄소 투여 직후 희생 (CCl
4+No treatment, n=3), 1x10
6개의 편도줄기세포를 48시간 동안 DMEM에 배양하여 수득한 조정배지를 20배 농축하여 꼬리정맥을 통해 2회 투여(CCl
4+T-CM, n=4)한 후, 2주 후 마우스를 희생하여 간조직을 획득하였다. 실험 및 절차는 이화여자대학교 동물 윤리위원회의 승인 하에 수행되었고, 모든 실험은 관련 구제 및 가이드라인 하에 수행되었다. Seven-week-old C57BL / 6 mice were randomly divided into untreated and carbon tetrachloride treated groups. The single dose of carbon tetrachloride was 10 μl / g, and the carbon tetrachloride was diluted to 10% in mineral oil and administered intraperitoneally twice a week for 3 weeks. The pretreated animals were divided into 3 groups, and sacrificed immediately after administration of carbon tetrachloride (CCl 4 + No treatment, n = 3), and the control medium obtained by culturing 1x10 6 amygdala stem cells in DMEM for 48 hours was concentrated 20 times. After two administrations (CCl 4 + T-CM, n = 4) through the tail vein, liver tissue was obtained at 2 weeks after sacrifice. The experiments and procedures were performed with the approval of the Ewha Womans University Animal Ethics Committee, and all experiments were performed under relevant remedies and guidelines.
1. 조직학적 분석1. Histological Analysis
조직학적 분석을 위하여 5 μm 두께의 간 조직 파라핀 절편을 제작하여 헤마톡실린과 에오신 (hematoxylin & eosin, H&E) 염색을 수행하였다. 간 조직의 섬유화는 콜라겐 축적을 시리우스 레드(Sirius Red) 염색으로 확인한 후, 콜라겐 섬유의 염색 정도를 단위 면적 당 화소밀도(pixel densities)로 수치화하여 콜라겐 인덱스를 계산하였다.For histological analysis, 5 μm-thick liver tissue paraffin sections were prepared and stained with hematoxylin and eosin (H & E). Fibrosis of liver tissue was confirmed by Sirius Red staining, and the collagen index was calculated by quantifying the degree of staining of collagen fibers by pixel densities per unit area.
그 결과를 도 2에 나타내었다. H&E 염색 결과, CCl
4 전처치로 손상된 간 조직이 편도줄기세포 조정배지(T-CM) 처리에 의해 회복됨을 확인할 수 있었고, 시리우스 레드 염색 결과, CCl
4 주입 그룹에서 확인된 광범위한 콜라겐 축적 병변이 편도줄기세포 조정배지 처리에 의해 대조군과 같은 수준으로 감소함을 확인할 수 있었다. The results are shown in FIG. As a result of H & E staining, it was confirmed that liver tissue damaged by CCl 4 pretreatment was recovered by tonsilocyte-mediated media (T-CM) treatment.Sirius red staining revealed extensive collagen accumulation lesions identified in CCl 4 injection group. Stem cell adjustment medium was confirmed to decrease to the same level as the control.
이를 콜라겐 인덱스로 수치화한 결과를 도3에 나타내었다. The result of quantifying the collagen index is shown in FIG. 3.
도 3에서 확인되는 바와 같이 편도 유래 중간엽 줄기세포의 조정 배지(T-CM)투여에 의해 통계학적으로 유의한 간 섬유화의 감소를 확인하였다(***p<0.001). As shown in FIG. 3, statistically significant reduction of liver fibrosis was confirmed by administration of toxin-mediated mesenchymal stem cells (T-CM) (*** p <0.001).
2. 간질환 표지 유전자 발현 확인2. Expression of liver disease marker gene
CCl
4 전처치 및 편도줄기세포 조정배지 주입에 의한 간조직에서의 간질환, 특히 간경변 관련 유전자 발현의 변화를 확인하기 위하여, 간 조직의 mRNA를 추출하여 cDNA로 합성 후, real-time PCR을 통하여 collagen type 1, alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1)과 metallopeptidase inhibitor 1 (Timp1)의 발현을 확인하였다. 사용한 프라이머의 서열을 다음과 같다.To identify changes in liver disease, especially liver cirrhosis-related gene expression in liver tissues by CCl 4 pretreatment and tonsilled stem cell-mediated media, liver mRNAs were extracted and synthesized into cDNA, followed by real-time PCR. Expression of collagen type 1, alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) was confirmed. The sequence of the used primer is as follows.
| 유전자gene | 서열 (5’→3’)Sequence (5 '→ 3') | Product lengthProduct length | |
| Col1a1 (NM_007742.4)Col1a1 (NM_007742.4) | ForwardForward | GCT CCT CTT AGG GGC CAC T (서열번호 1)GCT CCT CTT AGG GGC CAC T (SEQ ID NO: 1) | 103103 |
| ReverseReverse | CCA CGT CTC ACC ATT GGG G (서열번호 2)CCA CGT CTC ACC ATT GGG G (SEQ ID NO: 2) | ||
| Tgfb1 (NM_011577.2)Tgfb1 (NM_011577.2) | ForwardForward | CTC CCG TGG CTT CTA GTG C (서열번호 3)CTC CCG TGG CTT CTA GTG C (SEQ ID NO: 3) | 133133 |
| ReverseReverse | GCC TTA GTT TGG ACA GGA TC (서열번호 4)GCC TTA GTT TGG ACA GGA TC (SEQ ID NO: 4) | ||
| Timp1 (NM_003254.2)Timp1 (NM_003254.2) | ForwardForward | CTT GGT TCC CTG GCG TAC TC (서열번호 5)CTT GGT TCC CTG GCG TAC TC (SEQ ID NO: 5) | 150150 |
| ReverseReverse | ACC TGA TCC GTC CAC AAA CAG (서열번호 6)ACC TGA TCC GTC CAC AAA CAG (SEQ ID NO: 6) | ||
| Gapdh (NM_008084.3)Gapdh (NM_008084.3) | ForwardForward | AGG TCG GTG TGA ACG GAT TT (서열번호 7)AGG TCG GTG TGA ACG GAT TT (SEQ ID NO: 7) | 123123 |
| ReverseReverse | TGT AGA CCA TGT AGT TGA GG (서열번호 8)TGT AGA CCA TGT AGT TGA GG (SEQ ID NO: 8) |
위 분석 결과를 도 4에 나타내었다. The analysis results are shown in FIG. 4.
도 4에서 확인되는 바와 같이, 유전자 발현 분석 결과 CCl4 전처치 그룹에서 증가한 간경변 표지유전자(Col1a1, Tgfb1, Timp1)의 발현이 편도 유래 중간엽 줄기세포의 조정배지 처리에 의해 통계적으로 유의하게 감소한 것을 확인하였다 (*p<0.05, **p<0.01).As shown in FIG. 4, the gene expression analysis revealed that the expression of increased cirrhosis marker genes (Col1a1, Tgfb1, Timp1) in the CCl4 pretreatment group was statistically significantly reduced by the control medium of tonsil-derived mesenchymal stem cells. (* P <0.05, ** p <0.01).
상기 결과들로부터 본원 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지가 간질환의 예방 및 치료에 우수한 효과가 있음을 확인하였다. From the above results, it was confirmed that the control medium of tonsil-derived mesenchymal stem cells according to the present invention has an excellent effect on the prevention and treatment of liver disease.
Claims (17)
- 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating liver disease, including adjusting media of tonsil derived mesenchymal stem cells.
- 제1항에 있어서, 상기 간질환은 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 간질환 예방 또는 치료용 약학 조성물.The method of claim 1, wherein the liver disease is any one or more selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, hepatic coma, hepatic atrophy and liver cancer. Pharmaceutical composition.
- 제2항에 있어서, 상기 간질환은 간경변인 간질환 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating liver disease of claim 2, wherein the liver disease is cirrhosis.
- 제1항에 있어서, 상기 편도 유래 중간엽 줄기세포의 조정 배지는 무혈청 배지에서 배양된 배양액, 배양상층액 또는 이의 농축물인 간질환 예방 또는 치료용 약학 조성물.The pharmaceutical composition of claim 1, wherein the medium for adjusting tonsil-derived mesenchymal stem cells is a culture medium, a culture supernatant, or a concentrate thereof cultured in a serum-free medium.
- 제1항에 있어서, 상기 편도 유래 중간엽 줄기세포의 조정 배지는 IL-1Ra, proMMP-1 및 preprostromelysin로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것인 간질환 예방 또는 치료용 약학 조성물.The method of claim 1, wherein the tonsil-derived mesenchymal stem cells, the control medium comprises any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin liver disease prevention or treatment pharmaceutical composition.
- 제1항에 있어서, 상기 간질환 예방 또는 치료는 섬유화 억제에 의한 것인 간질환 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the prevention or treatment of liver disease is by inhibiting fibrosis.
- 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물.Food composition for preventing or improving liver disease, including the adjustment medium of tonsil derived mesenchymal stem cells.
- 제7항에 있어서, 상기 간질환은 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 간질환 예방 또는 개선용 식품 조성물.The method of claim 7, wherein the liver disease is at least one selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, hepatic coma, hepatic atrophy and liver cancer. Food composition.
- 제8항에 있어서, 상기 간질환은 간경변인 간질환 예방 또는 개선용 식품 조성물.The food composition of claim 8, wherein the liver disease is cirrhosis.
- 제7항에 있어서, 상기 편도 유래 중간엽 줄기세포의 조정 배지는 무혈청 배지에서 배양된 배양액, 배양상층액 또는 이의 농축물인 간질환 예방 또는 개선용 식품 조성물.The method of claim 7, wherein the tonsil-derived mesenchymal stem cells, the control medium is a culture medium, culture supernatant or concentrate thereof cultured in a serum-free medium, liver disease prevention or improvement food composition.
- 제7항에 있어서, 상기 편도 유래 중간엽 줄기세포의 조정 배지는 IL-1Ra, proMMP-1 및 preprostromelysin로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것인 간질환 예방 또는 개선용 식품 조성물.The method of claim 7, wherein the amygdala-derived mesenchymal stem cell control medium comprises any one or more selected from the group consisting of IL-1Ra, proMMP-1 and preprostromelysin liver disease prevention or food composition.
- 편도 유래 중간엽 줄기세포의 조정 배지를 치료학적 유효량으로 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 간질환의 치료방법.A method of treating liver disease comprising administering to the subject in need thereof a modulating medium of tonsil derived mesenchymal stem cells.
- 제12항에 있어서, 상기 간질환은 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 간질환의 치료방법.The method of claim 12, wherein the liver disease is at least one selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, hepatic coma, hepatic atrophy and liver cancer.
- 간질환의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도.Use of modulated media of tonsil-derived mesenchymal stem cells in the manufacture of a medicament for the treatment of liver disease.
- 제14항에 있어서, 상기 간질환은 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 용도. 15. The use according to claim 14, wherein the liver disease is at least one selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, hepatic coma, hepatic atrophy and liver cancer.
- 간질환의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물. A composition comprising a control medium of tonsil derived mesenchymal stem cells for use in the treatment of liver disease.
- 제16항에 있어서, 상기 간질환은 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 조성물. The composition of claim 16, wherein the liver disease is at least one selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, hepatic coma, hepatic atrophy and liver cancer.
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| KR101822153B1 (en) | 2016-04-25 | 2018-01-26 | (주) 노바렉스 | Pharmaceutical composition for preventing or treating liver disease comprising licorice extract having glycyrrhizin and liquiritin |
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2018
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140135367A (en) * | 2013-05-16 | 2014-11-26 | 이화여자대학교 산학협력단 | Differentiation method from tonsil-derived mesenchymal stem cells to hepatocytes and cell therapy composition comprising tonsil-derived mesenchymal stem cells for treatment of hepatopathy |
Non-Patent Citations (5)
| Title |
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| CHO, K.-A.: "Conditioned media from human palatine tonsil mesenchymal stem cells regulates the interaction between myotubes and fibroblasts by 11-IRa activity", JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, vol. 21, no. 1, 2017, pages 130 - 141, XP055639370 * |
| GAZDIC, M . ET AL.: "Mesenchymal stem cell -dependent modulation of liver diseases", INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES, vol. 13, no. 9, 2017, pages 1109 - 1117, XP055639366 * |
| KIM, Y.-H.: "Conditioned medium from tonsil-derived mesenchymal stem cells relieves CC14-induced liver fibrosis in mice", TISSUE ENG. REGEN. MED., vol. 16, no. 1, 19 October 2018 (2018-10-19), pages 51 - 58, XP036694608 * |
| PARK, M.: "Tonsil-derived mesenchymal stem cells ameliorate CCI4-induced liver fibrosis in mice via autophagy activation", SCIENTIFIC REPORTS, vol. 5, 8616, 8 May 2015 (2015-05-08), pages 1 - 7, XP055639377 * |
| SANG, J.-F.: "Combined mesenchymal stem cell transplantation and interleukin-1 receptor antagonism after partial hepatectomy", WORLD JOURNAL OF GASTROENTEROLOGY, vol. 22, no. 16, 2016, pages 4120 - 4135, XP055639374 * |
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| Publication number | Publication date |
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| KR20190114678A (en) | 2019-10-10 |
| KR102526447B1 (en) | 2023-04-26 |
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