WO2020101212A1 - Composition, comprising conditioned medium of tonsil-derived mesenchymal stem cell, for inhibiting cancer metastasis - Google Patents
Composition, comprising conditioned medium of tonsil-derived mesenchymal stem cell, for inhibiting cancer metastasis Download PDFInfo
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- WO2020101212A1 WO2020101212A1 PCT/KR2019/014042 KR2019014042W WO2020101212A1 WO 2020101212 A1 WO2020101212 A1 WO 2020101212A1 KR 2019014042 W KR2019014042 W KR 2019014042W WO 2020101212 A1 WO2020101212 A1 WO 2020101212A1
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- cancer
- tonsil
- mesenchymal stem
- stem cells
- derived mesenchymal
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Images
Classifications
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- 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
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- 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
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- 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
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
Definitions
- the present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
- the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising a mediator of the tonsil-derived mesenchymal stem cells.
- the present invention relates to a food composition for preventing or ameliorating cancer, comprising a media for mediating tonsil-derived mesenchymal stem cells.
- 'tumor' refers to a mass that is abnormally grown by autonomous overgrowth of body tissue, and can be divided into a benign tumor and a malignant tumor. While benign tumors are relatively slow to grow and metastasis (the tumors move away from where they originated), malignancies grow rapidly as they invade surrounding tissues and spread or metastasize to each part of the body to save lives. Threatens. Malignant tumors can be thought of as synonymous with cancer.
- EMT Epithelial-to-mesenchymal transition
- E-cadherin is a membranous glycoprotein, and the extracellular region binds to the E-cadherin molecule of adjacent cells to maintain adhesion between cells, and the intracellular region binds ⁇ -, ⁇ - and p120 caderin to form epithelial cells. Polarity and cytoskeleton formation. E-cadherin is regulated by various signaling systems involved in cell proliferation and apoptosis, including TGF- ⁇ , Integrin, Wnt, RTK (receptortyrosine kinase), and Notch.
- TGF- ⁇ acts on SMAD or PI3K / AKT (phosphatidylinositol 3-kinase / serine-threonine kinase), and of these, PI3K / AKT has been reported to play an important role in activating EMT.
- SMAD phosphatidylinositol 3-kinase / serine-threonine kinase
- Tonsil-derived mesenchymal stem cell conditioned medium is located on the inside of the neck and the back of the nose, primarily defending our body from substances, such as bacteria, which invade from the outside, and at the same time as lymphoid epithelial immune tissue.
- undifferentiated stem cells having the ability to differentiate into two or more new cells while having the ability to self-replicate derived from the amygdala, the tissue performing the action.
- tonsil-derived mesenchymal stem cells there have been reports on treatment efficacy against skin inflammatory diseases and treatment efficacy against chronic inflammatory bowel disease.
- the present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
- the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, including the adjusted medium of tonsil-derived mesenchymal stem cells.
- the present invention provides a food composition for preventing or ameliorating cancer, comprising a mediator for mediating tonsil-derived mesenchymal stem cells.
- the present inventors have identified a suitable type of cell-derived substances capable of preventing or treating cancer, and the media for the adjustment of the amygdala-derived mesenchymal stem cells has a different profile compared to the other-derived mesenchymal stem cells.
- the present invention was completed by confirming that it is effective in preventing and treating metastasis of cancer, in particular.
- the tonsil-derived mesenchymal stem cells are tissues that are located on the inside of the neck and the back of the nose, primarily defending our bodies from substances such as bacteria invading from the outside, and at the same time acting as lymphoid epithelial immune tissues.
- undifferentiated stem cells having the ability to differentiate into two or more new cells while having the ability to self-replicate derived from the tonsil.
- tonsil-derived mesenchymal stem cell control medium refers to a culture medium in which tonsil-derived mesenchymal stem cells are removed from a culture obtained by culturing tonsil-derived mesenchymal stem cells or a supernatant thereof, and "one-way derived Mesenchymal stem cell culture supernatant "," one way mesenchymal stem cell condition culture “or” one way mesenchymal stem cell culture medium "can be used interchangeably.
- prevention refers to all actions to suppress or delay the onset of cancer by administration of the adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention.
- treatment refers to all actions of improving or beneficially improving the symptoms of cancer by administering the adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention.
- improvement in the present invention means any act of improving the bad condition of cancer by administering or ingesting the composition of the present invention to an individual.
- cancer metastasis refers to a condition in which malignant tumors spread away from an affected organ to other tissues.
- the cancer may be metastatic cancer.
- it refers to cancer that is caused by cancer cells moving from the primary organ to another organ to multiply.
- the spread of cancer to other parts of the body can be largely divided into primary cancer, where cancerous tissue grows and directly invades surrounding organs and distant metastases along blood vessels or lymphatic vessels to other distant organs.
- primary cancers it may mean that the cancer has spread to the stomach organs, but is not limited thereto.
- the metastatic cancer may be metastasis and / or recurrence through the epithelial mesenchymal metastasis process, or may be a cancer that is difficult to treat because it has resistance to drugs for treating cancer.
- cancers may be any one or more selected from the group consisting of lung cancer, breast cancer, liver cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, kidney cancer and melanoma.
- the present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
- the media for the adjustment of mesenchymal stem cells derived from the amygdala of the present invention is characterized by inhibiting epithelial-mesenchymal transition (EMT), which is known as a pre-stage pathology, and cancer metastasis such as cancer cell migration and infiltration due to EMT suppression from the initial stage. Suppress.
- EMT epithelial-mesenchymal transition
- the media for the adjustment of tonsil-derived mesenchymal stem cells has a different protein profile compared to other mesenchymal stem cells and their media.
- the media for mediation of tonsil-derived mesenchymal stem cells unlike other derived mesenchymal stem cells and their media, contains a large amount of CD109 while competing with the TGF- ⁇ receptor and binding to TGF- ⁇ , thereby inhibiting normal TGF- ⁇ signaling. And, accordingly, the metastasis of cancer is suppressed through EMT inhibition.
- the tonsil-derived mesenchymal stem cells according to the present invention are easier to obtain the tonsils, which are the tissues of origin, compared to other derived stem cells, and it is possible to recycle the tonsil tissues that are discarded after surgery, and the adjustment to be injected is very high in initial yield.
- the amount of medium can be freely adjusted.
- the adjusted medium of tonsil-derived mesenchymal stem cells includes those produced through isolation, culture, and special manipulation from an individual, and can be used in cancer as medicines used for the purpose of treatment, diagnosis, and prevention. .
- cancer is as mentioned above.
- tonsil-derived mesenchymal stem cells can be extracted from the tonsils according to methods known in the art. For example, it can be extracted from one way according to the method described in Korean Patent Registration No. 10-1508413.
- the media for adjusting the amygdala-derived mesenchymal stem cells may be cultured tonsil-derived mesenchymal stem cells and removed from the cells, and then the obtained culture medium, culture supernatant or a concentrate thereof, or a lyophilized product thereof.
- the tonsil-derived mesenchymal stem cells can be cultured normally using a medium for culturing stem cells.
- the tonsil-derived mesenchymal stem cell culture may be obtained by culturing tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum or serum-free medium.
- the supernatant obtained after removing stem cells and macromolecules by centrifugation or filtration using a filter may be used.
- the obtained supernatant can be used as it is or as a concentrate obtained by concentration.
- the 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 can be appropriately selected or modified by those skilled in the art.
- the medium is a medium suitable for maintaining and storing the same cell type as the tonsil-derived mesenchymal stem cells, and may be DMEM medium supplemented with serum.
- Serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight based on the total weight of the serum medium.
- antibiotics, antifungal agents and mycoplasma inhibitors may be included, and these may be 1% by weight based on the total weight of the medium.
- Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include gentamicin, ciprofloxacin, tyrosine, etc. , But is not limited to this.
- the medium may be a serum-free medium.
- the serum-free medium may be low glucose DMEM medium.
- the tonsil-derived mesenchymal stem cell regulatory medium is concentrated by removing the tonsil-derived mesenchymal stem cells under serum-free low glucose DMEM to remove the cells.
- the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, including the adjusted medium of tonsil-derived mesenchymal stem cells.
- prevention means all actions to suppress or delay the onset of cancer by administration of the composition
- treatment refers to all actions that improve or benefit the symptoms of cancer by administration of the composition.
- the pharmaceutical composition for preventing or treating cancer according to the present invention suppresses EMT (epithelial-mesenchymal transition), thereby inhibiting cancer progression, thereby inducing cancer cell death and inhibiting cancer metastasis, thereby having excellent effects in preventing and treating cancer. .
- EMT epidermal-mesenchymal transition
- composition of the present invention can be formulated into various formulations such as liquids, suspensions, emulsions, lotions, ointments, lyophilizers according to conventional methods.
- the pharmaceutical composition of the present invention can be formulated and administered as a pharmaceutical preparation in a unit dosage form suitable for administration in a patient's body according to a conventional method in the pharmaceutical field, and the preparation is effectively administered by single or multiple administrations. Includes the amount.
- Formulations suitable for this purpose are preferably parenteral administration preparations such as injections, injections, and the like.
- the pharmaceutical composition for preventing or treating cancer may include a pharmaceutically acceptable inert carrier and a diluent.
- compositions of the present invention are excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin , Alginate, and binders such as polyvinyl pyrrolidone, talc, lubricants such as calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, interfaces such as polysorbate, 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 recipient to be transplanted.
- Diluents include, but are not limited to, brine, aqueous buffers, solvents and / or dispersion media.
- a preservative, a painless agent, a solubilizing agent, or a stabilizer, etc. in the case of a formulation for topical administration, may further include a base, excipient, lubricant, or preservative.
- the composition of the present invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (e.g. DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) can be added to the cell population prior to freezing.
- standard cryopreservatives e.g. DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)
- the dosage may vary depending on the degree of concentration, but may be administered by dividing the body weight from 10 ⁇ l / kg to 1 ml / kg once or several times.
- the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and sex, and thus, the dosage
- the scope of the present invention is not limited in any way.
- the present invention also provides a food composition for preventing or improving cancer, comprising a mediator media for 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 health functional food.
- health functional food refers to food manufactured and processed using ingredients or ingredients having useful functionality for the human body according to the Act on Health Functional Food, and "functional” refers to the structure and function of the human body. This means taking it for the purpose of controlling nutrients or obtaining useful effects for health purposes such as 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 rules and general test methods of food additives approved by the Ministry of Food and Drug Safety unless otherwise specified. It is judged according to the standards and standards for items.
- Items listed in the "Food Additives Revolution” include, for example, chemical additives such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as chromochromate, licorice extract, crystalline cellulose, high color pigment, and guar gum, And mixed preparations such as an L-sodium glutamate preparation, an alkali added additive, a preservative preparation, and a tar colorant.
- chemical additives such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid
- natural additives such as chromochromate, licorice extract, crystalline cellulose, high color pigment, and guar gum
- mixed preparations such as an L-sodium glutamate preparation, an alkali added additive, a preservative preparation, and a tar colorant.
- the food composition of the present invention may contain 0.01 to 95% by weight, preferably 5 to 90% by weight of the adjusted medium of tonsil-derived mesenchymal stem cells relative to the total weight of the composition.
- the food composition of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and / or improving cancer.
- the hard capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as an adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention, and excipients
- Soft capsules may be prepared by filling a capsule base such as gelatin with a mixture of additives such as food compositions and excipients according to the present invention.
- the soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if necessary.
- the present invention provides a method of treating cancer comprising the step of administering to the subject the media for adjustment of the tonsil-derived mesenchymal stem cells.
- the present invention provides a method for inhibiting metastasis of cancer comprising the step of administering to the subject a control medium of tonsil-derived mesenchymal stem cells.
- the subject refers to an animal, and may be a mammal capable of exerting a beneficial effect, typically by treatment with a control medium of tonsil-derived mesenchymal stem cells of the present invention.
- Preferred examples of such subjects may include primates such as humans.
- such subjects may include all subjects who have symptoms of cancer or are at risk of having such symptoms.
- the present invention also provides the use of a conditioned medium of tonsil derived mesenchymal stem cells in the manufacture of a medicament for the treatment of cancer.
- the present invention also provides a composition comprising a conditioned medium of tonsil derived mesenchymal stem cells for use in the treatment of cancer.
- the present invention also provides the use of a conditioned medium of tonsil-derived mesenchymal stem cells for the treatment of cancer.
- composition of the present invention has a different protein profile from other mesenchymal stem cells or their media, thereby suppressing cancer metastasis such as cancer cell migration and invasion due to EMT inhibition, and inducing cancer cell death. It has excellent effects on metastasis suppression and prevention, improvement and treatment of cancer.
- Figure 1 shows the results of confirming the amount of CD109 secretion in the control medium of mesenchymal stem cells of different origin.
- Figure 2 shows the results confirming the induction of EMT response in cancer cells by TGF- ⁇ treatment.
- Figure 3 shows the results of confirming the inhibition of proliferation and metastasis inhibition of cancer cells according to the control medium treatment of tonsil-derived mesenchymal stem cells.
- Figure 4 shows the results of confirming the change in expression of the EMT-related gene according to the control medium treatment of tonsil-derived mesenchymal stem cells.
- Figure 5 shows the results of confirming the inhibition of cancer tissue formation in a mouse animal model of cancer cell line transplantation according to the adjusted medium treatment of tonsil-derived mesenchymal stem cells.
- Figure 6 shows the results confirming the effect of inhibiting cancer cell proliferation according to the adjusted medium treatment of tonsil-derived mesenchymal stem cells.
- Example 1 Preparation of mesenchymal stem cells derived from fat, bone marrow, or tonsils or their media
- Tonsil-derived mesenchymal stem cells are derived from tonsil tissue (4-20 years old low-aged tissue) extracted from a patient performing tonsillectomy in the otolaryngology department of the otolaryngology department at Ewha Womens University Mokdong Hospital. Got. It was manufactured similarly to the manufacturing method of Korean Patent No. 10-1508413.
- tonsil-derived mesenchymal stem cells were cultured with Low glucose DMEM. When the confluency reached 70-80% in a 100 mm culture tissue plate, the medium was removed, and the cells were adhered to the plate, washed four times with PBS, and then serum-free medium (Low glucose DMEM) was added. After 48 hours, the medium was collected, centrifuged at 1300 rpm for 5 minutes, and the supernatant was filtered with a 0.2 uM filter. The filtered medium was concentrated 20 times with centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore) for use in experiments or stored at -80 ° C.
- Low glucose DMEM serum-free medium
- Adipose-derived mesenchymal stem cells used cells of the same passage provided by RNL Bio, and in the case of bone marrow mesenchymal stem cells, cells of the same passage from Seoul Yonsei University Severance Hospital Cell Therapy Center were used. . In the case of preparation of the conditioned medium, it was prepared under the same conditions as the conditioned medium from the mesenchymal stem cells derived from the stomach tonsil.
- FIG. 1A shows the results of confirming CD109 of the adjusted medium (T-MSC-CM1, CM2, CM3) of tonsil-derived mesenchymal stem cells from different donors. As can be seen in FIG. 1A, it was confirmed that CD109 was secreted at the same high level.
- B in Figure 1 shows the result of confirming the difference in secretion from the control medium of stem cells of different origin.
- AD-MSC-CM adipose-derived
- BM-MSC-CM bone marrow-derived
- CD109 a protein that inhibits the TGF- ⁇ signal, can effectively suppress the action of EMT, and this effect can be achieved only through the mediation of the amygdala-derived mesenchymal stem cells.
- HepG2 cells were seeded in 6 well plates and cultured in MEM medium (10% FBS, 1% penicillin / streptomycin). TGF- ⁇ was incubated for 72 hours by adding 0, 20, and 50 ng / ml concentrations, respectively.
- culture was performed by adding the control medium of the tonsil stem cell (amount obtained from 10 6 cells, 250 ul) to the group treated with TGF- ⁇ at a concentration of 20 ng / ml. Did.
- TGF- ⁇ (20 ng / ml) was treated and HepG2 cells cultured for 72 hours, TGF- ⁇ (20 ng / ml) and tonsil stem cell control medium (obtained from 10 6 cells, 250 ul) were added together. Then, HepG2 cells cultured for 72 hours and normal control HepG2 cells were collected and total RNA was isolated using Trizol reagent (Invitrogen). CDNA was synthesized by adding 5X Reverse transcription premix (ELPIS BIOTECH) to 1 ug of isolated RNA for 1 hour at 42 ° C.
- EMT marker genes human CD44, human CD133, human CD151, human EPCAM, human p53, and human K19 was confirmed by amplifying in StepOnePlus real-time PCR system (Applied biosystems) with specific primers in Table 1 below.
- the expression of each gene was normalized to the expression level of gapdh, a housekeeping gene.
- the relative expression level difference between each sample was calculated using the 2- ⁇ CT method.
- HepG2 cells were tagged with CFSE at a concentration of 5 uM, and then cultured for 72 hours with TGF- ⁇ (20 ng / ml).
- TGF- ⁇ (20 ng / ml)
- the culture medium of tonsil stem cells (amount obtained from 10 6 cells, 250 ul) was cultured with or without addition.
- the degree of CFSE fluorescence was measured by a flow cytometer.
- TGF- ⁇ was added to HepG2 cells, a liver cancer cell line, and cultured for 72 hours. As a result, prominent proliferation of cells was confirmed.
- CD44, CD133, CD151, and K19 increased significantly even at a concentration of 20 ng / ml.
- FIG. 3A shows the effect of inhibiting cell proliferation confirmed through a microscope
- FIG. 3B shows the effect of inhibiting cell proliferation according to CFSE fluorescence measurement.
- CFSE is stained on the cytoplasm of living cells to measure the degree of fluorescence, and it can be seen how many divisions have been made. Different colored peaks on the histogram of FIG. 3 indicate generation of proliferated cells.
- administration of tonsil-derived mesenchymal stem cells was confirmed to inhibit cell proliferation in hepatocellular carcinoma cells induced EMT response.
- the media for mediation of tonsil-derived mesenchymal stem cells suppresses EMT and shows excellent effects in the prevention and treatment of cancer.
- Figure 5 shows the results of confirming the size of cancer when 5 days (A) and 9 days (B) have passed after cell transplantation. It was confirmed that the size of the cancer tissue was significantly suppressed at the implantation site compared to the case where it was transplanted by being suspended in a normal DMEM medium.
- FIG. 6 shows that the tissue cells isolated from the tonsil stem cell control medium transplantation group proliferate slowly when in vitro culture of cancer tissues isolated from the mouse model of liver cancer cell line transplantation.
- FIG. 6A shows micrographs on the 2nd and 9th days of culture.
- cancerous tissue cells derived from the mouse transplanted together with the tonsil stem cell control medium slowly proliferated.
- B of FIG. 6 when the clusters of cells were checked through a microscope on the second day of culture, it was confirmed that the tissues cultured in the control medium of the amygdala-derived mesenchymal stem cells formed less clusters. This shows that while the cells grown in DMEM had active intercellular interactions, the proliferation proceeded rapidly, whereas the group treated with the control medium of tonsil-derived mesenchymal stem cells had little effect.
- the media for mediation of tonsil-derived mesenchymal stem cells suppresses the generation and progression of cancer by suppressing epithelial to mesenchymal transition (EMT) and has excellent anticancer effects.
- EMT epithelial to mesenchymal transition
- the effect of the mediation of the tonsil-derived mesenchymal stem cells is due to factors not identified in the bone marrow-derived or adipose-derived mesenchymal stem cells. It was confirmed that it has different characteristics from the derived mesenchymal stem cells and their media, and shows excellent effects in inhibiting cancer metastasis and preventing and treating cancer.
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Abstract
The present invention relates to a composition for inhibiting cancer metastasis, or preventing, alleviating, or treating cancer, comprising a conditioned medium of tonsil-derived mesenchymal stem cells. Having a protein profile different from those of mesenchymal stem cells derived from other tissues or conditioned media thereof, the composition of the present invention exhibits inhibitory activity against EMT to suppress cancer metastasis, such as cancer cell motility and penetration, at the early stage and to induce the apoptosis of cancer cells. Thus, the composition has excellent effects of inhibiting cancer metastasis and preventing, alleviating, and treating cancer.
Description
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 전이 억제용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising a mediator of the tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for preventing or ameliorating cancer, comprising a media for mediating tonsil-derived mesenchymal stem cells.
일반적으로 '종양(tumor)'은 신체 조직의 자율적인 과잉 성장에 의해 비정상적으로 자라난 덩어리를 의미하며, 양성종양(benign tumor)과 악성종양(malignant tumor)으로 구분할 수 있다. 양성종양이 비교적 성장 속도가 느리고 전이(metastasis; 종양이 원래 발생한 곳에서 멀리 떨어진 곳으로 이동함)되지 않는 것에 반해 악성종양은 주위 조직에 침윤하면서 빠르게 성장하고 신체 각 부위에 확산되거나 전이되어 생명을 위협하게 된다. 악성종양을 암과 동일한 의미로 생각할 수 있다.Generally, 'tumor' refers to a mass that is abnormally grown by autonomous overgrowth of body tissue, and can be divided into a benign tumor and a malignant tumor. While benign tumors are relatively slow to grow and metastasis (the tumors move away from where they originated), malignancies grow rapidly as they invade surrounding tissues and spread or metastasize to each part of the body to save lives. Threatens. Malignant tumors can be thought of as synonymous with cancer.
상피간엽이행(EMT; epithelial-to-mesenchymal transition)은 상피세포가 전이 능력과 침윤능력을 가지는 세포로 변환하는 과정이며 이는 척추동물과 무척추동물 모두에서의 태생기에 조직과 기관의 발생을 포함하는 형태학적 발생의 기본적인 현상으로 간주되었지만 과학의 발전과 더불어 성인의 상처 치유 및 암의 생성, 진행과정에 있어서도 EMT가 중요하게 작용함이 입증되었다. EMT에서 가장 초기에 일어나고 가장 중요한 과정은 E-카데린(E-cadherin)에서 N-카데린(N-cadherin)으로 전환하는 카데린 변환이다. E-카데린은 막성 당단백질로 세포외 영역은 인접 세포의 E-카데린 분자와 결합하여 세포 간의 부착을 유지하고, 세포 내 영역은 α-, β- 및 p120 카데린과 결합하여 상피 세포의 극성과 세포골격을 형성한다. E-카데린은 TGF-β,Integrin, Wnt, RTK(receptortyrosine kinase), Notch 등 세포증식과 세포소멸에 관여하는 다양한 신호체계에 의해 조절된다. 이중 TGF-β는 SMAD나 PI3K/AKT(phosphatidylinositol 3-kinase/serine-threonine kinase)에 작용하는데 이중 PI3K/AKT는 EMT를 활성화시키는데 중요한 역할을 담당한다고 보고되고 있다.Epithelial-to-mesenchymal transition (EMT) is the process by which epithelial cells are transformed into cells with metastatic and infiltrating abilities, which include the development of tissues and organs in the embryonic period in both vertebrates and invertebrates. Although it was regarded as a basic phenomenon of academic development, it has been proven that EMT plays an important role in the wound healing and cancer production and progression of adults with the advancement of science. The earliest and most important process in EMT is the conversion of Caderine from E-cadherin to N-cadherin. E-cadherin is a membranous glycoprotein, and the extracellular region binds to the E-cadherin molecule of adjacent cells to maintain adhesion between cells, and the intracellular region binds α-, β- and p120 caderin to form epithelial cells. Polarity and cytoskeleton formation. E-cadherin is regulated by various signaling systems involved in cell proliferation and apoptosis, including TGF-β, Integrin, Wnt, RTK (receptortyrosine kinase), and Notch. Among them, TGF-β acts on SMAD or PI3K / AKT (phosphatidylinositol 3-kinase / serine-threonine kinase), and of these, PI3K / AKT has been reported to play an important role in activating EMT.
편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell conditioned medium)는 목의 안쪽과 코의 뒷부분에 위치하여 외부에서 침입하는 세균 등의 물질로부터 일차적으로 우리 몸을 방어함과 동시에 림프 상피 면역 조직으로 작용을 수행하는 조직인 편도에서 유래된 자기 복제 능력을 가지면서 두개 이상의 새로운 세포로 분화하는 능력을 가진 미분화된 줄기세포를 의미한다. 편도 유래 중간엽 줄기세포에 관하여는, 피부 염증 질환에 대한 치료 효능, 만성 염증성 장질환에 대한 치료 효능 등에 관하여 보고된 바가 있다.Tonsil-derived mesenchymal stem cell conditioned medium is located on the inside of the neck and the back of the nose, primarily defending our body from substances, such as bacteria, which invade from the outside, and at the same time as lymphoid epithelial immune tissue. Refers to undifferentiated stem cells having the ability to differentiate into two or more new cells while having the ability to self-replicate derived from the amygdala, the tissue performing the action. Regarding tonsil-derived mesenchymal stem cells, there have been reports on treatment efficacy against skin inflammatory diseases and treatment efficacy against chronic inflammatory bowel disease.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 전이 억제용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, including the adjusted medium of tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or ameliorating cancer, comprising a mediator for mediating tonsil-derived mesenchymal stem cells.
본 발명자들은 암을 예방 또는 치료할 수 있는 적합한 종류의 세포 유래 물질들을 확인하던 중, 편도 유래 중간엽 줄기세포의 조정배지가 타 유래 중간엽 줄기세포들과 대비하여 상이한 프로파일을 가지며, 이에 의해 암의 예방 및 치료, 특히 암의 전이 억제에 효과적임을 확인하여 본 발명을 완성하였다.The present inventors have identified a suitable type of cell-derived substances capable of preventing or treating cancer, and the media for the adjustment of the amygdala-derived mesenchymal stem cells has a different profile compared to the other-derived mesenchymal stem cells. The present invention was completed by confirming that it is effective in preventing and treating metastasis of cancer, in particular.
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 tonsil-derived mesenchymal stem cells are tissues that are located on the inside of the neck and the back of the nose, primarily defending our bodies from substances such as bacteria invading from the outside, and at the same time acting as lymphoid epithelial immune tissues. Refers to undifferentiated stem cells having the ability to differentiate into two or more new cells while having the ability to self-replicate derived from the tonsil.
본 발명에 있어서, "편도 유래 중간엽 줄기세포 조정 배지"는 편도 유래 중간엽 줄기세포를 배양하여 수득된 배양액으로부터 편도 유래 중간엽 줄기세포가 제거된 배양액 또는 이의 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" refers to a culture medium in which tonsil-derived mesenchymal stem cells are removed from a culture obtained by culturing tonsil-derived mesenchymal stem cells or a supernatant thereof, and "one-way derived Mesenchymal stem cell culture supernatant "," one way mesenchymal stem cell condition culture "or" one way mesenchymal stem cell culture medium "can be used interchangeably.
본 발명에서 용어 “예방”이란 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지의 투여로 암의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지의 투여로 암의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명에서 용어 "개선"이란 본 발명의 조성물을 개체에 투여하거나 섭취시켜 암의 나쁜 상태를 좋게 하는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to all actions to suppress or delay the onset of cancer by administration of the adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention. In the present invention, the term "treatment" refers to all actions of improving or beneficially improving the symptoms of cancer by administering the adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention. The term "improvement" in the present invention means any act of improving the bad condition of cancer by administering or ingesting the composition of the present invention to an individual.
본 발명에서 용어 “암 전이”는 악성 종양이 발병한 장기에서 떨어져 다른 조직으로 전파하는 상태를 말한다.In the present invention, the term "cancer metastasis" refers to a condition in which malignant tumors spread away from an affected organ to other tissues.
본 발명에서 암은 전이 암일 수 있다. 즉, 암 세포가 원발 장기를 떠나 다른 장기로 이동하여 증식되어 발생된 암을 의미한다. 암이 신체 다른 부분으로 퍼지는 것은 크게 원발암에서 암 조직이 성장하여 직접적으로 주위 장기를 침습하는 것과 멀리 있는 다른 장기로 혈관이나 림프관을 따라 원격 전이를 하는 것으로 구분할 수 있다. 바람직하게는 다른 원발암에서 암이 위의 장기로 전이된 것을 의미할 수 있으나, 이에 제한되는 것은 아니다. 보다 상세하게 상기 전이암은 상피 중간엽 전이 과정을 통해 전이(metastasis) 및/또는 재발이 일어난 암이거나, 암 치료용 약물에 대한 내성을 가져 치료가 어려운 암일 수 있다.In the present invention, the cancer may be metastatic cancer. In other words, it refers to cancer that is caused by cancer cells moving from the primary organ to another organ to multiply. The spread of cancer to other parts of the body can be largely divided into primary cancer, where cancerous tissue grows and directly invades surrounding organs and distant metastases along blood vessels or lymphatic vessels to other distant organs. Preferably, in other primary cancers, it may mean that the cancer has spread to the stomach organs, but is not limited thereto. In more detail, the metastatic cancer may be metastasis and / or recurrence through the epithelial mesenchymal metastasis process, or may be a cancer that is difficult to treat because it has resistance to drugs for treating cancer.
이러한 암의 예로는 폐암, 유방암, 간암, 대장암, 위암, 뇌암, 췌장암, 갑상선암, 피부암, 골수암, 림프종, 자궁암, 자궁경부암, 신장암 및 흑색종로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.Examples of such cancers may be any one or more selected from the group consisting of lung cancer, breast cancer, liver cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, kidney cancer and melanoma.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 전이 억제용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a mediation medium for tonsil-derived mesenchymal stem cells.
본 발명의 편도 유래 중간엽 줄기세포의 조정 배지는 병리현상의 전단계로 알려진 EMT(epithelial-mesenchymal transition)를 억제하는 것을 특징으로 하며, EMT 억제작용으로 인한 암세포 이동 및 침투 등 암 전이를 초기 단계부터 억제한다.The media for the adjustment of mesenchymal stem cells derived from the amygdala of the present invention is characterized by inhibiting epithelial-mesenchymal transition (EMT), which is known as a pre-stage pathology, and cancer metastasis such as cancer cell migration and infiltration due to EMT suppression from the initial stage. Suppress.
편도 유래 중간엽 줄기세포의 조정배지는 타 유래 중간엽 줄기세포 및 이의 조정 배지와 대비하여 상이한 단백질 프로파일을 가진다.The media for the adjustment of tonsil-derived mesenchymal stem cells has a different protein profile compared to other mesenchymal stem cells and their media.
구체적으로, 편도 유래 중간엽 줄기세포의 조정 배지는 다른 유래 중간엽 줄기세포 및 이의 배지와 다르게 CD109를 다량 함유하면서 TGF-β수용체와 경쟁하여 TGF-β와 결합함으로써 정상적인 TGF-β신호전달을 저해하고, 이에 따라 EMT 억제를 통해 암의 전이를 억제한다.Specifically, the media for mediation of tonsil-derived mesenchymal stem cells, unlike other derived mesenchymal stem cells and their media, contains a large amount of CD109 while competing with the TGF-β receptor and binding to TGF-β, thereby inhibiting normal TGF-β signaling. And, accordingly, the metastasis of cancer is suppressed through EMT inhibition.
또한, EMT에 관여하는 유전자들의 발현을 크게 억제하여 EMT 진행을 억제함으로써 암의 전이를 억제한다.In addition, it suppresses metastasis of cancer by greatly suppressing the expression of genes involved in EMT and suppressing EMT progression.
또한, 본 발명에 따른 편도 유래 중간엽 줄기세포는 타 유래 줄기세포와 대비하여 기원 조직인 편도의 획득이 용이하며 수술 후 버려지는 편도 조직의 재활용이 가능하고 초기 수득율이 매우 높다는 점에서 주입해야 하는 조정 배지의 양을 자유롭게 조절할 수 있다.In addition, the tonsil-derived mesenchymal stem cells according to the present invention are easier to obtain the tonsils, which are the tissues of origin, compared to other derived stem cells, and it is possible to recycle the tonsil tissues that are discarded after surgery, and the adjustment to be injected is very high in initial yield. The amount of medium can be freely adjusted.
본 발명에 있어서, 편도 유래 중간엽 줄기세포의 조정배지는 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 것을 포함하며, 치료, 진단 및 예방의 목적으로 사용되는 의약품으로 사용되어 암에 사용될 수 있다.In the present invention, the adjusted medium of tonsil-derived mesenchymal stem cells includes those produced through isolation, culture, and special manipulation from an individual, and can be used in cancer as medicines used for the purpose of treatment, diagnosis, and prevention. .
본 발명에 있어서 암은 상기 언급된 바와 같다.In the present invention, cancer is as mentioned above.
본 발명에 있어서, 편도 유래 중간엽 줄기세포는 당업계에 알려진 방법에 따라 편도로부터 추출 가능하다. 예컨대, 대한민국등록특허 제10-1508413호 등에 기재된 방법에 따라 편도로부터 추출가능하다.In the present invention, tonsil-derived mesenchymal stem cells can be extracted from the tonsils according to methods known in the art. For example, it can be extracted from one way according to the method described in Korean Patent Registration No. 10-1508413.
편도 유래 중간엽 줄기세포의 조정 배지는 편도 유래 중간엽 줄기세포를 배양하고 세포를 제거한 후, 수득된 배양액, 배양 상층액 또는 이의 농축물이거나 이의 동결건조물일 수 있다.The media for adjusting the amygdala-derived mesenchymal stem cells may be cultured tonsil-derived mesenchymal stem cells and removed from the cells, and then the obtained culture medium, culture supernatant or a concentrate thereof, or a lyophilized product thereof.
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 또는 무혈청 배지에서 배양하여 수득된 것일 수 있다. 이를 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.The tonsil-derived mesenchymal stem cells can be cultured normally using a medium for culturing stem cells. The tonsil-derived mesenchymal stem cell culture may be obtained by culturing tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum or serum-free medium. The supernatant obtained after removing stem cells and macromolecules by centrifugation or filtration using a filter may be used. In addition, the obtained supernatant 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.The 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 can be appropriately selected or modified by those skilled in the art.
상기 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 medium is a medium suitable for maintaining and storing the same cell type as the tonsil-derived mesenchymal stem cells, and may be DMEM medium supplemented with serum. Serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents and mycoplasma inhibitors may be included, and these may be 1% by weight based on the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include gentamicin, ciprofloxacin, tyrosine, etc. , But is not limited to this.
상기 배지는 무혈청 배지일 수 있다. 바람직하게, 상기 무혈청 배지는 low glucose DMEM 배지일 수 있다.The medium may be a serum-free medium. Preferably, the serum-free medium may be low glucose DMEM medium.
본 발명의 일 실시양태에 따르면, 편도 유래 중간엽 줄기세포 조정배지는 편도 유래 중간엽 줄기세포를 무혈청 Low glucose DMEM 하에 배양하여 세포를 제거한 상층액을 농축한 것이다.According to one embodiment of the present invention, the tonsil-derived mesenchymal stem cell regulatory medium is concentrated by removing the tonsil-derived mesenchymal stem cells under serum-free low glucose DMEM to remove the cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, including the adjusted medium of tonsil-derived mesenchymal stem cells.
본 발명에서 예방은 상기 조성물의 투여로 암의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, 치료는 상기 조성물의 투여로 암의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.In the present invention, prevention means all actions to suppress or delay the onset of cancer by administration of the composition, and treatment refers to all actions that improve or benefit the symptoms of cancer by administration of the composition.
본 발명에 따른 암 예방 또는 치료용 약학 조성물은 EMT(epithelial-mesenchymal transition)를 억제하여 암의 진행을 억제함으로써 암 세포 사멸을 유도하고 암의 전이를 억제하여 암의 예방 및 치료에 우수한 효과를 가진다.The pharmaceutical composition for preventing or treating cancer according to the present invention suppresses EMT (epithelial-mesenchymal transition), thereby inhibiting cancer progression, thereby inducing cancer cell death and inhibiting cancer metastasis, thereby having excellent effects in preventing and treating cancer. .
본 발명의 약학 조성물은 통상의 방법에 따라 액제, 현탁액, 에멀젼, 로션, 연고, 동결건조제 등 다양한 제형으로 제제화될 수 있다. The pharmaceutical composition of the present invention can be formulated into various formulations such as liquids, suspensions, emulsions, lotions, ointments, lyophilizers according to 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 암 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 이를 이식받을 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 can be formulated and administered as a pharmaceutical preparation in a unit dosage form suitable for administration in a patient's body according to a conventional method in the pharmaceutical field, and the preparation is effectively administered by single or multiple administrations. Includes the amount. Formulations suitable for this purpose are preferably parenteral administration preparations such as injections, injections, and the like. In addition, the pharmaceutical composition for preventing or treating cancer may include a pharmaceutically acceptable inert carrier and a diluent. Pharmaceutically acceptable carriers and diluents that can be included in the pharmaceutical compositions of the present invention are excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin , Alginate, and binders such as polyvinyl pyrrolidone, talc, lubricants such as calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, interfaces such as polysorbate, 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 recipient to be transplanted. Diluents include, but are not limited to, brine, aqueous buffers, solvents and / or dispersion media. In addition, for example, in the case of an injection, a preservative, a painless agent, a solubilizing agent, or a stabilizer, etc., in the case of a formulation for topical administration, may further include a base, excipient, lubricant, or preservative. The composition of the present invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (e.g. DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) can be added to the cell population prior to 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 dosing method in the art, preferably it is possible to administer directly to the diseased area of the patient in need of treatment, but is not limited thereto. In addition, the administration is possible both by surgical methods such as non-surgical administration using a catheter and infusion after incision in a diseased area. The dosage may vary depending on the degree of concentration, but may be administered by dividing the body weight from 10 μl / kg to 1 ml / kg once or 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 various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and sex, and thus, the dosage The scope of the present invention is not limited in any way.
본 발명은 또한 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 예방 또는 개선용 식품 조성물을 제공한다.The present invention also provides a food composition for preventing or improving cancer, comprising a mediator media for 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 health functional food. The term "health functional food" refers to food manufactured and processed using ingredients or ingredients having useful functionality for the human body according to the Act on Health Functional Food, and "functional" refers to the structure and function of the human body. This means taking it for the purpose of controlling nutrients or obtaining useful effects for health purposes such as 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 rules and general test methods of food additives approved by the Ministry of Food and Drug Safety unless otherwise specified. It is judged according to the standards and standards for items.
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.Items listed in the "Food Additives Revolution" include, for example, chemical additives such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as chromochromate, licorice extract, crystalline cellulose, high color pigment, and guar gum, And mixed preparations such as an L-sodium glutamate preparation, an alkali added additive, a preservative preparation, and a tar colorant.
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 contain 0.01 to 95% by weight, preferably 5 to 90% by weight of the adjusted medium of tonsil-derived mesenchymal stem cells relative to the total weight of the composition.
또한, 본 발명의 식품 조성물은 암의 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 manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and / or improving cancer.
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, among the capsules of health functional foods, the hard capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as an adjusted medium of tonsil-derived mesenchymal stem cells according to the present invention, and excipients, Soft capsules may be prepared by filling a capsule base such as gelatin with a mixture of additives such as food compositions and excipients according to the present invention. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if 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 for the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, etc., are those described in the literature known in the art and include those having the same or similar functions (Korean Pharmacopoeia Commentary, Moonsungsa, Korea) College of Pharmacy, 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 functional foods in the ordinary sense.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 대상체에 투여하는 단계를 포함하는 암의 치료방법을 제공한다.The present invention provides a method of treating cancer comprising the step of administering to the subject the media for adjustment of the tonsil-derived mesenchymal stem cells.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 대상체에 투여하는 단계를 포함하는 암의 전이 억제 방법을 제공한다.The present invention provides a method for inhibiting metastasis of cancer comprising the step of administering to the subject a control medium of tonsil-derived mesenchymal stem cells.
본 발명에 "편도 유래 중간엽 줄기세포의 조정배지", "암", "투여" 등의 용어는 상기에서 설명한 바와 동일하다.In the present invention, terms such as "adjusted medium of tonsil-derived mesenchymal stem cells", "cancer", and "administration" are the same as described above.
상기 대상체는 동물을 말하며, 전형적으로 본 발명의 편도 유래 중간엽 줄기세포의 조정배지를 이용한 치료로 유익한 효과를 나타낼 수 있는 포유동물일 수 있다. 이러한 대상체의 바람직한 예로 인간과 같은 영장류가 포함될 수 있다. 또한 이와 같은 대상체들에는 암의 증상을 갖거나 이와 같은 증상을 가질 위험이 있는 대상체들이 모두 포함될 수 있다.The subject refers to an animal, and may be a mammal capable of exerting a beneficial effect, typically by treatment with a control medium of tonsil-derived mesenchymal stem cells of the present invention. Preferred examples of such subjects may include primates such as humans. In addition, such subjects may include all subjects who have symptoms of cancer or are at risk of having such symptoms.
본 발명은 또한 암의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다.The present invention also provides the use of a conditioned medium of tonsil derived mesenchymal stem cells in the manufacture of a medicament for the treatment of cancer.
본 발명은 또한 암의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물을 제공한다.The present invention also provides a composition comprising a conditioned medium of tonsil derived mesenchymal stem cells for use in the treatment of cancer.
본 발명은 또한 암의 치료를 위한 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다.The present invention also provides the use of a conditioned medium of tonsil-derived mesenchymal stem cells for the treatment of cancer.
본 발명의 조성물은 타 유래 중간엽 줄기세포 또는 이의 조정배지들과 상이한 단백질 프로파일을 가지고 이를 통해 EMT 억제작용으로 인한 암세포 이동 및 침투 등 암 전이를 초기 단계부터 억제하고 암세포 사멸을 유도하여, 암의 전이 억제와 암의 예방, 개선 및 치료에 우수한 효과를 가진다.The composition of the present invention has a different protein profile from other mesenchymal stem cells or their media, thereby suppressing cancer metastasis such as cancer cell migration and invasion due to EMT inhibition, and inducing cancer cell death. It has excellent effects on metastasis suppression and prevention, improvement and treatment of cancer.
도 1은 유래를 달리하는 중간엽 줄기세포의 조정배지에서의 CD109 분비량을 확인한 결과를 나타낸다.Figure 1 shows the results of confirming the amount of CD109 secretion in the control medium of mesenchymal stem cells of different origin.
도 2는 TGF-β처리에 의한 암 세포에서의 EMT 반응 유도를 확인한 결과를 나타낸다.Figure 2 shows the results confirming the induction of EMT response in cancer cells by TGF-β treatment.
도 3은 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 암 세포의 증식 억제 및 전이 억제를 확인한 결과를 나타낸다.Figure 3 shows the results of confirming the inhibition of proliferation and metastasis inhibition of cancer cells according to the control medium treatment of tonsil-derived mesenchymal stem cells.
도 4는 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 EMT 관련 유전자의 발현 변화를 확인한 결과를 나타낸다.Figure 4 shows the results of confirming the change in expression of the EMT-related gene according to the control medium treatment of tonsil-derived mesenchymal stem cells.
도 5는 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 암 세포주 이식 마우스 동물 모델에서의 암조직 형성 억제를 확인한 결과를 나타낸다.Figure 5 shows the results of confirming the inhibition of cancer tissue formation in a mouse animal model of cancer cell line transplantation according to the adjusted medium treatment of tonsil-derived mesenchymal stem cells.
도 6은 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 암세포 증식 억제 효과를 확인한 결과를 나타낸다.Figure 6 shows the results confirming the effect of inhibiting cancer cell proliferation according to the adjusted medium treatment of tonsil-derived mesenchymal stem cells.
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.In order to help the understanding of the present invention, examples and manufacturing examples are presented. The following examples and manufacturing examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the examples and the manufacturing examples.
<실시예 1> 지방, 골수 또는 편도 유래 중간엽 줄기세포 또는 이들의 조정배지의 제조<Example 1> Preparation of mesenchymal stem cells derived from fat, bone marrow, or tonsils or their media
편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell, T-MSC)는 이대목동병원 이비인후-두경부외과에서 편도적출술을 시행하는 환자로부터 적출된 편도조직(4-20세의 저연령층 조직)으로부터 얻었다. 이는 대한민국 등록 특허 제10-1508413호의 제조방법과 유사하게 제조되었다.Tonsil-derived mesenchymal stem cells (T-MSC) are derived from tonsil tissue (4-20 years old low-aged tissue) extracted from a patient performing tonsillectomy in the otolaryngology department of the otolaryngology department at Ewha Womans University Mokdong Hospital. Got. It was manufactured similarly to the manufacturing method of Korean Patent No. 10-1508413.
위 제조된 줄기세포로부터 조정 배지를 제조하기 위하여 Low glucose DMEM 로 편도 유래 중간엽 줄기세포를 배양하였다. 100 mm culture tissue plate에 confluency 70-80%가 되었을 때, 배지를 걷어내고 세포가 접시에 붙은 상태에서 PBS로 네 번 세척한 후 무혈청 배지 (Low glucose DMEM)를 첨가하였다. 48시간 후, 배지를 걷어 1300 rpm에서 5분간 원심분리 한 후 0.2 uM filter 로 상층액을 여과시킨다. 여과된 배지는 centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore)으로 20배 농축하여 실험에 이용하거나 -80℃에서 보관하였다.In order to prepare a conditioned medium from the above prepared stem cells, tonsil-derived mesenchymal stem cells were cultured with Low glucose DMEM. When the confluency reached 70-80% in a 100 mm culture tissue plate, the medium was removed, and the cells were adhered to the plate, washed four times with PBS, and then serum-free medium (Low glucose DMEM) was added. After 48 hours, the medium was collected, centrifuged at 1300 rpm for 5 minutes, and the supernatant was filtered with a 0.2 uM filter. The filtered medium was concentrated 20 times with centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore) for use in experiments or stored at -80 ° C.
지방 유래 중간엽 줄기세포(AT-MSC)는 RNL Bio에서 제공되는 동일 계대의 세포를 사용하였으며, 골수 중간엽 줄기세포의 경우 서울 연세대학교 세브란스 병원 세포치료센터로부터 제공되는 동일 계대의 세포를 사용하였다. 조정 배지의 제조의 경우 위 편도 유래 중간엽 줄기세포로부터의 조정 배지와 동일한 조건 하에 제조를 하였다.Adipose-derived mesenchymal stem cells (AT-MSC) used cells of the same passage provided by RNL Bio, and in the case of bone marrow mesenchymal stem cells, cells of the same passage from Seoul Yonsei University Severance Hospital Cell Therapy Center were used. . In the case of preparation of the conditioned medium, it was prepared under the same conditions as the conditioned medium from the mesenchymal stem cells derived from the stomach tonsil.
<실시예 2> 편도 유래 중간엽 줄기세포의 조정 배지의 특성 확인<Example 2> Confirmation of the characteristics of the media to adjust the tonsil-derived mesenchymal stem cells
유래를 달리하는 줄기세포의 조정배지의 특성을 확인하기 위하여 분비되는 단백질의 차이를 웨스턴 블랏을 통해 확인하였다.In order to confirm the characteristics of the control medium of stem cells having different origins, differences in secreted proteins were confirmed through Western blot.
구체적으로, 골수 중간엽 줄기세포, 지방 중간엽 줄기세포, 편도 줄기세포로부터 수득한 조정배지 각 25 ul를 10% SDS PAGE에서 전기영동하여 단백질을 분리시키고 PVDF membrane (Millipore)에 전이시켰다. 전이된 membrane은 5% 탈지분유액에 1시간 동안 블로킹한 후에 TBST 용액으로 3번 세척하였다. 일차 항체인 anti-CD109 antibody (B-9, mouse monoclonal, Santacruz Biotechnology)와 4℃에서 16시간 동안 반응시킨 후 TBST 용액으로 3번 세척하고 2차 항체인 anti-mouse antibody와 1시간 동안 상온에서 반응시켰다. TBST로 세 번 세척한 뒤 ECL kit (Thermo scientific)로 발색시켜 ImageQuant LAS 3000 (Fujifilm)로 촬영하였다.Specifically, 25 ul each of the adjusted medium obtained from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and tonsil stem cells was electrophoresed on 10% SDS PAGE to separate proteins and transfer them to a PVDF membrane (Millipore). The transferred membrane was blocked with 5% skim milk powder for 1 hour and then washed 3 times with TBST solution. After reacting with the primary antibody, anti-CD109 antibody (B-9, mouse monoclonal, Santacruz Biotechnology) for 16 hours at 4 ° C, wash 3 times with TBST solution and react with the secondary antibody, anti-mouse antibody, at room temperature for 1 hour. Ordered. After washing three times with TBST, color was developed with an ECL kit (Thermo scientific) and photographed with ImageQuant LAS 3000 (Fujifilm).
그 결과를 도 1에 나타내었다.The results are shown in FIG. 1.
도 1의 A는 서로 다른 공여자들로부터의 편도 유래 중간엽 줄기세포의 조정배지(T-MSC-CM1, CM2, CM3)의 CD109을 확인한 결과를 나타낸다. 도 1의 A에서 확인할 수 있는 바와 같이, 모두 동일하게 높은 수준으로 CD109가 분비된 것을 확인할 수 있었다.FIG. 1A shows the results of confirming CD109 of the adjusted medium (T-MSC-CM1, CM2, CM3) of tonsil-derived mesenchymal stem cells from different donors. As can be seen in FIG. 1A, it was confirmed that CD109 was secreted at the same high level.
반면, 도 1의 B는 유래가 다른 줄기세포의 조정 배지에서 분비 차이를 확인한 결과를 나타낸다. 도 1의 B에서 확인할 수 있는 바와 같이, 지방 유래(AD-MSC-CM) 및 골수 유래(BM-MSC-CM) 모두 CD109가 거의 확인되지 않고 편도 유래 중간엽 줄기세포의 조정 배지에서만 높게 나타나는 것을 확인할 수 있었다.On the other hand, B in Figure 1 shows the result of confirming the difference in secretion from the control medium of stem cells of different origin. As can be seen from B of FIG. 1, it was found that CD109 was rarely found in both adipose-derived (AD-MSC-CM) and bone marrow-derived (BM-MSC-CM), and was high only in the media for adjustment of tonsil-derived mesenchymal stem cells. I could confirm.
이는 TGF-β 시그널을 억제하는 단백질인 CD109 등이 EMT의 작용을 효과적으로 억제할 수 있고, 이러한 작용 효과가 편도 유래 중간엽 줄기세포의 조정배지를 통해서만 이루어질 수 있음을 보여준다.This shows that CD109, a protein that inhibits the TGF-β signal, can effectively suppress the action of EMT, and this effect can be achieved only through the mediation of the amygdala-derived mesenchymal stem cells.
<실시예 3> 편도 유래 중간엽 줄기세포의 조정배지 투여에 의한 TGF-β유도 EMT 반응 억제<Example 3> Inhibition of TGF-β-induced EMT response by administration of adjusted medium of tonsil-derived mesenchymal stem cells
HepG2 세포를 6 well plate에 seeding 하여 MEM 배지 (10% FBS, 1% penicillin/streptomycin)로 배양하였다. TGF-β를 각각 0, 20, 50 ng/ml 농도로 첨가하여 72시간 동안 배양하였다. 한편, 편도 줄기세포 조정배지의 첨가 효과를 확인하기 위해, 20 ng/ml 농도의 TGF-β를 처리한 군에 편도 줄기세포의 조정배지 (10
6 세포로부터 얻은 양, 250 ul)을 첨가하여 배양하였다.HepG2 cells were seeded in 6 well plates and cultured in MEM medium (10% FBS, 1% penicillin / streptomycin). TGF-β was incubated for 72 hours by adding 0, 20, and 50 ng / ml concentrations, respectively. On the other hand, in order to confirm the effect of the addition of the tonsil stem cell control medium, culture was performed by adding the control medium of the tonsil stem cell (amount obtained from 10 6 cells, 250 ul) to the group treated with TGF-β at a concentration of 20 ng / ml. Did.
또한, TGF-β(20 ng/ml)를 처리하여 72시간 동안 배양한 HepG2 세포, TGF-β(20 ng/ml)와 편도 줄기세포 조정배지 (10
6 세포로부터 수득, 250 ul)을 함께 첨가하여 72시간 동안 배양한 HepG2 세포, 정상 대조군 HepG2 세포를 모아서 Trizol reagent (Invitrogen)를 사용하여 전체 RNA를 분리하였다. 분리한 전체 RNA 1 ug에 5X Reverse transcription premix (ELPIS BIOTECH)를 첨가하여 42℃에서 1시간 동안 반응시켜 cDNA를 합성하였다. EMT 마커 유전자들인 human CD44, human CD133, human CD151, human EPCAM, human p53, human K19의 발현을 하기 표 1의 특이적인 프라이머로 StepOnePlus real-time PCR system (Applied biosystems)에서 증폭시켜 확인하였다. 각 유전자들의 발현은 housekeeping 유전자인 gapdh의 발현량으로 normalization 하였다. 각 샘플간의 상대적인 발현량 차이는 2-
ΔΔCT 방법을 이용하여 산출하였다.In addition, TGF-β (20 ng / ml) was treated and HepG2 cells cultured for 72 hours, TGF-β (20 ng / ml) and tonsil stem cell control medium (obtained from 10 6 cells, 250 ul) were added together. Then, HepG2 cells cultured for 72 hours and normal control HepG2 cells were collected and total RNA was isolated using Trizol reagent (Invitrogen). CDNA was synthesized by adding 5X Reverse transcription premix (ELPIS BIOTECH) to 1 ug of isolated RNA for 1 hour at 42 ° C. Expression of EMT marker genes human CD44, human CD133, human CD151, human EPCAM, human p53, and human K19 was confirmed by amplifying in StepOnePlus real-time PCR system (Applied biosystems) with specific primers in Table 1 below. The expression of each gene was normalized to the expression level of gapdh, a housekeeping gene. The relative expression level difference between each sample was calculated using the 2- ΔΔCT method.
[표 1][Table 1]
이와 별도로, HepG2 세포를 5uM 농도의 CFSE로 태깅한 후 TGF-β(20 ng/ml)를 넣어 72시간 동안 배양하였다. 여기에 편도 줄기세포의 조정배지 (10
6 세포로부터 얻은 양, 250 ul)을 첨가하거나 첨가하지 않고 배양하였다. 72시간 후 유세포 분석기로 CFSE 형광 정도를 측정하였다.Separately, HepG2 cells were tagged with CFSE at a concentration of 5 uM, and then cultured for 72 hours with TGF-β (20 ng / ml). Here, the culture medium of tonsil stem cells (amount obtained from 10 6 cells, 250 ul) was cultured with or without addition. After 72 hours, the degree of CFSE fluorescence was measured by a flow cytometer.
TGF-β 처리에 의한 EMT 반응 유도를 확인하여, 그 결과를 도 2에 나타내었다.The induction of EMT reaction by TGF-β treatment was confirmed, and the results are shown in FIG. 2.
도 2의 A에서 확인되는 바와 같이, 간암 세포주인 HepG2 세포에 TGF-β를 첨가하여 72시간 배양한 결과, 세포의 두드러진 증식을 확인할 수 있었다.2A, TGF-β was added to HepG2 cells, a liver cancer cell line, and cultured for 72 hours. As a result, prominent proliferation of cells was confirmed.
또한, EMT 관련 마커 유전자의 발현 변화를 본 결과, CD44, CD133, CD151, K19가 20 ng/ml의 농도에서도 뚜렷하게 증가한 것을 확인할 수 있었다.In addition, as a result of looking at the expression change of the EMT-related marker gene, it was confirmed that CD44, CD133, CD151, and K19 increased significantly even at a concentration of 20 ng / ml.
편도 줄기세포 조정배지에 의한 간암 세포주 증식 억제 효과를 확인하여, 그 결과를 도 3에 나타내었다.The effect of inhibiting the proliferation of the liver cancer cell line by the tonsil stem cell media was confirmed, and the results are shown in FIG. 3.
도 3의 A는 현미경을 통하여 확인한 세포 증식 억제 효과를 나타내며, 도 3의 B는 CFSE 형광 측정에 따른 세포 증식 억제 효과를 나타낸다. CFSE는 살아있는 세포의 세포질에 염색되어 형광 정도를 측정하면 몇 번의 분열을 거쳤는지 알 수 있는데, 도 3의 히스토그램 상의 서로 다른 색깔의 peak는 증식한 세포들의 generation을 의미한다. 도 3에서 확인되는 바와 같이 편도 유래 중간엽 줄기세포의 투여는 EMT 반응이 유도된 간암 세포에서 세포 증식을 억제하는 것을 확인할 수 있었다.3A shows the effect of inhibiting cell proliferation confirmed through a microscope, and FIG. 3B shows the effect of inhibiting cell proliferation according to CFSE fluorescence measurement. CFSE is stained on the cytoplasm of living cells to measure the degree of fluorescence, and it can be seen how many divisions have been made. Different colored peaks on the histogram of FIG. 3 indicate generation of proliferated cells. As can be seen in Figure 3, administration of tonsil-derived mesenchymal stem cells was confirmed to inhibit cell proliferation in hepatocellular carcinoma cells induced EMT response.
편도 줄기세포 조정배지에 의한 EMT 억제 효과를 확인하여, 그 결과를 도 4에 나타내었다. 도 4에서 확인되는 바와 같이, TGF-β처리에 의해 증가된 CD44, CD133, CD151, K19 및 EPCAM의 발현 수준이 모두 감소하는 것을 확인할 수 있었다.The effect of inhibiting EMT by tonsil stem cell media was confirmed, and the results are shown in FIG. 4. As can be seen in Figure 4, it was confirmed that the expression levels of CD44, CD133, CD151, K19 and EPCAM increased by TGF-β treatment are all decreased.
위 결과를 통해 편도 유래 중간엽 줄기세포의 조정배지가 EMT를 억제하여 암의 예방 및 치료에 우수한 효과를 나타내는 것을 확인할 수 있다.Through the above results, it can be seen that the media for mediation of tonsil-derived mesenchymal stem cells suppresses EMT and shows excellent effects in the prevention and treatment of cancer.
<실시예 3> 편도 유래 중간엽 줄기세포의 조정배지 투여에 의한 암 조직 형성 억제<Example 3> Inhibition of cancer tissue formation by administration of adjusted medium of tonsil-derived mesenchymal stem cells
Balb/c nude 마우스 (수컷, 5주령)의 옆구리에 HepG2 세포 3X10
6 개를 이식하였다. 이때, 세포를 DMEM 배지에 부유시켜 주사하거나 혹은 편도 세포 조정배지 (7X10
5 개 세포로부터 얻음)에 부유시켜 주사하였다. 그리고나서, Balb/c nude 마우스에 HepG2 세포 3X10
6 개를 이식하고 5일 후, 육안으로 보이는 암 조직을 분리하여 MEM 배지에 담아 잘게 chopping 하였다. Chopping한 세포 부유액을 그대로 100 mm plate에 seeding하여 3일 후 배지를 걷어내고 현미경으로 관찰하였다. Six HepG2 cells 3X10 were implanted in the flank of Balb / c nude mice (male, 5 weeks old). At this time, the cells were injected by being suspended in DMEM medium or injected into the tonsil cell control medium (obtained from 5 cells of 7X10). Then, 6 HepG2 cells 3X10 were transplanted into Balb / c nude mice, and after 5 days, the cancerous tissues visible to the naked eye were separated and chopping finely in MEM medium. The suspended cell suspension was seeded on a 100 mm plate, and after 3 days, the medium was removed and observed under a microscope.
위 결과를 도 5 및 6에 나타내었다.The above results are shown in FIGS. 5 and 6.
도 5는 세포 이식 후 5일(A) 및 9일(B)이 지났을 때 암의 크기를 확인한 결과를 나타낸다. 일반 DMEM 배지에 부유시켜 이식한 경우와 비교하여, 이식 부위에서 암 조직의 크기가 현저히 억제되었음을 확인하였다.Figure 5 shows the results of confirming the size of cancer when 5 days (A) and 9 days (B) have passed after cell transplantation. It was confirmed that the size of the cancer tissue was significantly suppressed at the implantation site compared to the case where it was transplanted by being suspended in a normal DMEM medium.
도 6은 간암 세포주 이식 마우스 모델에서 분리한 암 조직의 체외 배양시 편도 줄기세포 조정배지 이식군에서 분리한 조직 세포가 느리게 증식함을 보여준다.Figure 6 shows that the tissue cells isolated from the tonsil stem cell control medium transplantation group proliferate slowly when in vitro culture of cancer tissues isolated from the mouse model of liver cancer cell line transplantation.
도 6의 A는 배양 2일 및 9일째의 현미경 사진을 나타낸다. 마우스에 세포를 이식한 부위에 형성된 암조직을 분리하여 세포 부유액을 배양해본 결과 편도 줄기세포 조정배지를 함께 이식했던 마우스 유래의 암조직 세포들이 더디게 증식하는 것을 확인할 수 있었다. 또한, 도 6의 B에서 확인되는 바와 같이, 배양 이틀째 현미경을 통해 세포들의 cluster를 확인하여 보면, 편도 유래 중간엽 줄기세포의 조정배지에서 배양한 조직이 클러스터 형성이 더 적게 이루어짐을 확인할 수 있었다. 이는 DMEM에서 키운 세포들이 세포간 상호작용이 활발하여 증식이 빠르게 진행된 반면, 편도 유래 중간엽 줄기세포의 조정 배지를 처리한 그룹은 그러한 작용이 적음을 보여준다.6A shows micrographs on the 2nd and 9th days of culture. As a result of culturing the cell suspension by separating the cancerous tissue formed at the site where the cells were transplanted into the mouse, it was confirmed that cancerous tissue cells derived from the mouse transplanted together with the tonsil stem cell control medium slowly proliferated. In addition, as shown in B of FIG. 6, when the clusters of cells were checked through a microscope on the second day of culture, it was confirmed that the tissues cultured in the control medium of the amygdala-derived mesenchymal stem cells formed less clusters. This shows that while the cells grown in DMEM had active intercellular interactions, the proliferation proceeded rapidly, whereas the group treated with the control medium of tonsil-derived mesenchymal stem cells had little effect.
위 확인되는 바와 같이, 편도 유래 중간엽 줄기세포의 조정 배지는 EMT (epithelial to mesenchymal transition)를 억제함으로써 암의 생성 및 진행을 억제하고 우수한 항암 효과를 가진다. 특히, 편도 유래 중간엽 줄기세포의 조정배지의 효과는 골수 유래 또는 지방 유래 중간엽 줄기세포의 조정배지들에서 확인되지 않는 인자들에 의한 것으로, 이를 통해 편도 유래 중간엽 줄기세포의 조정배지가 타유래 중간엽 줄기세포 및 이의 조정배지와 다른 특성을 지니고 암의 전이 억제, 암의 예방 및 치료에 우수한 효과를 나타내는 것을 확인하였다.As can be seen from the above, the media for mediation of tonsil-derived mesenchymal stem cells suppresses the generation and progression of cancer by suppressing epithelial to mesenchymal transition (EMT) and has excellent anticancer effects. In particular, the effect of the mediation of the tonsil-derived mesenchymal stem cells is due to factors not identified in the bone marrow-derived or adipose-derived mesenchymal stem cells. It was confirmed that it has different characteristics from the derived mesenchymal stem cells and their media, and shows excellent effects in inhibiting cancer metastasis and preventing and treating cancer.
Claims (15)
- 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 암 전이 억제용 약학 조성물.A pharmaceutical composition for inhibiting cancer metastasis, including a control medium of tonsil-derived mesenchymal stem cells.
- 제1항에 있어서, 편도 유래 중간엽 줄기세포의 조정 배지는 무혈청 배지에서 배양된 배양액, 배양상층액 또는 이의 농축물인 암 전이 억제용 약학 조성물.The pharmaceutical composition for inhibiting cancer metastasis according to claim 1, wherein the mediation medium for tonsil-derived mesenchymal stem cells is a culture medium, a culture supernatant, or a concentrate thereof cultured in a serum-free medium.
- 제1항에 있어서, 편도 유래 중간엽 줄기세포의 조정 배지는 CD109를 포함하는 것인 암 전이 억제용 약학 조성물.The pharmaceutical composition for inhibiting cancer metastasis according to claim 1, wherein the median medium of tonsil-derived mesenchymal stem cells comprises CD109.
- 제1항에 있어서, 편도 유래 중간엽 줄기세포의 조정 배지는 EMT(epithelial-mesenchymal transition)를 억제하는 것인 암 전이 억제용 약학 조성물.The pharmaceutical composition for inhibiting cancer metastasis according to claim 1, wherein the median medium of tonsil-derived mesenchymal stem cells inhibits epithelial-mesenchymal transition (EMT).
- 제1항에 있어서, 암은 전이 암인 암 전이 억제용 약학 조성물.The pharmaceutical composition for inhibiting cancer metastasis according to claim 1, wherein the cancer is metastatic cancer.
- 제1항에 있어서, 암은 폐암, 유방암, 간암, 대장암, 위암, 뇌암, 췌장암, 갑상선암, 피부암, 골수암, 림프종, 자궁암, 자궁경부암, 신장암 및 흑색종으로 이루어진 군으로부터 선택되는 어느 하나인 암 전이 억제용 약학 조성물.The method of claim 1, wherein the cancer is any one selected from the group consisting of lung cancer, breast cancer, liver cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, kidney cancer and melanoma. Pharmaceutical composition for inhibiting cancer metastasis.
- 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 암 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prevention or treatment of cancer, comprising a mediating medium of tonsil-derived mesenchymal stem cells.
- 제7항에 있어서, 편도 유래 중간엽 줄기세포의 조정 배지는 무혈청 배지에서 배양된 배양액, 배양상층액 또는 이의 농축물인 암 예방 또는 치료용 약학 조성물.The pharmaceutical composition for the prevention or treatment of cancer according to claim 7, wherein the mediation medium for tonsil-derived mesenchymal stem cells is a culture medium, a culture supernatant, or a concentrate thereof cultured in a serum-free medium.
- 제7항에 있어서, 편도 유래 중간엽 줄기세포의 조정 배지는 CD109를 포함하는 것인 암 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating cancer of claim 7, wherein the media for mediating tonsil-derived mesenchymal stem cells comprises CD109.
- 제7항에 있어서, 암은 폐암, 유방암, 간암, 대장암, 위암, 뇌암, 췌장암, 갑상선암, 피부암, 골수암, 림프종, 자궁암, 자궁경부암, 신장암 및 흑색종으로 이루어진 군으로부터 선택되는 어느 하나인 암 예방 또는 치료용 약학 조성물.The method of claim 7, wherein the cancer is any one selected from the group consisting of lung cancer, breast cancer, liver cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, kidney cancer and melanoma. Pharmaceutical composition for preventing or treating cancer.
- 편도 유래 중간엽 줄기세포의 조정 배지를 포함하는 암 예방 또는 개선용 식품 조성물.A food composition for preventing or improving cancer, comprising a conditioned medium of tonsil-derived mesenchymal stem cells.
- 암의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도.Use of media for mediation of tonsil derived mesenchymal stem cells in the manufacture of a medicament for the treatment of cancer.
- 암의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물.A composition comprising a conditioned medium of tonsil-derived mesenchymal stem cells for use in the treatment of cancer.
- 편도 유래 중간엽 줄기세포의 조정배지를 대상체에 투여하는 단계를 포함하는 암의 치료방법.A method of treating cancer comprising the step of administering to the subject an adjusted medium of tonsil-derived mesenchymal stem cells.
- 편도 유래 중간엽 줄기세포의 조정배지를 대상체에 투여하는 단계를 포함하는 암의 전이 억제방법.A method for inhibiting metastasis of cancer, comprising administering to the subject a media to adjust the tonsil-derived mesenchymal stem cells.
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