WO2018186505A1 - Composition for treating scars, improving skin, and preventing or treating hair loss including stem cell-derived exosome, and method for preparing same - Google Patents

Composition for treating scars, improving skin, and preventing or treating hair loss including stem cell-derived exosome, and method for preparing same Download PDF

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Publication number
WO2018186505A1
WO2018186505A1 PCT/KR2017/003645 KR2017003645W WO2018186505A1 WO 2018186505 A1 WO2018186505 A1 WO 2018186505A1 KR 2017003645 W KR2017003645 W KR 2017003645W WO 2018186505 A1 WO2018186505 A1 WO 2018186505A1
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centrifugation
exosomes
skin
minutes
recovering
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PCT/KR2017/003645
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French (fr)
Korean (ko)
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김수
이원종
최은욱
서민구
우은영
박은주
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㈜프로스테믹스
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Publication of WO2018186505A1 publication Critical patent/WO2018186505A1/en

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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
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic 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
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth

Definitions

  • the present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.
  • Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, both embryonic and adult stem cells have the ability to differentiate into different cell types. Embryonic stem cells have very good differentiation ability and long telomeres, but they have ethical problems and difficult to obtain large amounts of cells, while adult stem cells can obtain a large number of cells but can be transplanted to others. The risk of infection or differentiation is relatively low.
  • the compounds include cyclosporin derivatives (Korean Patent No. 10-0439467, Korean Patent No. 10-0695611), N-heterocyclic carboxylic acid and carbamate (Korean Patent No. 10-2001-0052502), quina Zolinone derivatives (Korean Patent Laid-Open Publication No. 10-1999-0023754), chrysine 7-O-crotonate (Korean Patent Publication 10-2001-0009474) and 1,2-disubstituted benzenecarboxamide derivatives 1999-0037394) have been reported.
  • such a hair loss treatment or hair growth improving composition is a chemical composition, which can cause fatal side effects for women and infants, and also has side effects such as acceleration of hair loss due to dermatitis in men. Side effects that can be used with prolonged use can also increase patient pain.
  • One object of the present invention is to provide a composition for treating wounds that can stably treat or promote wounds using stem cells or culture-derived exosomes.
  • Still another object of the present invention is to provide a composition for preventing or treating hair loss having a safe but excellent hair growth and hair growth effect using the exosome derived from the stem cells or the culture medium.
  • Wounds are also called wounds, and depending on the cause, they are divided into intestines, jaws, insoles, acne, fissures, insoles, school windows, etc. Symptoms of the wound include pain, bleeding, dysfunction, and when the wound, the living body exhibits a variety of physiological reactions to heal. Normally, injured cells undergo denaturation and death processes, causing flow of cells and tissue fluid to flow out from surrounding tissues, and precipitation of fibrin and granulation tissue are formed to heal wounds.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • TGF- ⁇ TGF- ⁇
  • IGF-I Insulin-like Growth Factor-1
  • the skin consists of the epidermis, the dermis and the subcutaneous tissue.
  • the epidermis the outer layer of skin, consists of the stratified squamous epithelium and acts as a protective barrier for the body to the outside environment.
  • the epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale.
  • the dermis is the layer between the epidermis and the subcutaneous tissue, divided into papillary and reticular dermis, blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, Korean glands, various Sensory nerves, fibroblasts and macrophages are present and occupy the largest part of the skin.
  • the dermis consists primarily of collagen and elastin fibers, which support the skin. Therefore, when such a problem occurs in the dermis, wrinkles occur and skin elasticity is lost, thereby aging the skin.
  • Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function that can contain a large amount of moisture, which serves to supply moisture to the dermis. When aging, the collagen loses its water-containing function and wrinkles increase. Collagen can also heal wounds by filling fibroblasts with sustained collagen production when the wound is injured.
  • Hair loss is caused by a variety of factors, including the slow growth of hair and the lack of growth factors that promote hair root cell growth.
  • This hair loss reduces the quality of life due to mental stress and causes a lot of interpersonal or social life.
  • the image of appearance in social life is very spectacular, and interest in the cause and treatment of hair loss is increasing day by day because the importance of appearance becomes an invisible vitality of self-esteem or social life.
  • the stem cell or its culture-derived exosomes are excellent in skin permeability, and excellent in the effects of wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, in addition to hair growth and hair growth effects Discovering outstanding things led to the present invention.
  • compositions for wound treatment comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the wound pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with a high purity and content of exosomes excellent in wound healing through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • a cosmetic composition for skin improvement comprising the adult stem cells or culture medium derived from the exosome (exosome) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the cosmetic composition for skin improvement, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with high purity and content exosomes excellent skin improvement effect through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • a pharmaceutical composition for preventing or treating hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • a cosmetic composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • a food composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the hair loss prevention or treatment pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with a high purity and content of exosomes excellent in hair growth and hair growth effect through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • the adult stem cell refers to an undifferentiated cell having a multipotency derived from an adult human cell of a mammal, including a human, for example, bone marrow, blood, brain, skin, fat (ie , Adipose tissue or fat cells), umbilical cord blood, umbilical cord Barton's jelly, and the like.
  • the "adult stem cells” include mesenchymal stem cells derived from adult cells.
  • Adipose-derived stem cells are known from known methods (e.g., WO2000 / 53795 and WO2005 / 042730) from adipose tissue or adipocytes of mammals including humans, preferably humans. It can be obtained through processes such as liposuction and sedimentation, enzymatic treatment of collagenase and the like, and removal of floating cells such as red blood cells by centrifugation.
  • the adipose tissue includes brown or white tissue derived from subcutaneous, retinal, visceral, breast gonad or other adipose tissue sites and can be readily obtained from conventional liposuction.
  • Exosomes in the present invention refers to the small vesicles of the membrane structure secreted from various cells.
  • the diameter of the exosome is approximately 30 to 1,000 nm, which means that the vesicle is released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane.
  • Representative expression markers of exosomes correspond to HSP70, CD63 and CD9.
  • the pharmaceutical composition may further include a suitable carrier, excipient or diluent according to a conventional method.
  • Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, may be used, but is not limited thereto.
  • compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to a conventional method.
  • when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used.
  • Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed.
  • the dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
  • the food composition may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads, and the like. Since the food composition of the present invention is composed of plant extracts having little toxicity and no side effects, it can be used with confidence even for long-term use for prophylactic purposes.
  • the amount may be added at a ratio of 0.1 to 50% of the total weight.
  • natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).
  • compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • additives can be used independently or in combination.
  • the proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.
  • the cosmetic composition is a lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Creams, suntan creams, skin lotions, skin creams, sunscreen cosmetics, cleansing milk, depilatory ⁇ cosmetic ⁇ , face and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, cosmetic creams, jasmine Oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Cleanser (Hand Cleaner), Medicated Soap ⁇ Non-Medical ⁇ , Cream Soap, Facial Wash, Systemic Cleanser, Scalp Cleaner, Hairrin, Cosmetic Soap, Tooth Whitening Gel, Toothpaste Or the like.
  • the composition of the present invention may further comprise a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of the cosmetic composition.
  • the kind of the solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used, and as the carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like.
  • a whitening agent, a moisturizing agent, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.
  • the oil may be hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin Can be used.
  • Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as fatty acids, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol.
  • Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the fatty acid ester.
  • surfactants cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible.
  • it may include a hygroscopic agent, a thickener, an antioxidant, and the like, which are widely known in the cosmetic field, and their types and amounts are known in the art.
  • Stem cells or the culture-derived exosomes provided by the present invention promote cell migration to the wound site and have excellent wound healing or wound healing promoting activity.
  • the stem cells or the culture-derived exosomes provided by the present invention is excellent in the hair growth and hair growth effect is excellent in the prevention and treatment of hair loss, it is safe without side effects even when ingested or applied to the skin.
  • Figure 1 shows a TEM picture of the exosomes in Experimental Example 1
  • (b) shows a graphical analysis of the size of the particles present in the exosomes.
  • Figure 2 (a) is a photograph confirming the expression of HSP70, CD63 and CD9 markers of exosomes using Western blot in Experimental Example 1, (b) is a CD24 and AQP2 markers of exosomes using real-time PCR The degree of expression is shown in a graph, and (c) and (d) show the expression of CD63, CD9 and CD81 markers of exosomes using FACS.
  • Figure 3 is a photograph showing the change in the movement of human skin fibroblasts to the wound site after treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed.
  • Figure 4 is a graph showing the ratio of human skin fibroblasts moved to the wound site after the treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed .
  • FIG. 5 is a graph showing the expression levels of procollagen, KGF and CD34 after treatment of the exosomes obtained in Example 3 in Experimental Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed.
  • Figure 6 (a) and (b) is a graph showing the expression level of procollagen and elastin for each concentration after the treatment of the exosomes obtained in Example 3 in Experimental Example 4.
  • Figure 7 (a) to (c) is a graph showing the expression level of KGF, CD34 and VEGF by concentration after the exosome treatment obtained in Example 3 in Experimental Example 5.
  • Example 8 is a graph showing the expression levels of LEF-1, PTC-1 and KGF after treatment of the exosomes obtained in Example 3 and Experimental Example 6 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. will be.
  • the stem cell or its culture-derived exosomes have excellent skin permeability, and excellent effects such as wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, and also excellent hair growth and hair growth effects.
  • Consent was obtained from a patient who had undergone a subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction.
  • the extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes in an incubator at about 37 ° C. and at about 5% CO 2 , and then the resulting adipose tissue was centrifuged at about 1200 g for 5 minutes to form stromal cells containing high density stem cells.
  • Vascular fractions were obtained. The obtained fractions were washed with PBS, other tissues were removed through a 70 ⁇ m nylon cell strainer, and histopaque-1077 (Sigma Co.) was used to separate cell debris and mononuclear cells.
  • the isolated mononuclear cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin as a medium at about 37 ° C., about 5% CO 2 incubator. After culturing for 24 hours at, the adhering stem cells were isolated by removing non-adherent cells.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin / streptomycin penicillin / streptomycin
  • Adipose derived stem cells 1.25 ⁇ 10 5 cells obtained in Example 1 were prepared with 1000 mg / L D glucose, 584 mg / L L-glutamine, 110 mg / L sodium pyruvate, 10% by volume FBS, 1% by volume penicillin -Added to DMEM medium supplemented with streptomycin and passaged for 30 days in a 5% by volume CO 2 incubator under conditions of about 90% humidity and about 37 ° C.
  • the cultures were removed from the passaged culture using a pipette, washed three times with phosphorylation buffer solution, and then supplemented with 365 mg / L L-glutamine, 15 mM HEPES, 55 mg / L sodium pyruvate. Inoculated at a concentration of 1.2 ⁇ 10 6 cells / dish in a 1: 1 (weight ratio) mixed medium of DMEM and Ham's F-12 nutrient mixtures and incubated for 72 hours under hypoxia conditions.
  • Example 2 50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 g for 20 minutes at 4 ° C., and the obtained supernatant was transferred to a tube capable of a new high speed centrifuge, followed by high speed centrifugation at 10,000 g for 30 minutes. .
  • the supernatant was transferred to a new ultra-centrifugal polycarbonate bottle, and the supernatant was removed by ultrafast centrifugation using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C. .
  • the settled pellet was resuspended using a micropipette in a tube with 1000 ⁇ l PBS.
  • the resuspended tube was centrifuged for 70 minutes at 100,000 g at 4 ° C. using an OPTIMA MAX-XP ultracentrifuge from Beckman Coulter, and all possible supernatants were removed and the pellet was recovered.
  • a small amount of PBS was added to the recovered pellet, resuspended, separated into 100 ⁇ l, stored at ⁇ 80 ° C., and dissolved if necessary.
  • Example 2 50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and then centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 ° C. for 20 minutes at 4 ° C. The supernatant after 20 min centrifugation at 2,000 ° C. at 4 ° C. was transferred to a new high-speed centrifuge tube, followed by high-speed centrifugation at 10,000 ° C. for 30 minutes at 4 ° C.
  • the supernatant was transferred to a new polycarbonate bottle for ultrafast centrifuge, ultrafast centrifuged using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C.
  • the removed fat-derived stem cell culture was prepared.
  • Example 3 The exosomes obtained in Example 3 were observed through a transmission electron microscope (TEM), the photograph is shown in Figure 1 (a), and the particles present in the exosomes obtained as described above through the nanoparticle tracking analysis (NTA) The distribution of each size was analyzed and the results are shown graphically in FIG.
  • TEM transmission electron microscope
  • NTA nanoparticle tracking analysis
  • the exosomes isolated from the adipose-derived stem cell cultures can be confirmed to have a fine spherical structure in nanometers, and as shown in FIG. It can be seen that mainly distributed in 30 ⁇ 150nm.
  • exosomes isolated from the adipose derived stem cell cultures have the characteristics of typical exosomes.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well and then treated with the exosomes obtained in Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. After 48 hours of incubation, RNA was extracted from the cells of each group, and then complementary DNA (cDNA) was synthesized using intron premix and real-time PCR was performed using the same. As genes related to skin anti-aging and regeneration in the cells of each treatment group, the expression of procollagen, KGF and CD34 was confirmed and the results are shown graphically in FIG. 5.
  • cDNA complementary DNA
  • the procollagen maintains skin elasticity and corresponds to a gene related to collagen synthesis necessary for the formation of the main protein of the skin.
  • KGF keratinocyte growth factor
  • CD34 corresponds to a gene involved in skin regeneration as a factor involved in blood vessel regeneration.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well, and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 ⁇ g / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in increasing skin anti-aging elasticity in the cells of each treatment group, the expression level of procollagen and elastin was confirmed, and the results are shown graphically in FIG. 6. However, the control group was not treated with exosomes.
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in maintaining skin elasticity and preventing aging.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 ⁇ g / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in skin regeneration in the cells of each treatment group, the expression levels of KGF, CD34 and VEGF were confirmed and the results are shown graphically in FIG. 7. However, the control group was not treated with exosomes.
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in skin regeneration.
  • Example 3 After attaching 5 X 10 5 human dermal papilla cells to a 60 mm (SPL, Korea) culture dish, the exosomes obtained in Example 3 and the adipose derived stem cell cultures from which the exosomes obtained in Comparative Example 1 were removed were treated. . After 48 hours of incubation, the cells were recovered, RNA was isolated using RNA isolation kit (Quiagen, USA), and cDNA was synthesized for 1 ug of RNA isolated using cDNA synthesis kit (Intronbio, Korea). Each primer was synthesized in Cosmojintech, primers were used at a concentration of 10 pmol / ul, real-time PCR was detected by the addition of 2X Cyber Green Master Mix (Takara, Japan). As genes involved in hair follicle growth and development in cells of each treatment group, the expression levels of LEF-1, PTC-1 and KGF were confirmed, and the results are shown graphically in FIG. 8.
  • lymphoid enhancer-binding factor-1 (LEF-1) is a gene that promotes the formation and differentiation of hair follicles
  • PTC-1 patched protein, Sonic Hedgehog (Shh) receptor
  • KGF keratinocyte growth factor
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in the growth, development and differentiation of hair follicles.
  • the present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.

Abstract

The present invention relates to a composition which includes an exosome derived from stem cells or culture media thereof, and has the effects of treating scars, improving skin, and preventing or treating hair loss. The stem cell or stem cell culture medium-derived exosome provided in the present invention has excellent skin permeability, and also has excellent skin moisturizing, skin whitening, wrinkle improving, and anti-aging effects when absorbed, and does not irritate the skin or have side-effects on the skin, thus being safe. Moreover, the stem cell or stem cell culture medium-derived exosome has outstanding hair growth and hair restoration effects, and thus exhibits outstanding hair loss prevention and treatment effects. The exosome can be ingested or applied onto the skin without any side-effects, and is thus safe.

Description

줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법Wound treatment, skin improvement and hair loss prevention or treatment composition comprising stem cell-derived exosomes and method for producing same
본 발명은 줄기세포 또는 그 배양액 유래 엑소좀을 포함하며, 상처 치료, 피부 개선 및 탈모 방지 또는 치료의 효과를 갖는 조성물 및 그 제조방법에 관한 것이다.The present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.
줄기세포란 특정한 세포로 분화가 진행되지 않은 채 유지되다가, 필요한 경우 신경, 혈액, 연골 등 신체를 구성하는 모든 종류의 세포로 분화할 능력을 가진 세포를 말한다. 이러한 줄기세포를 얻을 수 있는 방법은 크게 두 가지가 있는데, 첫째는 수정란으로부터 발생한 배아로부터 얻는 것(배아줄기세포)이고 둘째는 성인이 된 우리 몸의 각 부분에 간직되어 있는 줄기세포(성체줄기세포)를 회수하는 것이다. 기능적인 면에서 차이는 있지만 배아줄기세포나 성체줄기세포는 모두 여러 종류의 세포로 분화할 수 있는 능력을 가지고 있다. 배아줄기세포는 분화능력이 매우 뛰어나고 텔로미어가 긴 장점이 있으나, 윤리적인 문제를 안고 있고 세포를 다량 획득하는 것이 어려운 단점이 있는 반면, 성체줄기세포는 세포 수를 많이 얻을 수는 있으나 타인에게 이식할 때 감염의 위험이나 분화능력이 상대적으로 떨어지는 단점이 있다. Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, both embryonic and adult stem cells have the ability to differentiate into different cell types. Embryonic stem cells have very good differentiation ability and long telomeres, but they have ethical problems and difficult to obtain large amounts of cells, while adult stem cells can obtain a large number of cells but can be transplanted to others. The risk of infection or differentiation is relatively low.
성체 줄기세포는 의학적으로 적용하기에 대단히 안전하다는 특징이 있다. 구체적으로, 장기 재생을 위해 몸 안에 이식하여도 암이 발생하지 않으며, 성인의 몸 속에 있었기 때문에 면역거부반응이 발생하지 않아 자기 자신의 세포를 사용하는 자가 이식(autologous transplantation)이 가능하다. 또한, 주변 조직의 특성에 자신을 맞추어 분화하는 조직 특이적 분화능력(site-specific differentiation)이 있고, 미분화 상태에서 주입하여도 암을 유발하지 않기 때문에 이식된 이후 당장 필요한 세포를 만들어내는 것 이외에도 나중에 필요한 미분화 상태의 줄기세포를 다시 만들어서 저장하는 자가 재생산(self-renewal) 능력이 있는 장점이 있다. 따라서 성체줄기세포는 최근에 그 중요성이 부각되고 있으며, 그 유용성을 밝히기 위한 여러 가지 연구가 진행 중에 있다.Adult stem cells are very safe for medical applications. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration, and since it is in an adult body, the immune rejection reaction does not occur, thereby enabling autologous transplantation using its own cells. In addition, there is a site-specific differentiation ability to differentiate itself according to the characteristics of the surrounding tissue, and since injection does not cause cancer even when injected in the undifferentiated state, in addition to producing the cells needed immediately after transplantation The advantage is the ability to self-renewal to regenerate and store the necessary undifferentiated stem cells. Therefore, adult stem cells have recently gained importance, and various studies are underway to reveal their usefulness.
최근 전세계적으로 대머리 및 탈모 방지를 위한 많은 발모제들이 시판되고 있으나 아직도 치료효과를 만족할 수 있는 약물이나 기술은 미미한 실정이다. 미국 식품의약청인 FDA가 임상실험을 거쳐 발모제로 유일하게 승인한 약물은 미녹시딜(minoxidil)로 원래 고혈압 치료제로써 개발되어 발견된 부작용의 일종인 발모를 상품화한 것이며 전세계에서 최초로 미국 연방정부가 공인한 최초의 대머리 및 탈모증 치료약이다. 또한, 현재 사용되는 다른 약물로 프로페시아(Propecia)가 있는데 이는 탈모의 원인 중 호르몬설에 근거를 둔 것으로 남성호르몬은 5-알파환원효소의 작용을 받아 대사산물인 디하이드로테스토스테론(DHT)으로 바뀌는데 이 물질이 모낭을 위축, 소멸시키는 것으로 알려져 있다. 상기 프로페시아는 5-알파환원효소를 차단함으로써 DHT생성을 억제하여 남성형 탈모가 진행되는 것을 최대한 늦추는 효과가 있는 것으로 이 약물의 효과는 발모가 아니라 탈모의 추가방지에 있는 것이다.Recently, many hair repellents for preventing baldness and hair loss are commercially available, but there are still few drugs or technologies that can satisfy the therapeutic effect. The only drug approved by the US Food and Drug Administration (FDA) as a hair regrowth agent is minoxidil, a commercial product developed for the treatment of hypertension, which was originally developed as a drug for the treatment of hypertension. Medicine for baldness and alopecia. Another drug currently used is Propecia, which is based on hormonal theory of hair loss, and male hormones are converted to the metabolite dihydrotestosterone (DHT) under the action of 5-alpha-reductase. The substance is known to shrink and destroy hair follicles. The propecia inhibits DHT production by blocking 5-alpha-reductase, thereby slowing down the progression of masculine hair loss, and the effect of the drug is not in hair growth but in further prevention of hair loss.
그 외에도 발모 및 탈모 방지에 효과 있다고 알려진 물질들이 보고되고 있으다. 예를 들어, 화합물로는 사이클로스포린(Cyclosporin) 유도체(대한민국 등록특허 10-0439467, 대한민국 등록특허 10-0695611), N-헤테로사이클릭 카복실산 및 카르밤산염(대한민국 공개특허 10-2001-0052502), 퀴나졸리논 유도체(대한민국 공개특허10-1999-0023754), 크라이신 7-Ｏ-크로토네이트(대한민국 공개특허 10-2001-0009474) 및 1,2-이치환벤젠카르복사미드 유도체(대한민국 공개특허 10-1999-0037394) 등이 보고된 바 있다.In addition, there are reported substances known to be effective in preventing hair loss and hair loss. For example, the compounds include cyclosporin derivatives (Korean Patent No. 10-0439467, Korean Patent No. 10-0695611), N-heterocyclic carboxylic acid and carbamate (Korean Patent No. 10-2001-0052502), quina Zolinone derivatives (Korean Patent Laid-Open Publication No. 10-1999-0023754), chrysine 7-O-crotonate (Korean Patent Publication 10-2001-0009474) and 1,2-disubstituted benzenecarboxamide derivatives 1999-0037394) have been reported.
그러나 이와 같은 탈모 치료 또는 발모 개선용 조성물은 화학성분에 의한 조성물로서 여성이나 유아에게 치명적인 부작용이 일어날 수 있으며, 남성에게 있어서도 피부염증에 의한 탈모 가속 등의 부작용 등이 나타나고 있고, 많은 경우 평생에 걸쳐 사용해야 하며 장기간의 사용시에 나타날 수 있는 부작용들도 환자의 고통을 증가시킬 수 있다.However, such a hair loss treatment or hair growth improving composition is a chemical composition, which can cause fatal side effects for women and infants, and also has side effects such as acceleration of hair loss due to dermatitis in men. Side effects that can be used with prolonged use can also increase patient pain.
본 발명의 일 목적은 줄기세포 또는 배양액 유래 엑소좀을 이용하여 안정적으로 상처를 치료 또는 촉진할 수 있는 상처 치료용 조성물을 제공하고자 한다. One object of the present invention is to provide a composition for treating wounds that can stably treat or promote wounds using stem cells or culture-derived exosomes.
본 발명의 다른 목적은 상기한 줄기세포 또는 그 배양액 유래 엑소좀을 이용하여 안전하면서도 피부 보습, 피부 미백, 주름 개선 및 노화 방지 등의 효과가 우수한 피부 개선용 조성물을 제공하고자 한다. It is another object of the present invention to provide a composition for improving skin having excellent effects such as safe skin moisturizing, skin whitening, wrinkle improvement and anti-aging by using the stem cells or the culture-derived exosomes.
본 발명의 또 다른 목적은 상기한 줄기세포 또는 그 배양액 유래 엑소좀을 이용하여 안전하면서도 육모 및 발모 효과가 우수한 탈모 방지 또는 치료용 조성물을 제공하고자 한다.Still another object of the present invention is to provide a composition for preventing or treating hair loss having a safe but excellent hair growth and hair growth effect using the exosome derived from the stem cells or the culture medium.
상처는 창상이라고 칭해지기도 하며, 그 원인에 따라 절창, 자창, 할창, 좌창, 열창, 사창, 교창 등으로 나뉘고, 형상에 따라 선상창, 판상창, 결손창 등으로 나뉜다. 상처에 따른 증세로는 통증, 출혈, 기능장애 등이 있으며, 상처를 입는 경우, 생체는 이를 치유하기 위하여 다양한 생리적 반응을 나타낸다. 통상, 상해를 받은 세포의 변성, 사멸 과정을 거처 주위의 조직으로부터의 유주세포, 조직액의 유출되고, 섬유소의 석출과 육아조직이 형성되어 상처를 치유하게 된다.Wounds are also called wounds, and depending on the cause, they are divided into intestines, jaws, insoles, acne, fissures, insoles, school windows, etc. Symptoms of the wound include pain, bleeding, dysfunction, and when the wound, the living body exhibits a variety of physiological reactions to heal. Normally, injured cells undergo denaturation and death processes, causing flow of cells and tissue fluid to flow out from surrounding tissues, and precipitation of fibrin and granulation tissue are formed to heal wounds.
일반적으로 EGF(Epidermal Growth Factor), FGF(Fibroblast Growth Factor), TGF-α(Transforming Growth Factor-α), TGF-β, IGF-I (Insulin-like Growth Factor-1) 등의 성장인자들이 상처를 치료하거나 상처치료를 촉진시키는 것으로 알려져 있다.In general, growth factors such as EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), TGF-α (Transforming Growth Factor-α), TGF-β, and IGF-I (Insulin-like Growth Factor-1) It is known to cure or promote wound healing.
그러나, EGF 등의 특정 성장인자를 분리하여 임상에 적용할지라도 아직까지 만족할 만한 상처 치료 또는 상처 치료 촉진 효과를 기대하기가 곤란하다. 또한, MSC 등의 줄기세포를 직접 사용할 경우에는 세포의 생존율을 높게 유지하면서 제제화하는 것이 곤란할 뿐만 아니라 세포의 원천(source)에 따라 다양한 개체편차를 나타내어 목적하는 치료효과를 동일하게 나타내는 것이 여전히 곤란하다.However, even if a specific growth factor such as EGF is isolated and applied to the clinic, it is difficult to expect satisfactory wound treatment or wound healing promoting effect. In addition, when using stem cells such as MSC directly, it is difficult to formulate while maintaining a high survival rate of the cells, and it is still difficult to display various individual deviations according to the source of the cells, thus showing the same therapeutic effect. .
최근 의료기술 및 공중위생 혜택이 개선됨에 따라 초고령화 사회가 급속히 도래하고 있다. 이에 따라, 생활의 질적인 향상이 도모되고 있으며 젊음의 유지에 대한 욕구도 증가하고 있다. 외모, 특히 피부에서 가장 먼저 나타내게 되는 노화를 지연시키고 예방하기 위하여 여러 영역에서 많은 연구가 진행되고 있다. Recently, with the improvement of medical technology and public health benefits, an aging society is rapidly arriving. As a result, the quality of life is being improved and the desire for the maintenance of youth is also increasing. Many studies have been conducted in various areas to delay and prevent aging, which is the first to appear on the skin.
피부(skin)는 표피(epidermis), 진피(dermis) 및 피하조직(subcutaneous tissue)으로 이루어진다. 피부의 바깥층인 표피는 중층편평상피(stratified squamous epithelium)로 구성되며, 외부 환경에 대한 신체의 보호 장벽으로 작용한다. 표피는 바깥에서부터 각질층(stratum corneum), 투명층(stratum lucidum), 과립층(stratum granulosum), 유극층(stratum spinosum) 및 기저층(stratum basale)으로 나뉜다. 진피는 표피와 피하조직(subcutaneous tissue) 사이의 층으로서, 유두층(papillary dermis) 및 망상층(reticular dermis)으로 나뉘며, 표피에는 없는 혈관, 콜라겐, 엘라스틴 섬유, 모공, 입모근, 피지선, 한선, 여러 가지 감각신경, 섬유아세포 및 대식세포 등이 존재하며 피부 중 가장 많은 부위를 차지한다. The skin consists of the epidermis, the dermis and the subcutaneous tissue. The epidermis, the outer layer of skin, consists of the stratified squamous epithelium and acts as a protective barrier for the body to the outside environment. The epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is the layer between the epidermis and the subcutaneous tissue, divided into papillary and reticular dermis, blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, Korean glands, various Sensory nerves, fibroblasts and macrophages are present and occupy the largest part of the skin.
진피는 주로 콜라겐과 엘라스틴 섬유로 이루어지는데, 이들은 피부를 지지하는 역할을 한다. 따라서 이러한 진피에 문제가 발생하면 주름이 생기고 피부탄력이 없어져서 피부노화가 진행된다. 콜라겐은 피부재생, 피부 함수율, 상처치유 및 주름개선 등에 있어서 가장 중요한 역할을 담당하는 것으로 알려져 있으며, 섬유아세포로부터 생성된다. 콜라겐은 수분을 다량 함유할 수 있는 기능이 있어서 진피에 수분을 공급하는 역할을 하는데, 노화가 되면 콜라겐의 수분 함유 기능이 떨어지게 되어 주름이 늘어나게 된다. 콜라겐은 또한 상처가 생겼을 때 섬유아세포의 지속적인 콜라겐 생성으로 상처 부위를 메워주어 상처치유 작용도 한다. The dermis consists primarily of collagen and elastin fibers, which support the skin. Therefore, when such a problem occurs in the dermis, wrinkles occur and skin elasticity is lost, thereby aging the skin. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function that can contain a large amount of moisture, which serves to supply moisture to the dermis. When aging, the collagen loses its water-containing function and wrinkles increase. Collagen can also heal wounds by filling fibroblasts with sustained collagen production when the wound is injured.
또한, 현대 사회는 많은 스트레스로 인해 생체가 서서히 손상을 받고 있으며 이로 인한 모발손실이 커다란 사회 이슈가 되고 있다. 모발 손실은 모발 성장의 부진과 모근 세포의 성장촉진을 하는 성장인자의 결핍 등 다양한 인자에 의해 유발된다.Also, in modern society, the living body is gradually damaged by many stresses, and the hair loss caused by this is becoming a big social issue. Hair loss is caused by a variety of factors, including the slow growth of hair and the lack of growth factors that promote hair root cell growth.
이러한 탈모는 정신적 스트레스로 삶의 질을 저하시키며 대인관계나 사회생활에 많은 지장을 준다. 사회생활에 있어서 외모에서 풍기는 이미지는 매우 인상적으로 다가가며, 그 외모의 중요성이 자존감이나 사회생활의 보이지 않는 활력소가 된다는 점에서 탈모 원인과 치료에 관심이 날로 높아지고 있다.This hair loss reduces the quality of life due to mental stress and causes a lot of interpersonal or social life. The image of appearance in social life is very impressive, and interest in the cause and treatment of hair loss is increasing day by day because the importance of appearance becomes an invisible vitality of self-esteem or social life.
본 발명의 발명자들은 줄기세포 또는 그 배양액 유래 엑소좀은 피부 투과도가 우수하고, 흡수 시 상처 치료, 피부 보습, 피부 미백, 주름 개선 및 노화 방지 등의 효과가 우수하며, 그 외에 발모 및 육모 효과 또한 뛰어난 것을 발견하여 본 발명에 이르게 되었다. The inventors of the present invention, the stem cell or its culture-derived exosomes are excellent in skin permeability, and excellent in the effects of wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, in addition to hair growth and hair growth effects Discovering outstanding things led to the present invention.
본 발명의 일 구현 예에 따르면, 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 상처 치료용 약학적 조성물을 제공한다.According to one embodiment of the present invention, it provides a pharmaceutical composition for wound treatment comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
본 발명에서 상기 엑소좀은 1 ~ 100㎍/ml, 또는 1 ~ 50㎍/ml의 양으로 포함되는 것이 바람직하다. 상기 엑소좀이 1㎍/ml 미만의 양으로 포함되는 경우 상처 치료 효과가 미미할 수 있고, 100㎍/ml를 초과하여 포함되는 경우 경제적으로 문제될 수 있다.In the present invention, the exosomes are preferably contained in an amount of 1 ~ 100㎍ / ml, or 1 ~ 50㎍ / ml. When the exosomes are included in an amount of less than 1 μg / ml, the wound healing effect may be insignificant, and when included in excess of 100 μg / ml, it may be economically problematic.
또한, 상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함될 수 있다. 상기 엑소좀의 입자수가 1 X 107/ml 미만인 경우 상처 치료 효과가 미미할 수 있고, 입자수가 1 X 1012/ml를 초과하는 경우 경제적으로 문제될 수 있다.In addition, the exosomes may be included in the number of particles of 1 X 10 7 ~ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
본 발명의 다른 구현 예에 따르면, 상기한 상처 치료용 약학적 조성물을 제조하는 방법에 관한 것으로, 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; 상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; 상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; 상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및 상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함한다. According to another embodiment of the present invention, the present invention relates to a method for preparing the wound pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
본 발명은 5단계의 원심 분리 공정을 통하여 상처 치료 효과가 뛰어난 엑소좀을 높은 순도 및 함량으로 획득할 수 있다. The present invention can be obtained with a high purity and content of exosomes excellent in wound healing through a five-step centrifugation process.
본 발명에서 상기 성체 줄기세포 배양액은 성체 줄기세포를 800~1,200 mg/L의 D 글루코스, 500~600 mg/L의 L-글루타민, 100~200 mg/L의 소디움피루베이트, 5~15 부피%의 우태아혈청(Fetal Bovine Serum, FBS) 및 0.5~1.5부피%의 페니실린-스트렙토마이신이 보충된 DMEM 배지에 접종한 뒤 80~95 %의 습도 및 30~40 ℃ 온도 조건 하에서 5~10 % CO2 배양기에서 계대 배양하는 단계; 및 계대 배양된 배양물을 350~400 mg/L의 L-글루타민, 10~20 mM의 HEPES 및 50~60 mg/L의 소듐 피루베이트로 보충된 DMEM와 Ham's F-12 영양소 혼합액의 1:0.5~2 중량비의 혼합 배지에 접종한 뒤 1~5부피%의 산소를 포함하는 저산소(Hypoxia) 조건으로 60~120 시간 동안 배양하는 단계에 의하여 제조될 수 있다. In the present invention, the adult stem cell culture medium of adult stem cells 800 ~ 1,200 mg / L D glucose, 500 ~ 600 mg / L L- glutamine, 100 ~ 200 mg / L sodium pyruvate, 5 ~ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C. Passage in two incubators; And 1: 0.5 of DMEM and Ham's F-12 nutrient mixtures supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate After inoculating the mixed medium in a weight ratio of ~ 2 may be prepared by incubating for 60 to 120 hours under hypoxia (Hypoxia) conditions containing 1 to 5% by volume of oxygen.
본 발명의 다른 구현 예에 따르면, 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 피부 개선용 화장료 조성물을 제공한다. According to another embodiment of the present invention, it provides a cosmetic composition for skin improvement comprising the adult stem cells or culture medium derived from the exosome (exosome) as an active ingredient.
본 발명에서 상기 엑소좀은 1 ~ 100㎍/ml, 또는 1 ~ 50㎍/ml의 양으로 포함되는 것이 바람직하다. 상기 엑소좀이 1㎍/ml 미만의 양으로 포함되는 경우 상처 치료 효과가 미미할 수 있고, 100㎍/ml를 초과하여 포함되는 경우 경제적으로 문제될 수 있다.In the present invention, the exosomes are preferably contained in an amount of 1 ~ 100㎍ / ml, or 1 ~ 50㎍ / ml. When the exosomes are included in an amount of less than 1 μg / ml, the wound healing effect may be insignificant, and when included in excess of 100 μg / ml, it may be economically problematic.
또한, 상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함될 수 있다. 상기 엑소좀의 입자수가 1 X 107/ml 미만인 경우 상처 치료 효과가 미미할 수 있고, 입자수가 1 X 1012/ml를 초과하는 경우 경제적으로 문제될 수 있다.In addition, the exosomes may be included in the number of particles of 1 X 10 7 ~ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
본 발명의 다른 구현 예에 따르면, 상기한 피부 개선용 화장료 조성물을 제조하는 방법에 관한 것으로, 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; 상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; 상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; 상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및 상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함한다. According to another embodiment of the present invention, the present invention relates to a method for preparing the cosmetic composition for skin improvement, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
본 발명은 5단계의 원심 분리 공정을 통하여 피부 개선 효과가 뛰어난 엑소좀을 높은 순도 및 함량으로 획득할 수 있다. The present invention can be obtained with high purity and content exosomes excellent skin improvement effect through a five-step centrifugation process.
본 발명에서 상기 성체 줄기세포 배양액은 성체 줄기세포를 800~1,200 mg/L의 D 글루코스, 500~600 mg/L의 L-글루타민, 100~200 mg/L의 소디움피루베이트, 5~15 부피%의 우태아혈청(Fetal Bovine Serum, FBS) 및 0.5~1.5부피%의 페니실린-스트렙토마이신이 보충된 DMEM 배지에 접종한 뒤 80~95 %의 습도 및 30~40 ℃ 온도 조건 하에서 5~10 % CO2 배양기에서 계대 배양하는 단계; 및 계대 배양된 배양물을 350~400 mg/L의 L-글루타민, 10~20 mM의 HEPES 및 50~60 mg/L의 소듐 피루베이트로 보충된 DMEM와 Ham's F-12 영양소 혼합액의 1:0.5~2 중량비의 혼합 배지에 접종한 뒤 1~5부피%의 산소를 포함하는 저산소(Hypoxia) 조건으로 60~120 시간 동안 배양하는 단계에 의하여 제조될 수 있다. In the present invention, the adult stem cell culture medium of adult stem cells 800 ~ 1,200 mg / L D glucose, 500 ~ 600 mg / L L- glutamine, 100 ~ 200 mg / L sodium pyruvate, 5 ~ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C. Passage in two incubators; And 1: 0.5 of DMEM and Ham's F-12 nutrient mixtures supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate After inoculating the mixed medium in a weight ratio of ~ 2 may be prepared by incubating for 60 to 120 hours under hypoxia (Hypoxia) conditions containing 1 to 5% by volume of oxygen.
본 발명의 또 다른 구현 예에 따르면, 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 치료용 약학적 조성물을 제공한다. According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
본 발명에서 상기 엑소좀은 1 ~ 100㎍/ml, 또는 1 ~ 50㎍/ml의 양으로 포함되는 것이 바람직하다. 상기 엑소좀이 1㎍/ml 미만의 양으로 포함되는 경우 상처 치료 효과가 미미할 수 있고, 100㎍/ml를 초과하여 포함되는 경우 경제적으로 문제될 수 있다.In the present invention, the exosomes are preferably contained in an amount of 1 ~ 100㎍ / ml, or 1 ~ 50㎍ / ml. When the exosomes are included in an amount of less than 1 μg / ml, the wound healing effect may be insignificant, and when included in excess of 100 μg / ml, it may be economically problematic.
또한, 상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함될 수 있다. 상기 엑소좀의 입자수가 1 X 107/ml 미만인 경우 상처 치료 효과가 미미할 수 있고, 입자수가 1 X 1012/ml를 초과하는 경우 경제적으로 문제될 수 있다.In addition, the exosomes may be included in the number of particles of 1 X 10 7 ~ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
본 발명의 또 다른 구현 예에 따르면, 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 화장료 조성물을 제공한다. According to another embodiment of the present invention, there is provided a cosmetic composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
본 발명에서 상기 엑소좀은 1 ~ 100㎍/ml, 또는 1 ~ 50㎍/ml의 양으로 포함되는 것이 바람직하다. 상기 엑소좀이 1㎍/ml 미만의 양으로 포함되는 경우 상처 치료 효과가 미미할 수 있고, 100㎍/ml를 초과하여 포함되는 경우 경제적으로 문제될 수 있다.In the present invention, the exosomes are preferably contained in an amount of 1 ~ 100㎍ / ml, or 1 ~ 50㎍ / ml. When the exosomes are included in an amount of less than 1 μg / ml, the wound healing effect may be insignificant, and when included in excess of 100 μg / ml, it may be economically problematic.
또한, 상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함될 수 있다. 상기 엑소좀의 입자수가 1 X 107/ml 미만인 경우 상처 치료 효과가 미미할 수 있고, 입자수가 1 X 1012/ml를 초과하는 경우 경제적으로 문제될 수 있다.In addition, the exosomes may be included in the number of particles of 1 X 10 7 ~ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
본 발명의 또 다른 구현 예에 따르면, 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 식품 조성물을 제공한다. According to another embodiment of the present invention, there is provided a food composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
본 발명에서 상기 엑소좀은 1 ~ 100㎍/ml, 또는 1 ~ 50㎍/ml의 양으로 포함되는 것이 바람직하다. 상기 엑소좀이 1㎍/ml 미만의 양으로 포함되는 경우 상처 치료 효과가 미미할 수 있고, 100㎍/ml를 초과하여 포함되는 경우 경제적으로 문제될 수 있다.In the present invention, the exosomes are preferably contained in an amount of 1 ~ 100㎍ / ml, or 1 ~ 50㎍ / ml. When the exosomes are included in an amount of less than 1 μg / ml, the wound healing effect may be insignificant, and when included in excess of 100 μg / ml, it may be economically problematic.
또한, 상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함될 수 있다. 상기 엑소좀의 입자수가 1 X 107/ml 미만인 경우 상처 치료 효과가 미미할 수 있고, 입자수가 1 X 1012/ml를 초과하는 경우 경제적으로 문제될 수 있다.In addition, the exosomes may be included in the number of particles of 1 X 10 7 ~ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
본 발명의 다른 구현 예에 따르면, 상기한 탈모 방지 또는 치료용 약학적 조성물을 제조하는 방법에 관한 것으로, 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; 상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; 상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; 상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및 상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함한다. According to another embodiment of the present invention, the present invention relates to a method for preparing the hair loss prevention or treatment pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
본 발명은 5단계의 원심 분리 공정을 통하여 발모 및 육모 효과가 뛰어난 엑소좀을 높은 순도 및 함량으로 획득할 수 있다. The present invention can be obtained with a high purity and content of exosomes excellent in hair growth and hair growth effect through a five-step centrifugation process.
본 발명에서 상기 성체 줄기세포 배양액은 성체 줄기세포를 800~1,200 mg/L의 D 글루코스, 500~600 mg/L의 L-글루타민, 100~200 mg/L의 소디움피루베이트, 5~15 부피%의 우태아혈청(Fetal Bovine Serum, FBS) 및 0.5~1.5부피%의 페니실린-스트렙토마이신이 보충된 DMEM 배지에 접종한 뒤 80~95 %의 습도 및 30~40 ℃ 온도 조건 하에서 5~10 % CO2 배양기에서 계대 배양하는 단계; 및 계대 배양된 배양물을 350~400 mg/L의 L-글루타민, 10~20 mM의 HEPES 및 50~60 mg/L의 소듐 피루베이트로 보충된 DMEM와 Ham's F-12 영양소 혼합액의 1:0.5~2 중량비의 혼합 배지에 접종한 뒤 1~5부피%의 산소를 포함하는 저산소(Hypoxia) 조건으로 60~120 시간 동안 배양하는 단계에 의하여 제조될 수 있다. In the present invention, the adult stem cell culture medium of adult stem cells 800 ~ 1,200 mg / L D glucose, 500 ~ 600 mg / L L- glutamine, 100 ~ 200 mg / L sodium pyruvate, 5 ~ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C. Passage in two incubators; And 1: 0.5 of DMEM and Ham's F-12 nutrient mixtures supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate After inoculating the mixed medium in a weight ratio of ~ 2 may be prepared by incubating for 60 to 120 hours under hypoxia (Hypoxia) conditions containing 1 to 5% by volume of oxygen.
본 발명에서 상기 성체 줄기세포는 인간을 포함한 포유동물, 바람직하게는 인간의 성체 세포로부터 유래된 중복성(multipotency)을 갖는 미분화 세포를 말하며, 예를 들어, 골수, 혈액, 뇌, 피부, 지방(즉, 지방조직 또는 지방세포), 제대혈, 제대의 바르톤 젤리(Wharton's jelly) 등의 다양한 성체 세포로부터 유래될 수 있다. 본 명세서에 있어서, 상기 "성체 줄기세포"는 성체 세포로부터 유래된 중간엽 줄기세포(mesenchymal stem cell)를 포함한다.In the present invention, the adult stem cell refers to an undifferentiated cell having a multipotency derived from an adult human cell of a mammal, including a human, for example, bone marrow, blood, brain, skin, fat (ie , Adipose tissue or fat cells), umbilical cord blood, umbilical cord Barton's jelly, and the like. In the present specification, the "adult stem cells" include mesenchymal stem cells derived from adult cells.
상기 성체 줄기세포로서, 통상적으로 흔히 시행되는 지방흡입 과정에서 폐기되는 지방조직을 사용하여 얻을 수 있어, 침습적 시술이 필요 없는 지방 유래 줄기세포를 바람직하게 사용할 수 있다. 지방 유래 줄기세포는 인간을 포함한 포유동물, 바람직하게는 인간의 지방조직 또는 지방세포로부터 공지의 방법(예를 들어, 국제특허공개 제WO2000/53795호 및 제WO2005/042730호)에 개시된 바에 따라, 지방흡입(liposuction) 및 침강, 콜라게나제(collagenase) 등의 효소처리, 원심분리에 의한 적혈구 등의 부유 세포 제거 등의 과정을 통하여 얻을 수 있다. 상기 지방조직은 피하, 그물막, 내장, 유방 생식선 또는 그 밖의 지방 조직 부위로부터 유래된 갈색 또는 백색 조직을 포함하며, 통상의 지방흡입술로부터 손쉽게 얻을 수 있다. As the adult stem cells, it can be obtained using adipose tissue discarded in a commonly performed liposuction process, it is possible to preferably use adipose derived stem cells that do not require invasive procedures. Adipose-derived stem cells are known from known methods (e.g., WO2000 / 53795 and WO2005 / 042730) from adipose tissue or adipocytes of mammals including humans, preferably humans. It can be obtained through processes such as liposuction and sedimentation, enzymatic treatment of collagenase and the like, and removal of floating cells such as red blood cells by centrifugation. The adipose tissue includes brown or white tissue derived from subcutaneous, retinal, visceral, breast gonad or other adipose tissue sites and can be readily obtained from conventional liposuction.
본 발명에서 엑소좀(exosome)은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭을 의미한다. 엑소좀의 직경은 대략 30 ~ 1,000 nm이며, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 엑소좀의 대표적인 발현 마커는 HSP70, CD63 및 CD9에 해당한다. Exosomes in the present invention (exosome) refers to the small vesicles of the membrane structure secreted from various cells. The diameter of the exosome is approximately 30 to 1,000 nm, which means that the vesicle is released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane. Representative expression markers of exosomes correspond to HSP70, CD63 and CD9.
또한, 본 발명에서 상기 약학 조성물은 통상의 방법에 따른 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 메틸히드록시 벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 사용할 수 있으나, 이에 제한되지 않는다. In addition, in the present invention, the pharmaceutical composition may further include a suitable carrier, excipient or diluent according to a conventional method. Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, may be used, but is not limited thereto.
또한, 본 발명에 따른 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상세하게는, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 본 발명의 약학 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose), 락토오스 (lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다. In addition, the pharmaceutical compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to a conventional method. Can be used. Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명에 따른 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. 본 발명에서 사용된 용어 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
본 발명의 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50mg/kg 또는 0.001 내지 50mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 약학적 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형될 수 있다.The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
본 발명에서 식품 조성물은 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 분말, 과립, 정제, 캡슐, 과자, 떡, 빵 등의 형태로 제조될 수 있다. 본 발명의 식품 조성물은 독성 및 부작용이 거의 없는 식물추출물로 구성된 것이므로 예방 목적으로 장기간 복용 시에도 안심하고 사용할 수 있다.In the present invention, the food composition may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads, and the like. Since the food composition of the present invention is composed of plant extracts having little toxicity and no side effects, it can be used with confidence even for long-term use for prophylactic purposes.
본 발명에서 유효 성분으로 포함되는 엑소좀이 식품 조성물에 포함될 때 그 양은 전체 중량의 0.1 내지 50%의 비율로 첨가할 수 있다.When the exosomes included as an active ingredient in the present invention is included in the food composition, the amount may be added at a ratio of 0.1 to 50% of the total weight.
여기서, 상기 식품 조성물이 음료 형태로 제조되는 경우 지시된 비율로 상기 식품 조성물을 함유하는 것 외에 특별한 제한점은 없으며 통상의 음료와 같이 여러가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 즉, 천연 탄수화물로서 포도당 등의 모노사카라이드, 과당 등의 디사카라이드, 슈크로스 등의 및 폴리사카라이드, 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜 등을 포함할 수 있다. 상기 향미제로서는 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등) 등을 들 수 있다. Here, when the food composition is prepared in the form of a drink, there is no particular limitation other than the containing the food composition in the ratio indicated, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).
그 외 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.Other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 식품 조성물 100 중량부 당 0.1 내지 약 50 중량부의 범위에서 선택되는 것이 일반적이다.These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.
또한, 본 발명에서 화장료 조성물은 화장수, 영양로션, 영양에센스, 마사지 크림, 미용목욕물첨가제, 바디로션, 바디밀크, 배스오일, 베이비오일, 베이비파우더, 샤워겔, 샤워크림, 선스크린로션, 선스크린크림, 선탠크림, 스킨로션, 스킨크림, 자외선차단용 화장품, 크렌징밀크, 탈모제{화장용}, 페이스 및 바디로션, 페이스 및 바디크림, 피부미백크림, 핸드로션, 헤어로션, 화장용크림, 쟈스민오일, 목욕비누, 물비누, 미용비누, 샴푸, 손세정제(핸드클리너), 약용비누{비의료용}, 크림비누, 페이셜 워시, 전신 세정제, 두피 세정제, 헤어린스, 화장비누, 치아미백용 겔, 치약 등의 형태로 제조될 수 있다. 이를 위해 본 발명의 조성물은 화장료 조성물의 제조에 통상적으로 사용하는 용매나, 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.In the present invention, the cosmetic composition is a lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Creams, suntan creams, skin lotions, skin creams, sunscreen cosmetics, cleansing milk, depilatory {cosmetic}, face and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, cosmetic creams, jasmine Oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Cleanser (Hand Cleaner), Medicated Soap {Non-Medical}, Cream Soap, Facial Wash, Systemic Cleanser, Scalp Cleaner, Hairrin, Cosmetic Soap, Tooth Whitening Gel, Toothpaste Or the like. To this end, the composition of the present invention may further comprise a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of the cosmetic composition.
본 발명의 화장료 조성물 내에 더 추가될 수 있는 용매의 종류는 특별히 한정하지 않으나, 예를 들어, 물, 식염수, DMSO 또는 이들의 조합을 사용할 수 있고, 담체, 부형제 또는 희석제로는 정제수, 오일, 왁스, 지방산, 지방산 알콜, 지방산 에스테르, 계면활성제, 흡습제(humectant), 증점제, 항산화제, 점도 안정화제, 킬레이팅제, 완충제, 저급 알콜 등이 포함되지만, 이에 제한되는 것은 아니다. 또한, 필요에 따라 미백제, 보습제, 비타민, 자외선 차단제, 향수, 염료, 항생제, 항박테리아제, 항진균제를 포함할 수 있다. The kind of the solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used, and as the carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. In addition, a whitening agent, a moisturizing agent, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.
상기 오일로서는 수소화 식물성유, 피마자유, 면실유, 올리브유, 야자인유, 호호바유, 아보카도유가 이용될 수 있으며, 왁스로는 밀랍, 경랍, 카르나우바, 칸델릴라, 몬탄, 세레신, 액체 파라핀, 라놀린이 이용될 수 있다.The oil may be hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin Can be used.
지방산으로는 스테아르산, 리놀레산, 리놀렌산, 올레산이 이용될 수 있고, 지방산 알콜로는 세틸 알콜, 옥틸 도데칸올, 올레일 알콜, 판텐올, 라놀린 알콜, 스테아릴 알콜, 헥사데칸올이 이용될 수 있으며 지방산 에스테르로는 이소프로필 미리스테이트, 이소프로필 팔미테이트, 부틸 스테아레이트가 이용될 수 있다. 계면 활성제로는 당업계에 알려진 양이온 계면활성제, 음이온 계면활성제 및 비이온성 계면활성제가 사용가능하며 가능한 한 천연물 유래의 계면활성제가 바람직하다. Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as fatty acids, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol. Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the fatty acid ester. As surfactants, cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible.
그 외에도 화장품 분야에서 널리 알려진 흡습제, 증점제, 항산화제 등을 포함할 수 있으며, 이들의 종류와 양은 당업계에 공지된 바에 따른다. In addition, it may include a hygroscopic agent, a thickener, an antioxidant, and the like, which are widely known in the cosmetic field, and their types and amounts are known in the art.
본 발명에서 제공하는 줄기세포 또는 그 배양액 유래 엑소좀은 상처 부위로 세포 이동을 촉진하여 우수한 상처 치료 또는 상처 치료 촉진 활성을 갖는다.Stem cells or the culture-derived exosomes provided by the present invention promote cell migration to the wound site and have excellent wound healing or wound healing promoting activity.
피부 투과도가 우수하면서, 흡수 시 피부 보습, 피부 미백, 주름 개선 및 노화 방지 등의 피부 개선 효과가 우수할 뿐만 아니라, 피부에 대한 자극이나 부작용이 없고 안전하다.While excellent in skin permeability, it is excellent in skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, and is safe without irritation or side effects on the skin.
또한, 본 발명에서 제공하는 줄기세포 또는 그 배양액 유래 엑소좀은 발모 및 육모 효과가 우수하여 탈모의 방지 및 치료 효과가 뛰어나며, 이를 섭취하거나 피부에 도포하여도 부작용이 없이 안전하다.In addition, the stem cells or the culture-derived exosomes provided by the present invention is excellent in the hair growth and hair growth effect is excellent in the prevention and treatment of hair loss, it is safe without side effects even when ingested or applied to the skin.
도 1의 (a)는 실험예 1에서 엑소좀의 TEM 사진을 도시한 것이고, (b)는 엑소좀 내 존재하는 입자의 크기 별 분석을 그래프로 나타낸 것이다. Figure 1 (a) shows a TEM picture of the exosomes in Experimental Example 1, (b) shows a graphical analysis of the size of the particles present in the exosomes.
도 2의 (a)는 실험예 1에서 웨스턴 블럿을 이용하여 엑소좀의 HSP70, CD63 및 CD9 마커의 발현 여부를 확인한 사진이고, (b)는 실시간 PCR을 이용하여 엑소좀의 CD24 및 AQP2 마커의 발현 정도를 그래프로 나타낸 것이며, (c) 및 (d)는 FACS를 이용하여 엑소좀의 CD63, CD9 및 CD81 마커의 발현 여부를 나타낸 것이다.Figure 2 (a) is a photograph confirming the expression of HSP70, CD63 and CD9 markers of exosomes using Western blot in Experimental Example 1, (b) is a CD24 and AQP2 markers of exosomes using real-time PCR The degree of expression is shown in a graph, and (c) and (d) show the expression of CD63, CD9 and CD81 markers of exosomes using FACS.
도 3은 실험예 2에서 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액의 처리 후 상처 부위로 인간 피부 섬유아세포의 이동 변화를 나타낸 사진이다.Figure 3 is a photograph showing the change in the movement of human skin fibroblasts to the wound site after treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed.
도 4는 실험예 2에서 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액의 처리 후 상처 부위로 이동한 인간 피부 섬유아세포의 비율을 그래프로 나타낸 것이다. Figure 4 is a graph showing the ratio of human skin fibroblasts moved to the wound site after the treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed .
도 5는 실험예 3에서 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액의 처리 후 프로콜라겐, KGF 및 CD34의 발현 정도를 그래프로 나타낸 것이다.FIG. 5 is a graph showing the expression levels of procollagen, KGF and CD34 after treatment of the exosomes obtained in Example 3 in Experimental Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed.
도 6의 (a) 및 (b)는 실험예 4에서 실시예 3에서 얻어진 엑소좀의 처리 후 농도 별 프로콜라겐 및 엘라스틴의 발현 정도를 그래프로 나타낸 것이다. Figure 6 (a) and (b) is a graph showing the expression level of procollagen and elastin for each concentration after the treatment of the exosomes obtained in Example 3 in Experimental Example 4.
도 7의 (a) 내지 (c)는 실험예 5에서 실시예 3에서 얻어진 엑소좀 처리 후 농도 별 KGF, CD34 및 VEGF의 발현 정도를 그래프로 나타낸 것이다.Figure 7 (a) to (c) is a graph showing the expression level of KGF, CD34 and VEGF by concentration after the exosome treatment obtained in Example 3 in Experimental Example 5.
도 8은 실험예 6에서 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액의 처리 후 LEF-1, PTC-1 및 KGF 의 발현 정도를 그래프로 나타낸 것이다.8 is a graph showing the expression levels of LEF-1, PTC-1 and KGF after treatment of the exosomes obtained in Example 3 and Experimental Example 6 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. will be.
본 발명에서 줄기세포 또는 그 배양액 유래 엑소좀은 피부 투과도가 우수하고, 흡수 시 상처 치료, 피부 보습, 피부 미백, 주름 개선 및 노화 방지 등의 효과가 우수하며, 그 외에 발모 및 육모 효과 또한 뛰어나다. In the present invention, the stem cell or its culture-derived exosomes have excellent skin permeability, and excellent effects such as wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, and also excellent hair growth and hair growth effects.
이하, 본 발명의 바람직한 실시형태들을 설명한다. 그러나 본 발명의 실시형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. 또한, 본 발명의 실시형태는 당해 기술분야에서 평균적인 지식을 가진 자에게 본 발명을 더욱 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, preferred embodiments of the present invention will be described. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
[실시예 1] 지방 유래 줄기세포의 수득Example 1 Obtaining Adipose-Derived Stem Cells
피하지방 제거 시술을 받은 환자로부터 동의를 얻고, 해당 환자로부터 지방 흡인(liposuction) 방법을 통하여 제거되어 버려지는 지방조직을 수거하였다. 지방조직의 세포외 기질을 약 37℃, 약 5% CO2 배양기에서 45분간 0.075% 콜라게나아제로 처리한 다음, 얻어진 지방조직을 약 1200g에서 5분간 원심 분리하여 고밀도의 줄기세포를 포함한 스트로마성 혈관 분획을 수득하였다. 얻어진 분획을 PBS로 세척하고 70㎛ 나일론 세포 여과기를 통하여 다른 조직을 제거한 후 Histopaque-1077(Sigma Co.)로 적혈구를 포함한 세포파편과 단핵 세포를 분리하였다.Consent was obtained from a patient who had undergone a subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction. The extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes in an incubator at about 37 ° C. and at about 5% CO 2 , and then the resulting adipose tissue was centrifuged at about 1200 g for 5 minutes to form stromal cells containing high density stem cells. Vascular fractions were obtained. The obtained fractions were washed with PBS, other tissues were removed through a 70 μm nylon cell strainer, and histopaque-1077 (Sigma Co.) was used to separate cell debris and mononuclear cells.
분리된 단핵세포를 10% 소 태아 혈청((fetal bovine serum, FBS), 1% 페니실린/스트렙토마이신으로 보충된 DMEM(Dulbecco's Modified Eagle's Medium)을 배지로 사용하여 약 37℃, 약 5% CO2 배양기에서 24 시간 동안 배양한 후, 비접착성 세포들을 제거하여 지방유래 줄기세포를 단리하였다.The isolated mononuclear cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin as a medium at about 37 ° C., about 5% CO 2 incubator. After culturing for 24 hours at, the adhering stem cells were isolated by removing non-adherent cells.
[실시예 2] 지방 유래 줄기세포의 배양Example 2 Culture of Adipose-derived Stem Cells
실시예 1에서 얻어진 지방유래 줄기세포 1.25 X 105 cells를 1000 mg/L의 D 글루코스, 584 mg/L의 L-글루타민, 110 mg/L의 소디움피루베이트, 10 부피% FBS, 1부피% 페니실린-스트렙토마이신이 보충된 DMEM 배지에 가하고, 약 90 %의 습도 및 약 37℃의 온도 조건 하에서 5부피% CO2 배양기에서 30일 동안 계대 배양하였다.Adipose derived stem cells 1.25 × 10 5 cells obtained in Example 1 were prepared with 1000 mg / L D glucose, 584 mg / L L-glutamine, 110 mg / L sodium pyruvate, 10% by volume FBS, 1% by volume penicillin -Added to DMEM medium supplemented with streptomycin and passaged for 30 days in a 5% by volume CO 2 incubator under conditions of about 90% humidity and about 37 ° C.
파이펫을 사용하여 계대 배양된 배양물로부터 배양액을 제거한 후, 인산화 완충 용액으로 3 회 세척한 후, 365 mg/L의 L-글루타민, 15mM의 HEPES, 55 mg/L의 소듐 피루베이트로 보충된 DMEM 및 Ham's F-12 영양소 혼합액의 1:1 (중량비) 혼합 배지 중에서 1.2 X 106 cell/dish의 농도로 접종하여 저산소(Hypoxia) 조건으로 72 시간 동안 배양하였다.The cultures were removed from the passaged culture using a pipette, washed three times with phosphorylation buffer solution, and then supplemented with 365 mg / L L-glutamine, 15 mM HEPES, 55 mg / L sodium pyruvate. Inoculated at a concentration of 1.2 × 10 6 cells / dish in a 1: 1 (weight ratio) mixed medium of DMEM and Ham's F-12 nutrient mixtures and incubated for 72 hours under hypoxia conditions.
[실시예 3] 지방 유래 줄기세포 배양액 유래 엑소좀 분리Example 3 Isolation of Adipose-derived Stem Cell Culture-derived Exosomes
실시예 2에서 얻어진 지방 줄기세포 배양액 50ml를 원심분리기 튜브에 옮긴 후, 4℃, 300g에서 10분간 원심 분리하였다. 상등액을 모아서 새로운 원심분리기 튜브에 옮긴 후, 이를 다시 4℃, 2,000 g에서 20분간 원심 분리하고 얻어진 상등액을 새로운 고속원심분리기가 가능한 튜브에 옮겨 다시 4℃, 10,000 g에서 30분간 고속원심분리를 하였다. 상등액을 새로운 초고속원심분리용 폴리카보네이트 보틀(bottle)에 옮기고, 이를 4℃ 110,000 g에서 70분간 Beckman Coulter 사의 OPTIMA XE-90 초원심분리기(Ultracentrifuge)를 이용하여 초고속원심분리를 실시하여 상등액을 제거하였다. 가라앉은 펠렛을 1000 ㎕ PBS로 튜브내에서 마이크로피펫을 이용하여 재현탁시켰다. 재현탁시킨 튜브를 Beckman Coulter 사의 OPTIMA MAX-XP 초원심분리기를 이용하여 4℃, 100,000 g에서 70분간 원심 분리하고, 가능한 상등액을 전부 제거하고 펠렛을 회수하였다. 회수된 펠렛에 소량의 PBS를 추가하고 재현탁한 뒤 100 ㎕로 분액하여 -80 ℃에 보관한 후 필요한 경우 녹여 사용하였다.50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 g for 20 minutes at 4 ° C., and the obtained supernatant was transferred to a tube capable of a new high speed centrifuge, followed by high speed centrifugation at 10,000 g for 30 minutes. . The supernatant was transferred to a new ultra-centrifugal polycarbonate bottle, and the supernatant was removed by ultrafast centrifugation using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C. . The settled pellet was resuspended using a micropipette in a tube with 1000 μl PBS. The resuspended tube was centrifuged for 70 minutes at 100,000 g at 4 ° C. using an OPTIMA MAX-XP ultracentrifuge from Beckman Coulter, and all possible supernatants were removed and the pellet was recovered. A small amount of PBS was added to the recovered pellet, resuspended, separated into 100 μl, stored at −80 ° C., and dissolved if necessary.
[비교예 1] 지방 유래 줄기세포 배양액으로부터 엑소좀의 제거Comparative Example 1 Removal of Exosomes from Adipose-derived Stem Cell Culture
실시예 2에서 얻어진 지방 줄기세포 배양액 50ml를 원심분리기 튜브에 옮긴 후, 4℃, 300g에서 10분간 원심분리를 하였다. 상등액을 모아서 새로운 원심분리기 튜브에 옮긴 후 이를 다시 4℃, 2,000 g에서 20분간 원심분리를 하였다. 4℃, 2,000g에서 20분간 원심분리를 마친 상등액을 새로운 고속원심분리기가 가능한 튜브에 옮겨 다시 4℃, 10,000g에서 30분간 고속원심분리를 하였다. 상등액을 새로운 초고속원심분리기용 폴리카보네이트 보틀에 옮기고, 이를 4℃ 110,000 g에서 70분간 Beckman Coulter 사의 OPTIMA XE-90 초원심분리기를 사용하여 초고속원심분리를 한 후, 펠렛을 제거하고 상등액 만을 취하여 엑소좀이 제거된 지방 유래 줄기세포 배양액을 준비하였다.50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and then centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 ° C. for 20 minutes at 4 ° C. The supernatant after 20 min centrifugation at 2,000 ° C. at 4 ° C. was transferred to a new high-speed centrifuge tube, followed by high-speed centrifugation at 10,000 ° C. for 30 minutes at 4 ° C. The supernatant was transferred to a new polycarbonate bottle for ultrafast centrifuge, ultrafast centrifuged using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C. The removed fat-derived stem cell culture was prepared.
[실험예 1] 지방 유래 줄기세포 배양액 유래 엑소좀의 특성 분석Experimental Example 1 Characterization of Adipose-derived Stem Cell Culture-derived Exosomes
실시예 3에서 얻어진 엑소좀을 투과 전자 현미경(TEM)을 통해 관찰하고, 그 사진을 도 1의 (a)에 나타내었고, NTA(Nanoparticle tracking analysis)를 통해 상기와 같이 얻어진 엑소좀 내에 존재하는 입자의 크기 별 분포를 분석해 그 결과를 도 1의 (b)에 그래프로 나타내었다.The exosomes obtained in Example 3 were observed through a transmission electron microscope (TEM), the photograph is shown in Figure 1 (a), and the particles present in the exosomes obtained as described above through the nanoparticle tracking analysis (NTA) The distribution of each size was analyzed and the results are shown graphically in FIG.
웨스턴 블럿(Western blot)을 이용하여 실시예 3에서 얻어진 엑소좀과, 대조군으로 지방 유래 줄기세포의 용해물에 대하여 대표적인 엑소좀 마커인 HSP70, CD63 및 CD9의 발현을 확인하여 그 결과를 도 2의 (a)에 나타내었다. 또한, 실시간 PCR을 이용하여 실시예 3에서 얻어진 엑소좀과, 대조군으로 지방 유래 줄기세포에 대하여 대표적인 엑소좀 마커인 CD24과 AQP2의 발현 수준을 확인하여 그 결과를 도 2의 (b)에 나타내었다. 마지막으로, 유세포분석기 (FACS)를 이용하여 실시예 3에서 얻어진 엑소좀에 대하여 엑소좀 표면 마커인 CD63, CD9 및 CD81의 발현 여부를 확인해 그 결과를 도 2의 (c) 및 (d)에 나타내었다. Western blot using the exosomes obtained in Example 3, and the control of the expression of the exosome markers HSP70, CD63 and CD9 for the lysate of fat-derived stem cells as a control to confirm the results of Figure 2 It is shown to (a). In addition, by using real-time PCR, the expression levels of the exosomes obtained in Example 3 and the representative exosome markers CD24 and AQP2 for the adipose derived stem cells as a control was confirmed and the results are shown in (b) of FIG. . Finally, the expression of the exosome surface markers CD63, CD9 and CD81 was confirmed with respect to the exosomes obtained in Example 3 using a flow cytometer (FACS), and the results are shown in FIGS. 2C and 2D. It was.
도 1의 (a)에서 보는 바와 같이, 지방 유래 줄기세포 배양액에서 분리된 엑소좀은 나노미터 단위의 미세한 구형 구조를 갖는 것을 확인할 수 있고, 도 1의 (b)에서 보는 바와 같이, 그 직경 크기는 주로 30 ~ 150nm에 분포하는 것을 확인할 수 있다. As shown in (a) of FIG. 1, the exosomes isolated from the adipose-derived stem cell cultures can be confirmed to have a fine spherical structure in nanometers, and as shown in FIG. It can be seen that mainly distributed in 30 ~ 150nm.
또한, 도 2의 (a) 내지 (d)에서 보는 바와 같이, 지방 유래 줄기세포 배양액에서 분리된 엑소좀은 엑소좀의 대표적인 마커를 모두 갖고 있는 것을 확인할 수 있다. In addition, as shown in (a) to (d) of Figure 2, it can be confirmed that the exosomes separated from the adipose-derived stem cell culture have all the representative markers of the exosomes.
이를 통해 상기 지방 유래 줄기세포 배양액으로부터 분리된 엑소좀은 전형적인 엑소좀의 특성을 갖는 것을 알 수 있다. It can be seen that the exosomes isolated from the adipose derived stem cell cultures have the characteristics of typical exosomes.
[실험예 2] 상처 부위에서 인간 피부 섬유아세포의 이동(migration) 효과Experimental Example 2 Migration Effect of Human Skin Fibroblasts at the Wound Site
인간 피부 섬유아세포를 5 X 104 세포/웰의 농도로 배양 접시에 부착시키고 일정한 거리 간격으로 상처(scratch)를 만든 뒤, 상처 부분에 상기 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리한 후, 48시간 후에 세포 이동 정도를 확인하여 그 결과를 도 3 및 도 4에 나타내었다. Human skin fibroblasts were attached to the culture dish at a concentration of 5 × 10 4 cells / well, and the wounds were made at regular distance intervals, and then the exosomes obtained in Example 3 on the wounds were obtained in Comparative Example 1 After treating the adipose derived stem cell culture medium from which the exosomes were removed, the degree of cell migration was confirmed after 48 hours, and the results are shown in FIGS. 3 and 4.
도 3에서 보는 바와 같이, 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리한 경우보다 엑소좀을 처리한 경우 많은 세포가 상처 부위로 이동함을 시각적으로 확인할 수 있다. As shown in Figure 3, it can be visually confirmed that many cells move to the wound site when treated with exosomes than when treated with adipose-derived stem cell culture medium from which the exosomes were removed.
또한, 도 4에서도 엑소좀을 처리한 경우, 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리한 경우보다 세포 이동 효과가 2배 이상 증가됨을 확인할 수 있다. In addition, in the case of treating the exosomes in Figure 4, it can be seen that the cell migration effect is increased by more than two times than when the exosomes from the fat-derived stem cell culture solution.
[실험예 3] 피부 노화방지 및 재생 관련 유전자 발현 증가 효과 Experimental Example 3 Gene Expression Increase Effects of Anti-Aging and Regeneration
인간 피부 섬유아세포를 5 X 104 세포/웰의 농도로 배양 접시에 부착시킨 후 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리하였다. 48시간 배양 후에 각 군의 세포에서 RNA를 추출한 후 인트론 프레믹스(intron premix)를 이용하여 상보적 DNA(cDNA)를 합성하고 이를 이용하여 실시간 PCR(Real-time PCR)을 수행하였다. 각각의 처리 군의 세포에서 피부 노화방지 및 재생 관련하는 유전자로, 프로콜라겐, KGF 및 CD34의 발현을 확인하여 그 결과를 도 5에 그래프로 나타내었다. Human skin fibroblasts were attached to the culture dish at a concentration of 5 × 10 4 cells / well and then treated with the exosomes obtained in Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. After 48 hours of incubation, RNA was extracted from the cells of each group, and then complementary DNA (cDNA) was synthesized using intron premix and real-time PCR was performed using the same. As genes related to skin anti-aging and regeneration in the cells of each treatment group, the expression of procollagen, KGF and CD34 was confirmed and the results are shown graphically in FIG. 5.
단, 상기 프로콜라겐은 피부 탄력을 유지하여 주며, 피부의 주된 단백질 형성에 필요한 콜라겐 합성에 관련된 유전자에 해당한다. KGF(keratinocyte growth factor)는 피부세포의 분화와 성장 및 생리활성을 자극하는 주요한 성장인자로 피부 노화를 방지하고 콜라겐을 합성하며, 피부 재생을 조절하고, 항노화에 관여하는 유전자에 해당한다. 마지막으로 CD34는 혈관 재생에 관여하는 인자로서 피부 재생에 관여하는 유전자에 해당한다. However, the procollagen maintains skin elasticity and corresponds to a gene related to collagen synthesis necessary for the formation of the main protein of the skin. KGF (keratinocyte growth factor) is a major growth factor that stimulates the differentiation, growth and physiological activity of skin cells. It is a gene that prevents skin aging, synthesizes collagen, regulates skin regeneration, and is involved in anti-aging. Finally, CD34 corresponds to a gene involved in skin regeneration as a factor involved in blood vessel regeneration.
도 5에서 보는 바와 같이, 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리한 경우보다 엑소좀을 처리한 경우 프로콜라겐, KGF 및 CD34의 발현율이 현저히 증가하는 것을 확인할 수 있다. As shown in Figure 5, it can be seen that the expression rate of procollagen, KGF and CD34 significantly increased when treated with exosomes than when treated with adipose-derived stem cell culture medium from which the exosomes were removed.
[실험예 4] 피부 탄력 관련 유전자의 발현 증가 효과 Experimental Example 4 Effect of Increased Expression of Skin Elasticity-related Genes
인간 피부 섬유아세포를 5 X 104 세포/웰의 농도로 배양 접시에 부착시킨 후, 실시예 3에서 얻어진 엑소좀을 5 및 10㎍/ml의 농도로 각각 처리하였다. 48시간 배양 후에 각 군의 세포에서 RNA를 추출한 후 인트론 프레믹스를 이용하여 cDNA를 합성하고 이를 이용하여 실시간 PCR을 수행하였다. 각각의 처리 군의 세포에서 피부 노화방지 탄력 증가에 관여하는 유전자로, 프로콜라겐과 엘라스틴의 발현 정도를 확인하여 그 결과를 도 6에 그래프로 나타내었다. 단, 대조군으로는 엑소좀을 처리하지 않았다. Human skin fibroblasts were attached to the culture dish at a concentration of 5 × 10 4 cells / well, and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 μg / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in increasing skin anti-aging elasticity in the cells of each treatment group, the expression level of procollagen and elastin was confirmed, and the results are shown graphically in FIG. 6. However, the control group was not treated with exosomes.
도 6의 (a) 및 (b)에서 보는 바와 같이, 엑소좀을 처리한 경우 대조군 대비 프로콜라겐과 엘라스틴의 mRNA 발현 수준이 현저히 높아지는 것을 확인할 수 있고, 처리 농도가 증가할수록 발현 수준이 더욱 높아지는 것을 확인할 수 있다. As shown in (a) and (b) of Figure 6, when treated with exosomes it can be seen that the mRNA expression level of procollagen and elastin is significantly higher than the control group, the expression level is higher as the treatment concentration increases You can check it.
이를 통하여 본 발명에 따르는 지방 유래 줄기세포 배양액으로부터 얻어진 엑소좀은 피부 탄력 유지 및 노화 방지에 중요한 역할을 하는 것을 알 수 있다.Through this, it can be seen that the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in maintaining skin elasticity and preventing aging.
[실험예 5] 피부 재생 관련 유전자의 발현 증가 효과 Experimental Example 5 Effect of Increased Expression of Skin Regeneration-Related Genes
인간 피부 섬유아세포를 5 X 104 세포/웰의 농도로 배양접시에 부착시킨 후 실시예 3에서 얻어진 엑소좀을 5 및 10㎍/ml의 농도로 각각 처리하였다. 48시간 배양 후에 각 군의 세포에서 RNA를 추출한 후 인트론 프레믹스를 이용하여 cDNA를 합성하고 이를 이용하여 실시간 PCR을 수행하였다. 각각의 처리 군의 세포에서 피부 재생에 관여하는 유전자로, KGF, CD34 및 VEGF의 발현 정도를 확인하여 그 결과를 도 7에 그래프로 나타내었다. 단, 대조군으로는 엑소좀을 처리하지 않았다. Human skin fibroblasts were attached to the culture dish at a concentration of 5 × 10 4 cells / well and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 μg / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in skin regeneration in the cells of each treatment group, the expression levels of KGF, CD34 and VEGF were confirmed and the results are shown graphically in FIG. 7. However, the control group was not treated with exosomes.
도 7의 (a) 내지 (c)에서 보는 바와 같이, 엑소좀을 처리한 경우 대조군 대비 KGF, CD34 및 VEGF의 mRNA 발현 수준이 현저히 높아지는 것을 확인할 수 있고, 처리 농도가 증가할수록 발현 수준이 더욱 높아지는 것을 확인할 수 있다. As shown in Figure 7 (a) to (c), when treated with exosomes it can be seen that the mRNA expression level of KGF, CD34 and VEGF is significantly higher than the control group, the expression level is higher as the treatment concentration increases You can see that.
이를 통하여 본 발명에 따르는 지방 유래 줄기세포 배양액으로부터 얻어진 엑소좀은 피부 재생에 중요한 역할을 하는 것을 알 수 있다.Through this, it can be seen that the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in skin regeneration.
[실험예 9] 모유두 세포의 모낭 성장 및 발달 관련 유전자의 발현 증가 효과Experimental Example 9 Expression of Genes Related to Hair Follicle Growth and Development
인간 모유두세포 5 X 105개를 60 mm (SPL, Korea) 배양 접시에 부착시킨 후, 실시예 3에서 얻어진 엑소좀과, 비교예 1에서 얻어진 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리하였다. 48시간 배양 후, 세포를 회수하여 RNA 분리 키트(Quiagen, USA)를 사용해 RNA를 분리하고, cDNA 합성 키트(Intronbio, Korea)를 사용하여 분리된 RNA 1ug에 대해 cDNA를 합성하였다. 각각의 프라이머는 코스모진텍에서 합성하였고, 프라이머는 10 pmol/ul의 농도로 사용하였으며, 실시간 PCR은 2X 사이버 그린 마스터 믹스(Takara, Japan)를 첨가하여 검출하였다. 각각의 처리 군의 세포에서 모낭의 성장 및 발달에 관여하는 유전자로, LEF-1, PTC-1 및 KGF 의 발현 정도를 확인하여 그 결과를 도 8에 그래프로 나타내었다.After attaching 5 X 10 5 human dermal papilla cells to a 60 mm (SPL, Korea) culture dish, the exosomes obtained in Example 3 and the adipose derived stem cell cultures from which the exosomes obtained in Comparative Example 1 were removed were treated. . After 48 hours of incubation, the cells were recovered, RNA was isolated using RNA isolation kit (Quiagen, USA), and cDNA was synthesized for 1 ug of RNA isolated using cDNA synthesis kit (Intronbio, Korea). Each primer was synthesized in Cosmojintech, primers were used at a concentration of 10 pmol / ul, real-time PCR was detected by the addition of 2X Cyber Green Master Mix (Takara, Japan). As genes involved in hair follicle growth and development in cells of each treatment group, the expression levels of LEF-1, PTC-1 and KGF were confirmed, and the results are shown graphically in FIG. 8.
단, 상기 LEF-1(lymphoid enhancer-binding factor-1)은 모낭의 형태 형성과 분화를 촉진하는 유전자이고, PTC-1(patched protein, Sonic Hedgehog(Shh) receptor)는 모낭의 형태를 형성하고 그 성장을 조절하는 유전자이고, KGF(keratinocyte growth factor)는 모낭의 성장, 발달 및 분화에 중요한 영향을 미치는 유전자에 해당한다. However, the lymphoid enhancer-binding factor-1 (LEF-1) is a gene that promotes the formation and differentiation of hair follicles, and the PTC-1 (patched protein, Sonic Hedgehog (Shh) receptor) forms the hair follicle and It is a gene that regulates growth, and KGF (keratinocyte growth factor) corresponds to a gene that has an important effect on the growth, development and differentiation of hair follicles.
도 8에서 보는 바와 같이, 엑소좀이 제거된 지방 유래 줄기세포 배양액을 처리한 경우보다 엑소좀을 처리한 경우 LEF-1, PTC-1 및 KGF의 발현율이 현저히 증가하는 것을 확인할 수 있다. As shown in Figure 8, it can be seen that the expression rate of LEF-1, PTC-1 and KGF significantly increased when treated with exosomes than when treated with adipose-derived stem cell culture medium from which the exosomes were removed.
이를 통하여 본 발명에 따르는 지방 유래 줄기세포 배양액으로부터 얻어진 엑소좀은 모낭의 성장, 발달 및 분화에 중요한 역할을 하는 것을 알 수 있다.Through this, it can be seen that the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in the growth, development and differentiation of hair follicles.
이상, 본 발명을 상기 실시예를 중심으로 하여 설명하였으나 이는 예시에 지나지 아니하며, 본 발명은 본 발명의 기술분야에서 통상의 지식을 가진 자에게 자명한 다양한 변형 및 균등한 기타의 실시 예를 이하에 첨부한 청구범위 내에서 수행할 수 있다는 사실을 이해하여야 한다.In the above, the present invention has been described with reference to the above embodiment, which is merely an example, and the present invention includes various modifications and other equivalent embodiments that are obvious to those skilled in the art. It should be understood that it can be carried out within the scope of the appended claims.
본 발명은 줄기세포 또는 그 배양액 유래 엑소좀을 포함하며, 상처 치료, 피부 개선 및 탈모 방지 또는 치료의 효과를 갖는 조성물 및 그 제조방법에 관한 것이다.The present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.

Claims (24)

  1. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하며,Adult stem cells or culture medium derived from the exosomes (exosomes) as an active ingredient,
    상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함되는 상처 치료용 약학적 조성물.The exosomes are wound composition comprising 1 X 10 7 ~ 1 X 10 12 / ml of the number of particles of the pharmaceutical composition for wound treatment.
  2. 제1항에 있어서,The method of claim 1,
    상기 성체 줄기세포는 골수, 혈액, 뇌, 피부, 지방, 제대혈 및 제대의 바르톤 젤리(Wharton's jelly) 중 적어도 하나로부터 유래된 것인 상처 치료용 약학적 조성물.The adult stem cells are wound marrow, blood, brain, skin, fat, umbilical cord blood and umbilical cord of Barton's jelly (Wharton's jelly) is a pharmaceutical composition for wound treatment.
  3. 제1항에 있어서,The method of claim 1,
    상기 엑소좀은 1 ~ 100㎍/ml의 양으로 포함되는 상처 치료용 약학적 조성물.The exosome is a pharmaceutical composition for treating wounds contained in an amount of 1 ~ 100㎍ / ml.
  4. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하며,Adult stem cells or culture medium derived from the exosomes (exosomes) as an active ingredient,
    상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함되는 피부 개선용 화장료 조성물.The exosome is a cosmetic composition for skin improvement comprising a number of particles of 1 X 10 7 ~ 1 X 10 12 / ml.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 성체 줄기세포는 골수, 혈액, 뇌, 피부, 지방, 제대혈 및 제대의 바르톤 젤리(Wharton's jelly) 중 적어도 하나로부터 유래된 것인 피부 개선용 화장료 조성물.The adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood and umbilical cord Barton's jelly (Wharton's jelly).
  6. 제4항에 있어서,The method of claim 4, wherein
    상기 엑소좀은 1 ~ 100㎍/ml의 양으로 포함되는 피부 개선용 화장료 조성물.The exosome is a cosmetic composition for improving the skin contained in the amount of 1 ~ 100㎍ / ml.
  7. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 치료용 약학적 조성물.Hair loss prevention or therapeutic pharmaceutical composition comprising an adult stem cell or its culture-derived exosomes (exosomes) as an active ingredient.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 성체 줄기세포는 골수, 혈액, 뇌, 피부, 지방, 제대혈 및 제대의 바르톤 젤리(Wharton's jelly) 중 적어도 하나로부터 유래된 것인 탈모 방지 또는 치료용 약학적 조성물.The adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood and umbilical cord Barton's jelly (Wharton's jelly) pharmaceutical composition for preventing or treating hair loss.
  9. 제7항에 있어서,The method of claim 7, wherein
    상기 엑소좀은 1 ~ 100㎍/ml의 양으로 포함되는 탈모 방지 또는 치료용 약학적 조성물.The exosome is a pharmaceutical composition for preventing or treating hair loss is contained in the amount of 1 ~ 100㎍ / ml.
  10. 제7항에 있어서,The method of claim 7, wherein
    상기 엑소좀은 1 X 107 ~ 1 X 1012/ml의 입자수로 포함되는 탈모 방지 또는 치료용 약학적 조성물.The exosomes are 1 X 10 7 ~ 1 X 10 12 / ml of the hair loss prevention or therapeutic pharmaceutical composition comprising a particle number.
  11. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 식품 조성물.Food composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  12. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 화장료 조성물.Hair loss prevention or improvement cosmetic composition comprising an adult stem cell or its culture-derived exosomes (exosomes) as an active ingredient.
  13. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 식품 조성물.Food composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  14. 성체 줄기세포 또는 그 배양액 유래 엑소좀(exosome)을 유효성분으로 포함하는 탈모 방지 또는 개선용 화장료 조성물.Hair loss prevention or improvement cosmetic composition comprising an adult stem cell or its culture-derived exosomes (exosomes) as an active ingredient.
  15. 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; Centrifuging adult stem cell culture at 100-500 g for 5-30 minutes;
    상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
    상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g;
    상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
    상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함하는 상처 치료용 약학적 조성물의 제조방법.Recovering the pellets after the fourth centrifugation to recover the pellets by recovering the pellets by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  16. 제15항에 있어서,The method of claim 15,
    상기 성체 줄기세포 배양액은 성체 줄기세포를 800~1,200 mg/L의 D 글루코스, 500~600 mg/L의 L-글루타민, 100~200 mg/L의 소디움피루베이트, 5~15 부피%의 우태아혈청(Fetal Bovine Serum, FBS) 및 0.5~1.5부피%의 페니실린-스트렙토마이신이 보충된 DMEM 배지에 접종한 뒤 80~95 %의 습도 및 30~40 ℃ 온도 조건 하에서 5~10 % CO2 배양기에서 계대 배양하는 단계; 및 계대 배양된 배양물을 350~400 mg/L의 L-글루타민, 10~20 mM의 HEPES 및 50~60 mg/L의 소듐 피루베이트로 보충된 DMEM와 Ham's F-12 영양소 혼합액의 1:0.5~2 중량비의 혼합 배지에 접종한 뒤 1~5부피%의 산소를 포함하는 저산소(Hypoxia) 조건으로 60~120 시간 동안 배양하는 단계에 의하여 제조되는, 상처 치료용 약학적 조성물의 제조방법.The adult stem cell culture medium contained adult stem cells at 800-1,200 mg / L D glucose, 500-600 mg / L L-glutamine, 100-200 mg / L sodium pyruvate, 5-15% by volume fetal calf Inoculated in DMEM medium supplemented with serum (Fetal Bovine Serum (FBS)) and 0.5-1.5% by volume penicillin-streptomycin, and then incubated in a 5-10% CO 2 incubator under conditions of 80-95% humidity and 30-40 ° C Passaging; And 1: 0.5 of DMEM and Ham's F-12 nutrient mixtures supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate Inoculated in a mixed medium in a weight ratio of ~ 2 and prepared by culturing for 60 to 120 hours under hypoxia (Hypoxia) conditions containing 1 to 5% by volume of oxygen, a method for preparing a pharmaceutical composition for wound treatment.
  17. 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; Centrifuging adult stem cell culture at 100-500 g for 5-30 minutes;
    상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
    상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g;
    상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
    상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함하는 피부 개선용 화장료 조성물의 제조방법.Recovering the pellets after the fourth centrifugation, the method for producing a cosmetic composition for skin improvement comprising recovering the pellets by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  18. 성체 줄기세포 배양액을 100~500g에서 5~30분 동안 1차 원심 분리하는 단계; Centrifuging adult stem cell culture at 100-500 g for 5-30 minutes;
    상기 1차 원심 분리 후 상등액을 회수하여 1,000~3,000g에서 10~30분 동안 2차 원심 분리하는 단계; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
    상기 2차 원심 분리 후 상등액을 회수하여 5,000~20,000 g에서 20~60분 동안 3차 원심 분리하는 단계; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g;
    상기 3차 원심 분리 후 상등액을 회수하여 100,000~120,000 g에서 60~120분 동안 4차 원심 분리하는 단계; 및Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
    상기 4차 원심 분리 후 펠렛을 회수하여 50,000~150,000 g에서 60~120분간 5차 원심 분리하여 펠렛을 회수하는 단계를 포함하는 탈모 방지 또는 치료용 약학적 조성물의 제조방법.Recovering the pellets after the fourth centrifugation to recover the pellets by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g for recovering the pellets.
  19. 상처를 치료하기 위하여, 치료를 필요로 하는 대상에 청구항 제1항 내지 제3항 중 어느 한 항의 약학 조성물을 투여하는 단계를 포함하는, 상처 치료 방법. A method of treating a wound, comprising administering the pharmaceutical composition of claim 1 to a subject in need thereof for treating the wound.
  20. 피부를 개선하기 위하여, 개선을 필요로 하는 대상에 청구항 제4항 내지 제6항 중 어느 한 항의 화장료 조성물을 투여하는 단계를 포함하는, 피부 개선 방법. In order to improve the skin, comprising the step of administering the cosmetic composition of any one of claims 4 to 6 to the subject in need of improvement, skin improvement method.
  21. 탈모를 방지 또는 치료하기 위하여, 치료를 필요로 하는 대상에 청구항 제7항 내지 제10항 중 어느 한 항의 약학적 조성물을 투여하는 단계를 포함하는, 탈모의 방지 또는 치료 방법. A method for preventing or treating hair loss, comprising administering the pharmaceutical composition of any one of claims 7 to 10 to a subject in need thereof in order to prevent or treat hair loss.
  22. 상처를 치료하기 위한 청구항 제1항 내지 제3항 중 어느 한 항의 약학적 조성물의 용도. Use of the pharmaceutical composition of claim 1 for treating a wound.
  23. 피부를 개선하기 위한 청구항 제4항 내지 제6항 중 어느 한 항의 화장료 조성물의 용도. Use of the cosmetic composition of claim 4 for improving the skin.
  24. 탈모를 방지 또는 치료하기 위한 청구항 제7항 내지 제10항 중 어느 한 항의 약학적 조성물의 용도. Use of the pharmaceutical composition of claim 7 for preventing or treating hair loss.
PCT/KR2017/003645 2017-04-03 2017-04-03 Composition for treating scars, improving skin, and preventing or treating hair loss including stem cell-derived exosome, and method for preparing same WO2018186505A1 (en)

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