WO2021104214A1 - Use of exosome derived from carcass in skin regulation product - Google Patents

Use of exosome derived from carcass in skin regulation product Download PDF

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WO2021104214A1
WO2021104214A1 PCT/CN2020/130931 CN2020130931W WO2021104214A1 WO 2021104214 A1 WO2021104214 A1 WO 2021104214A1 CN 2020130931 W CN2020130931 W CN 2020130931W WO 2021104214 A1 WO2021104214 A1 WO 2021104214A1
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exosomes
skin
products
product
fetal
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PCT/CN2020/130931
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French (fr)
Chinese (zh)
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傅文燕
胡适
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沣潮医药科技（上海）有限公司
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    • 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
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/982—Reproductive organs; Embryos, Eggs
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/34—Animal material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C15/00—Butter; Butter preparations; Making thereof
    • A23C15/12—Butter preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/09—Other cheese preparations; Mixtures of cheese with other foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G4/00—Chewing gum
    • A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
    • A23G4/12—Chewing gum characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
    • A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the technical field of biological skin conditioning products, and in particular relates to the use of exosomes derived from carcass in skin conditioning products and skin conditioning products containing the exosomes.
  • the descriptive literature does not describe the actual composition of sheep placenta. At present, most of the sheep placenta products on the market use physical and chemical means to break and crack the fetus, and then further obtain the protein and other molecules in it. On the one hand, its biological effects need to be further observed, which is generally considered to be more controversial. On the other hand, it is very different from the sheep fetal therapy described by Paul Nihan. Therefore, the active ingredients of sheep placenta are not clear, which has always been a scientific problem in the field.
  • exosome living cells can secrete a large number of exosomes (exosome), exosomes are endosomes that reverse budding to form multivesicular endosomes, and multivesicular endosomes fuse with cell membranes. Released outside the cell to form exosomes. Almost all cells can secrete exosomes.
  • the characteristics of these exosomes are: the diameter is between 30-150nm; the density is between 1.13-1.19g/mL; they express specific proteins and carry important signal molecules of the parent cell, including proteins, lipids and RNA, etc. ; Maintain the life activity similar to the parental cells. In particular, once the cell dies, the amount of exosomes secreted immediately decreases, indicating that exosomes are an active effector secreted by living cells.
  • Mammalian umbilical cord, placenta, etc. are important sources of exosomes. There have been many studies on extracting exosomes from mammalian umbilical cord or placenta and using them for skin conditioning, but animal fetuses have not been seen as the source of such exosomes. Research. In a small number of studies using animal carcass as a raw material for material extraction, the extracts are mostly concentrated in small molecular weight water-soluble molecules. For example, the patent document CN 103462864B discloses the separation of water-soluble molecules with a molecular weight of less than 4000D from the internal organs of animals. Used in cosmetics and food production.
  • patent document CN 103462865 B discloses the extraction and use of water extracts with molecular weights below 5000D from animal carcass skin and muscle. It is a water-soluble molecule with a molecular weight of less than 5000D, not an exosome.
  • the present invention was made to solve the above-mentioned technical problems, and aims to provide the use of exosomes derived from carcass in skin conditioning products, a method for preparing the exosomes, and skin conditioning products using the exosomes as active ingredients.
  • the first aspect of the present invention is to provide the use of carcass-derived exosomes in skin conditioning products.
  • the exosomes are exosomes extracted from the carcass of non-human viviparous animals, new internal organs or physiological fluids.
  • the fetus refers to larvae and new-born animals in the mothers of non-human viviparous animals, excluding fetal appendages.
  • sheep, cattle, deer and camels namely fetal sheep, fetal cattle, fetal deer and fetal camels
  • animal larvae are preferably fetal sheep at least 2 months old, fetal cattle at least 4 months old, fetal deer at least 3 months old, Fetal camels that are at least 6 months old
  • newborns refer to newborn animals within 24 hours of natural delivery.
  • the organs include lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and accessories, uterus, skin, kidney, sinus, colon, rectum, esophagus, blood, brain and its covering, spinal cord and its covering , Muscle, connective tissue, adrenal gland, parathyroid, thyroid, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymphatic system and other organs; preferably liver, bone marrow.
  • the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph , Plasma, serum, synovial fluid, cerebrospinal fluid, uterine cavity and appendages, urine, and interstitial, intracellular and extracellular fluids; preferably blood, plasma and serum.
  • exosomes can promote cell proliferation, improve cell survival, promote cell ATP synthesis, increase mitochondrial activity, reduce inflammation and cell senescence, and increase the expression of some local targets. It is effective in aging. That is, in terms of application, the inventor found that exosomes have cosmetic and dermatological uses.
  • the cosmetic and dermatological uses include anti-aging, enhancing skin elasticity, fighting acne, strengthening hair follicles, and anti-hair loss.
  • the skin conditioning products of the present invention include direct application skin care products, oral skin conditioning products, or dermatological use products.
  • the direct application type skin care product is an anti-aging or skin elasticity skin care product
  • the oral skin conditioning product includes dairy products containing the exosomes, fat-based products, beverage products or cereal products
  • products for dermatological purposes are Dermatological products for fighting acne or strengthening hair follicles and anti-hair loss.
  • the anti-aging or skin elasticity skin care products are one or more combinations of skin care products that promote cell collagen expression, increase cell myofibril protein synthesis, maintain stem cell stemness, and stimulate stem cell proliferation; fight acne
  • the dermatological use products of keratinocytes are products that inhibit the gene expression of inflammatory mediators or matrix proteases in keratinocytes, and one or a combination of products that inhibit VEGF synthesis and release in keratinocytes; enhance hair follicles and anti-hair loss skin
  • the products for disease use are any one or two of products that promote ATP synthesis in hair follicle papillary fibroblasts or enhance mitochondrial metabolic activity, increase cell viability of microhair follicles, or reduce microhair follicle membrane damage.
  • the exosomes derived from the fetus of the present invention are intended to resist aging and promote healing by stimulating proliferation and maintaining the activity of stem cells. Therefore, the examples presented below show that exosomes have the following effects on human mesenchymal stem cells: increase the ratio of ALDH-positive cells and the expression of Ki67 ratio.
  • Exosomes specifically target the inflammatory process of acne that plays a central role in the development and aggravation of the disease. Therefore, the examples presented below show that exosomes have the following effects on keratinocytes:
  • Anti-inflammatory effect Inhibition of early mediators of inflammation (IL-1 ⁇ , IL-1 ⁇ ) and late mediators (IL-8); inhibition of keratinocyte-induced MMP (MMP-2 and MMP-9); inhibition of VEGF release effect.
  • Exosomes also have the following effects on P. acnes: inhibit P. acnes from inducing IL-8; inhibit P. acnes from inducing MMP-9.
  • topical or oral administration of the fetus-derived exosomes is used to maintain and/or increase the expression of type V collagen and/or myofibrin-1 and/or to increase the proliferation of hair follicle fibroblasts and/or It is used to increase cell viability and/or ATP synthesis and/or mitochondrial activity and/or reduce cell damage and/or reduce cell aging, and is used to strengthen hair follicles, thereby reducing the loss of skin appendages, such as hair loss.
  • the topical or oral administration of exosomes derived from the carcass of the present invention is used to increase the antioxidant enzymes NQO1 (Entrez Gene: 1728) and GSTT1 (Entrez Gene: 2952). Expression to eliminate and eliminate toxins; increase the expression of the natural immune factor skin antimicrobial peptide hBD3 (Entrez Gene: 55894) to achieve skin protection; and increase the antioxidant protein GPX1 (Entrez Gene: 2876) and catalase (catalase, Entrez Gene: 847), TRX1 (Entrez Gene: 7295) and PRDX1 (Entrez Gene: 5052) expression to inhibit the production of reactive oxygen species. Therefore, the composition can be used as a pharmaceutical composition or a cosmetic composition for preventing skin problems caused by Asian sand dust (PM2.5), protecting the skin, and preventing and treating skin aging.
  • PM2.5 Asian sand dust
  • the second aspect of the present invention is to provide a method for preparing carcass-derived exosomes, which are prepared by a method including the following steps:
  • the separation and purification of exosomes includes: centrifuging the culture solution or fluid at 4°C, 1000g for 10 minutes, and collecting the supernatant; collecting the supernatant at 4°C and centrifuging at 2000g for 20 minutes to collect the supernatant; and collecting the supernatant at 4°C Centrifuge at 10,000g for 30min to collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer. Filtration with 0.45 ⁇ m filter membrane to obtain exosomes.
  • the exosomes prepared according to the above method have the following characteristics: a diameter of about 30-150 nm, a lipid membrane, and genetic materials such as protein, mRNA, and microRNA, etc. are encapsulated in the exosomes.
  • Protein analysis contains CD63, CD81, Alix and other protein molecules.
  • the third aspect of the present invention provides a composition containing exosomes as an active ingredient.
  • the composition contains at least one pharmaceutically acceptable excipient, especially cosmetically and dermatologically acceptable excipients.
  • the excipient may be an excipient suitable for topical application in vitro, or an excipient that can be applied to the human body.
  • formulations are suitable for topical application, and include creams, gels, emulsifiers, emulsions, ointments, lotions, oils, aqueous solutions or hydrogen-alcohol or glycolic acid solutions, Powder, patch, spray or any other product for external application.
  • Such formulations are presented in the examples below.
  • various formulations are suitable for oral administration, wherein the exosomes can be included in a dietary supplement or dietary composition.
  • dietary supplements can be provided in the form of hard or soft gelatin or vegetable capsules.
  • the dietary supplement may thus contain 1% to 100% exosomes by weight.
  • exosomes of the present invention can also be incorporated into dietary compositions such as foods, beverages and nutraceuticals, and the dietary compositions include the following products:
  • Dairy products such as cheese, butter, milk and other milky beverages, mixtures and pastes containing milk products, ice cream and yogurt;
  • Fat-based products such as margarine, pasty food, mayonnaise, cooking fat, frying oil and vinaigrette;
  • Cereal-based products composed of grains, such as bread and pasta products, regardless of whether these foods have been cooked, baked or processed;
  • Candies such as chocolate, confectionery, chewing gum, desserts, toppings, refreshing fruit drinks (sorbet), cake icing and other garnishes;
  • Alcoholic or non-alcoholic beverages including soda and other soft drinks, juices, dietary supplements, meal substitutes in the form of beverages such as those sold under the brand names Boost TM and Ensure TM, and;
  • exosomes can be incorporated into food, nutraceuticals, dietary products, especially high-protein products or beverages directly with the help of techniques such as mixing, infusion, injection, blending, adsorption, kneading and spraying without other modifications.
  • the fourth aspect of the present invention also relates to a method for the cosmetic treatment of acne, which is characterized in that the composition of the present invention is applied to a diseased skin area or a diseased individual orally takes the nutraceutical composition of the present invention.
  • the present invention may also relate to the use of exosomes for the preparation of cosmetics, nutraceuticals or dermatological compositions for the treatment or prevention of acne.
  • composition will preferably be formulated in a formulation suitable for topical application.
  • composition in the context of use in food, for nutritional or cosmetic purposes (cosmetic food), the composition will preferably be formulated in a formulation suitable for oral administration.
  • the cosmetic care method of the present invention includes topical application of the exosomes of the present invention or a composition containing the same to all or part of the body selected from the group consisting of legs, feet, armpits, hands, thighs, abdomen, neckline, The neck, arms, trunk, back, face, skin appendages, preferably hair, and/or scalp, more advantageously the scalp.
  • composition of the present invention may also contain at least one pharmaceutical adjuvant known to those skilled in the art, such as thickeners, preservatives, fragrances, colorants, chemical or mineral sunscreens, wetting agents, and hot spring water. Wait.
  • pharmaceutical adjuvant known to those skilled in the art, such as thickeners, preservatives, fragrances, colorants, chemical or mineral sunscreens, wetting agents, and hot spring water. Wait.
  • composition of the present invention may also comprise a sebum regulator, antibacterial agent, antifungal agent, keratolytic agent, stratum corneum regulator, astringent, anti-inflammatory/anti-irritant, antioxidant/radical scavenger, At least one anti-aging compound of scarring agent, anti-aging agent and/or wetting agent.
  • sodium regulator refers, for example, to 5- ⁇ -reductase inhibitors, such as active substances Zinc and its gluconate, salicylate and pyroglutamic acid also have sebum inhibitory activity. Mention can also be made of spironolactone, an antiandrogen and aldosterone antagonist, which significantly reduces the rate of sebum secretion after 12 weeks of application. Other extracted molecules (such as seeds from Cucurbita pepo, pumpkin seed oil, and palm cabbage) limit sebum production by inhibiting 5- ⁇ -reductase transcription and activity. Other lipid-derived sebum regulators that affect the properties of sebum, such as linoleic acid, can also be used.
  • antibacterial agent and “antifungal agent” refer to molecules that restrict the growth of or destroy pathogenic microorganisms such as certain bacteria such as Propionibacterium acnes or certain fungi (Malassezia furfur).
  • preservatives commonly used in cosmetics or nutraceuticals, that is, molecules with antibacterial activity (pseudopreservatives) such as caprylic acid derivatives (caprylylglycine, caprylic glyceride, etc.), such as hexylene glycol and sodium levulinate , Zinc and copper derivatives (gluconate and PCA), phytosphingosine and its derivatives, benzoyl peroxide, piroctone ethanolamine, zinc pyrithione, selenium disulfide, econazole, ketone Conazole, or topical antibiotics such as erythromycin and clindamycin.
  • stratum corneum regulator and “keratolytic agent” refer to substances that regulate or assist the elimination of dead cells in the epidermal stratum corneum.
  • Commonly used stratum corneum regulators/stratum corneum separation agents include: fruit alpha-hydroxy acid (AHAS) (citric acid, glycolic acid, malic acid, lactic acid, etc.) ), the combination of AHA ester, AHAS and other molecules such as the combination of malic acid and marzipan
  • AHAS fruit alpha-hydroxy acid
  • AHA ester AHAS and other molecules
  • glycolic acid or lactic acid and arginine or hydroxy acid and lipid molecules such as (Lipo-hydroxy acid) combination, amphoteric hydroxy acid complex (AHCare), willow bark (white willow bark extract), azelaic acid and its salts and esters, salicylic acid and its derivatives such as caprylyl salicylic acid Or in combination with other molecules such as salicylic acid and polysaccharides ( ⁇
  • astringent refers to substances that help narrow pores. The most commonly used are polyphenols, zinc derivatives, and North American witch hazel.
  • anti-inflammatory/anti-irritant refers to a substance that limits the inflammatory response caused by cytokines or arachidonic acid metabolism mediators and has smooth and anti-irritant properties.
  • the most common are glycyrrhetinic acid (glycyrrhizin derivatives) and its salts and esters, ⁇ -bisabolol, Ginkgo biloba, Calendula, lipoic acid, ⁇ -carotene, vitamin B3 (niacinamide) Nicolamide), vitamin E, vitamin C, vitamin B12, flavonoids (green tea, quercetin, etc.), lycopene or lutein, avocado sugar, avocado oleodistillate, arabic gal Glycan, lupin peptide, total lupin extract, quinoa peptide extract, circulating ceramide (Oxazoline derivatives), anti-glycation agents such as carnosine, N-acetylcysteine, isoflavones
  • Antioxidant refers to molecules that reduce or prevent the oxidation of other chemicals.
  • Antioxidant/radical scavengers that can be used in combination are preferably selected from the group consisting of mercaptans and phenols, licorice derivatives such as glycyrrhetinic acid and its salts and esters, ⁇ -bisabolol, ginkgo extract, marigold Flower (Calendula) extract, circulating ceramide (Oxazoline derivatives), avocado peptides, trace elements such as copper, zinc and selenium, lipoic acid, vitamin B12, vitamin B3 (niacinamide, nicotiamide), vitamin C, vitamin E, coenzyme Q10, krill oil (krill), glutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), lycopene or lutein, ⁇ -carotene, polyphenol family such as tannins, phenolic acids
  • the antioxidant group also includes anti-glycation agents such as carnosine or certain peptides, N-acetylcysteine, and antioxidant or free radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, glutathione Peroxidase, thioredoxin reductase and its antagonists.
  • anti-glycation agents such as carnosine or certain peptides, N-acetylcysteine, and antioxidant or free radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, glutathione Peroxidase, thioredoxin reductase and its antagonists.
  • SOD superoxide dismutase
  • catalase glutathione Peroxidase
  • thioredoxin reductase and its antagonists.
  • the substance that can be used in combination to generate scars/repair barrier function is preferably vitamin A, panthenol (vitamin B5), avocado furan Lupin, Macatide Extract, Quinoa Peptide Extract, Arabinogalactan, Zinc Oxide, Magnesium, Silicon, Asiatic Acid or Asiatic Acid, Dextran Sulfate, Coenzyme Q10, Glucosamine And its derivatives, chondroitin sulfate and total glycosaminoglycans (GAGs), dextran sulfate, ceramide, cholesterol, squalane, phospholipids, fermented or unfermented soybean peptides, plant peptides, marine polysaccharides, plants Polysaccharides or biotechnological polysaccharides, such as algae extracts or fern extracts, trace elements, tannin-rich plant extracts such as tannins derived from gallic acid, which are called gallic tannins or hydrolyzable tannins, It was originally found in oak gall, and
  • Anti-aging agents that can be used in combination to treat acne in mature subjects are antioxidants, especially vitamin C, vitamin A, retinol, retinal, hyaluronic acid of any molecular weight, avocado furan Lupin peptide and maca peptide extract.
  • humectants/emollients are glycerin or its derivatives, urea, pyrrolidone carboxylic acid and its derivatives, hyaluronic acid of any molecular weight, glycosaminoglycans and those of marine, plant or biotechnological origin Any other polysaccharides, such as xanthan gum, Certain fatty acids such as lauric acid, myristic acid, monounsaturated and polyunsaturated omega-3, -6, -7 and -9 fatty acids (linoleic acid, palmitoleic acid, etc.), sunflower oleodistillate , Avocado peptides and cupuacu butter.
  • the combination of the present invention includes a composition comprising exosomes and plant and animal unsaponifiables, for example, avocado and soybean unsaponifiables and unsaponifiable vegetable oils or animal oil concentrates
  • unsaponifiables for example, avocado and soybean unsaponifiables and unsaponifiable vegetable oils or animal oil concentrates
  • sunflower oil or palm oil concentrate or vegetable oils containing unsaponifiables, such as soybean oil and rapeseed oil, and derivatives of unsaponifiables such as avocado furan, sterol esters, and vitamin derivatives.
  • the combination of the present invention includes a composition comprising exosomes and avocado sugar (see application WO2005/115421).
  • the composition is particularly suitable for the treatment of skin barrier repair and inflammation.
  • the combination of the present invention includes a composition comprising exosomes and avocado peptides (see International Application WO2005/105123).
  • the composition is particularly suitable for treating irritation and inflammation.
  • the combination of the present invention includes a composition comprising exosomes and avocado oil (see international applications WO2004/012496, WO2004/012752, WO2004/016106, WO2007/057439).
  • the combination of the present invention includes a composition comprising exosomes and avocado furan (Avocado furan, which can be obtained by the method described in the international application WO01/21605).
  • the composition is particularly suitable for treating inflammation, promoting scar formation and suitable for its anti-aging properties.
  • the combination of the present invention includes a composition comprising exosomes and avocado and soy unsaponifiables.
  • the avocado and soybean unsaponifiables that can be used in combination are preferably a mixture of avocado furanic unsaponifiables and soybean unsaponifiables in a ratio of about 1:3-2:3, respectively.
  • the combination of the present invention includes a composition comprising exosomes and lupeol (FR2822821, FR2857596).
  • the composition is particularly suitable for supporting scar formation.
  • the combination of the present invention includes a composition comprising exosomes and lupin peptides, such as lupin peptides obtained according to the method described in application WO2005/102259.
  • the composition is particularly suitable for treating inflammation and is used because of its anti-aging properties.
  • the combination of the present invention includes a composition comprising exosomes and total lupin extract (see International Application WO2005/102259).
  • the composition is particularly suitable for treating irritation.
  • the combination of the present invention includes a composition comprising exosomes and lupin oil, preferably sweet white lupin oil, as described in the international application WO98/47479 White lupin oil.
  • the combination of the present invention includes a composition comprising exosomes and a maca peptide extract (see International Application WO2004/112742).
  • the composition is particularly preferred due to its scarring properties and anti-aging properties.
  • the combination of the present invention includes a composition comprising exosomes and rice peptides (see International Application 2008/009709).
  • the composition is particularly preferred due to its properties related to the stimulation of melanin formation and the transfer of melanin.
  • the combination of the present invention includes a composition comprising exosomes and circulating ceramide (Oxazoline derivatives), as described in international applications WO2004/050052, WO2004/050079 and WO2004/112741.
  • the composition is particularly suitable for the treatment of inflammatory reactions.
  • the combination of the present invention includes a composition comprising exosomes and quinoa extract, especially peptide extract (see International Application WO2008/080974).
  • the composition is particularly suitable for the treatment of inflammatory diseases and skin barrier repair.
  • the combination of the present invention includes a composition comprising exosomes and cupuacu butter.
  • the composition is particularly preferred because of its wetting properties.
  • the combination of the present invention includes a composition comprising exosomes and rapeseed oil essence (rapeseed oleodistillate).
  • the combination of the present invention includes a composition comprising exosomes and corn oleodistillate.
  • all of these combinations contain at least one other active compound, and may contain two, three, four or more active compounds as described above.
  • the exosomes of the present invention can be combined with sunscreen active substances known to those skilled in the art such as UVB and/or UVA filters or shades or Any inorganic and/or organic light-shielding material or filter is used in combination, and the skilled person will adjust their selection and concentration according to the degree of protection sought.
  • sunscreen active substances known to those skilled in the art such as UVB and/or UVA filters or shades or Any inorganic and/or organic light-shielding material or filter is used in combination, and the skilled person will adjust their selection and concentration according to the degree of protection sought.
  • sunscreen active substances titanium dioxide, zinc oxide, methylene bis-benzotriazole tetramethyl butyl phenol (brand name: TIINOORB M) and bis-ethylhexyloxyphenol methoxybenzene can be specifically mentioned.
  • Triazine brand name: TINOSORB S
  • octyl-salicylic acid butylmethoxydibenzoylmethane
  • terephthalmethylene dicamphorsulfonic acid 4-methylbenzylidene camphor
  • benzophenone Ethylhexyl methoxycinnamate
  • aging and the term “skin aging” are used to describe visible changes in the appearance of the skin and those visible changes that can be detected by touch, such as wrinkles, fine lines, and wrinkles in a non-limiting sense.
  • Roughness, expression lines, stretch marks, discontinuities, deep wrinkles, sagging, sagging skin (such as sagging cheeks, eye bags, double chin), increase in pore size, loss of elasticity, loss of resilience, loss of firmness, elastic tissue degeneration , Abnormal differentiation, hyperkeratosis, keratosis, skin color changes (such as spots, redness, or bags under the eyes), formation of hyperpigmented areas (such as age spots, melasma, or freckles), loss of smoothness, orange peel Skin-like skin, loss of collagen structure and other histological changes in the stratum corneum, dermis, epidermis, vascular system (such as the formation of spider veins or telangiectasia), or these tissues near the skin.
  • Skin aging is a process with two main parts.
  • the two main parts are: temporal aging, which is attributed to the passage of time; and light-induced aging, which is attributed to the level of exposure to ultraviolet radiation and is called light Ageing.
  • the sum of several environmental factors such as exposure to tobacco smoke, exposure to pollution, and climatic conditions (such as cold and/or wind) also contribute to skin aging.
  • skin anti-aging treatment is a treatment for preventing, delaying, and/or reducing the aging of human skin.
  • the present invention provides the use of carcass-derived exosomes in skin conditioning products.
  • the exosomes can promote cell collagen expression, increase cell myofibrillar protein synthesis, maintain stem cell stemness, and stimulate stem cells. Proliferation to achieve anti-aging function; products that can inhibit inflammatory mediators or matrix protease gene expression in keratinocytes, and inhibit the synthesis and release of VEGF in keratinocytes to achieve pharmaceutical functions against acne; can promote hair follicle papillary fibroblasts ATP synthesis or products that enhance the metabolic activity of mitochondria, increase the cell viability of the microhair follicles or reduce the damage of the microhair follicle membrane, realize the dermatological use of enhancing hair follicles and anti-hair loss; it can achieve clearance and elimination by increasing the expression of antioxidant enzymes NQO1 and GSTT1 Eliminate toxins; increase the expression of the skin antimicrobial peptide hBD3, which is a natural immune factor, to protect the skin; and increase the
  • the present invention provides a new approach for skin care such as anti-aging skin care, acne fighting, anti-hair loss, and anti-Asian dust effects.
  • Figure 1 is the electron microscope detection result of the exosomes derived from the carcass of the present invention
  • Figure 2 shows the results of the particle size distribution of exosomes detected by NTA.
  • the comparison statistics between multiple groups uses analysis of variance and between-group tests to calculate the P value.
  • the non-parametric T test is used to calculate the P value.
  • the comparison of multiple rates uses the chi-square test to calculate the P value. .
  • the 4-month-old Alpine black sheep fetal serum was filtered with a 0.45 ⁇ m filter, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant Centrifuge the supernatant at 10,000g for 30min at 4°C, and collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet in phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend in a small amount of phosphate buffer.
  • exosomes Precipitate, filter with 0.45 ⁇ m filter membrane to obtain exosomes.
  • the Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent).
  • the exosomes were lyophilized and stored at -80°C.
  • Adult goat serum was used to extract exosomes in the same way as control exosomes.
  • the 11-month-old Australian alpaca serum was filtered with a 0.45 ⁇ m filter, centrifuged at 4°C, 1000g for 10min, and collected the supernatant; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant was 4°C Centrifuge at 10,000g for 30min to collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer.
  • exosomes were lyophilized and stored at -80°C.
  • Adult alpaca serum was used to extract exosomes in the same way as control exosomes.
  • the 8-month-old Australian fetal bovine serum was filtered with a 0.22 ⁇ m sterile membrane, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant 4 Centrifuge the supernatant at 10,000g for 30min at °C, and collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer , 0.45 ⁇ m filter membrane to obtain exosomes.
  • the Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80
  • the liver of 4-month-old alpine black sheep fetal sheep was washed repeatedly with phosphate buffered saline (PBS) and cut into tissue pieces with a diameter of about 1-2 mm; after digestion with type 2 collagenase and trypsin, the supernatant was centrifuged Put the cell pellet into a culture flask, and use DMEM/F12 medium containing 10% fetal bovine serum, 5% CO 2 , 37°C saturated humidity; remove non-adherent cells, and replace with no serum after 80% of adherent cells are fused
  • the culture medium was cultured for 48 hours; the culture supernatant was collected; the culture solution rich in exosomes was obtained by filtering with a 0.45 ⁇ m filter membrane.
  • the particle size detection method of exosomes derived from fetal sheep serum obtained in Example 1 by NanoFCM Flow NanoAnalyzer includes the following steps:
  • Particle size detection The particle size standard, blank control and sample (uterine cavity fluid) need to be sampled under the same detection conditions (laser power and attenuation coefficient of the scattering channel are exactly the same); all samples are under lower injection pressure (Sampling is not more than 1.0kPa) detection; under appropriate conditions, detect the particle size standard (the scattered light signal of the smallest particle size silicon ball is completely separated from the background and the largest silicon ball signal is not saturated, and the detector saturation value is 3.6k ); Detection of the buffer used to resuspend the sample to be tested, used to deduct background particles, as a blank control; to detect the sample to be tested, if the signal intensity of more than 20% of the particles in the sample to be tested reaches saturation in the scattering channel, it indicates that the current The particle size standard product is not the best choice for the particle size characterization of the sample; after the above steps, it is detected that most of the particle size in the sample is around 85nm, which is in the range of 30-150nm in exosomal diameter
  • the fetal goat serum exosomes (0.02% mass-volume ratio) described in Example 1 were added to the culture medium (DMEM medium, 1% antibiotics) of human fibroblasts to confirm that the exosomes promote 1 at the cellular level.
  • Type collagen synthesis The synthetic collagen was measured using the PICP kit (Procollagen Type I C Peptidase Immunoassay Kit) for quantification.
  • human fibroblast culture medium human fibroblasts (human fibroblast) were divided into 6-well plates at 2 ⁇ 10 5 cells/well. After confirming cell adhesion, fetal goat serum exosomes as the active ingredient were dissolved in the culture medium.
  • the control group used adult goat serum exosomes with the same concentration.
  • the blank group did not add any exosomes, only the culture medium. After culturing for 24 hours in an incubator at 37°C and 5% CO2, the content of type I collagen in the cell lysate was determined. The results are shown in Table 1. The results show that fetal goat serum exosomes can promote collagen expression .
  • Example 7 The effect of exosomes on artificial skin
  • This example was performed using the fetal camel serum exosomes and control exosomes described in Example 2.
  • EFT-400 fetal sheep serum exosomes described in Example 1 at a concentration of 0.02% (mass volume ratio) was applied as an active ingredient.
  • Exosomes containing 1% serum from adult goats were used as a control (control). No exosomes were added to the blank group, only culture medium.
  • Example 8 Exosomes enhance the expression of ALDH in mesenchymal stem cells.
  • This example was performed using the fetal goat serum exosomes and control exosomes described in Example 1.
  • the mesenchymal stem cells (MSC) derived from human bone marrow were divided into 6-well plates at 2 ⁇ 10 5 cells/well. After confirming cell adhesion, fetal goat serum exosomes as the active ingredient were dissolved in culture medium. Adult goat serum exosomes were used in the control group, and the concentration of exosomes in each well was 200 ⁇ g/ml. No exosomes were added to the blank group, only culture medium. After culturing in an incubator at 37°C and 5% CO 2 for 24 hours, flow cytometry was used to detect the ALDH positive rate and Ki67 positive rate of MSC cells. The results are shown in Table 4.
  • Example 9 The effect of exosomes on the prevention and treatment of acne
  • Table 8 Analysis of IL-8 in keratinocytes/P. acnes co-culture
  • MMP-9 plays a particularly important role.
  • this protease is positively regulated by pro-inflammatory cytokines and also positively regulated by P. acnes.
  • Table 11 MMP-9 gene expression in keratinocyte/P. acnes co-culture
  • PMA stimulates keratinocytes for 24 hours to induce a large and significant release of VEGF (+226%).
  • pretreatment of keratinocytes with exosomes for 24 hours had this effect and significantly inhibited this effect.
  • the stomach of a 3-month-old fetal pig was washed repeatedly with phosphate buffered saline (PBS) and cut into tissue pieces with a diameter of about 1-2mm; after digestion with type 2 collagenase and trypsin, the supernatant was centrifuged and the cells were collected Put the precipitate in a culture flask, use DMEM/F12 medium containing 10% fetal bovine serum, 5% CO 2 , 37°C saturated humidity; remove non-adherent cells, replace the serum-free medium after the adherent cells are 80% fused 48h; collect the culture supernatant; filter with a 0.45 ⁇ m filter membrane to obtain a culture solution rich in exosomes.
  • PBS phosphate buffered saline
  • the fetal gastric juice of newborn black sheep was filtered with a 0.45 ⁇ m filter, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant was 4°C, 10000g Centrifuge for 30 min, collect the supernatant; centrifuge the collected supernatant at 110,000 g for 90 min, discard the supernatant, and resuspend the pellet in phosphate buffer; centrifuge again at 110,000 g for 90 min, discard the supernatant, and resuspend the pellet in a small amount of phosphate buffer, 0.45 ⁇ m Filter membrane to obtain exosomes.
  • the Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80°C.
  • Example 1 The fetal sheep serum exosomes described in Example 1 and the fetal sheep liver exosomes described in Example 4 were used for this example.
  • the exosomes increased the expression of type V collagen by at least 22% and 35%, showing the properties of exosomes in enhancing the expression of collagen, which can be used to strengthen hair follicles and reduce The nature of hair loss.
  • Example 14 Exosomes promote increased ATP synthesis in hair follicle papilla fibroblasts
  • Non-pathological human fibroblasts from hair follicle papilla were cultured on DMEM medium for 24 hours, and then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 6 On days, the final concentration/volume is 0.02% or 0.05% by volume relative to the total volume of the culture medium. After 6 days, the ATP content was measured by enzymatic method (luciferin/luciferase complex; ATP Bioluminescence kit Roche Diagnostics) (Table 15).
  • the fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 135 15.95 p ⁇ 0.05
  • the fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 157 20.33 p ⁇ 0.01
  • Example 15 Exosomes enhance the mitochondrial metabolic activity of dermal papilla fibroblasts
  • the non-pathological human fibroblasts from the hair follicle papilla were cultured for 24 hours, and the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 were treated for 6 days.
  • the total volume of the medium is 0.02% and 0.05%.
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • the resulting precipitate was extracted with DMSO, and then the optical density of the DMSO solution was measured at 540 nm (Table 16).
  • the exosomes of the present invention show increased mitochondrial activity of hair follicle papilla fibroblasts. This mitochondrial activity participates in the enhancement of hair follicles.
  • Example 16 The effect of exosomes on microhair follicles
  • the micro hair follicle model includes papillary fibroblasts, keratinocytes from the outer sheath of hair follicles and melanocytes co-cultured in three dimensions. Due to the ability to integrate the neuro-epidermal-mesenchymal interactions between various cell types, this cell model constitutes the closest reconstructed organ model to hair follicles.
  • Non-pathological papillary fibroblasts were cultured for 3 days. Then, melanocytes and keratinocytes of the outer sheath of hair follicles are added to the new type of papilla to form micro hair follicles. After culturing for 24 hours, the fetal goat serum exosomes described in Example 1 were added, and the final concentration was 0.05% by volume relative to the total volume of the culture medium. The same medium was cultured in the case of adult goat serum exosomes (control). After 48 hours of treatment, the culture medium and microfollicles were sampled for analysis.
  • the cell viability of microhair follicles was measured by the PrestoBlue method (Thermo Fisher Scientific). The measurement is carried out after 48 hours of treatment. The value is expressed as the mean% of the normalized control (Table 17).
  • the exosomes of the present invention have the ability to increase the cell viability of microhair follicles, making them active exosomes for enhancing hair follicles.
  • lactate dehydrogenase detection method cell damage is measured by a colorimetric method, so that cell damage can be quantified based on the measurement of lactate dehydrogenase activity in damaged cells in the culture medium. Increased cell membrane damage and cell lysis lead to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells. After 48 hours of treatment, lactate dehydrogenase activity was measured in the culture medium (Table 18).
  • the exosomes of the present invention have the ability to reduce membrane damage, making them the exosomes of the present invention that are active in enhancing hair follicles, and enhancing hair follicles promotes the reduction of hair loss.
  • Example 17 Exosomes respond to the production of reactive oxygen species in human keratinocytes caused by Asian dust
  • Asian dust is a climatic phenomenon that originated in the deserts of my country, Mongolia, Central Asia and Russia. In these places, sand or yellow dust blows eastward with the wind and falls to the ground. Almost every spring, Beijing and other northern regions are affected by Asian dust.
  • Asian dust contains organic and inorganic substances, and their physical properties and composition are different. It even contains metals that may have biological effects.
  • the large particles contained in the Asian dust remain in or around the place, while the particles with a diameter of 10 ⁇ m or less (particulate matter 10; PM10), the continuous outbreak of PM2.5 in recent years also belong to this type of climate pollution.
  • Asian dust storms are known to cause dry skin, keratinization, itching, acne, atopy, etc. by depriving the skin of moisture.
  • Asian dust causes an increase in keratinocytes including the cytokines interleukin 6 and interleukin 8 pro-inflammatory factors (Choi et al,. Toxicol Lett. 15; 200(1-2): 92 -9.2010).
  • Human epidermal neonatal keratinocyte cells were cultured for 24 hours under the same culture conditions as those described in the Lonza company's instructions. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% or 0.05% volume relative to the total volume of the culture medium. , And then add 25 ⁇ g/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season.
  • Beijing China World Trade Center Phase III
  • Example 18 Expression of Exosomes on Toxin Detoxification and Elimination Factors and Antimicrobial Peptides
  • Example 17 the human epidermal keratinocyte cells were cultured for 24 hours, and the culture conditions were the same as those described in the instructions of Lonza Company. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% and 0.05% volume relative to the total volume of the culture medium. , And then add 25 ⁇ g/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season.
  • Beijing China World Trade Center Phase III
  • RNA kit Qiagen
  • RT Superscript reverse transcriptase II kit
  • qPCR real-time reverse transcription polymerase chain reaction
  • the fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 331.97 33.29 p ⁇ 0.05
  • the fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 743.04 97.00 p ⁇ 0.05
  • the results show that the extrafetal body of the present invention can increase the expression of detoxified protein NQO1, skin antimicrobial peptide hBD3 of natural immune factors, and GSTT1 of detoxifying enzyme in normal human keratinocytes.
  • Example 17 the human epidermal keratinocyte cells were cultured for 24 hours, and the culture conditions were the same as those described in the instructions of Lonza Company. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% and 0.05% volume relative to the total volume of the culture medium. , And then add 25 ⁇ g/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season.
  • Beijing China World Trade Center Phase III
  • RNA kit Qiagen
  • RT Superscript reverse transcriptase II kit
  • qPCR real-time reverse transcription polymerase chain reaction
  • TaqMan gene expression assay kit (TaqMan gene expression assay kit, A plied Biosystems) to detect the expression levels of GPX1 (Hs00829989_gH), catalase (Hs00156308_m1), TRX1 (Hs01555212_g1) and PRDX1 (Hs00602020_mH), and the results are shown in the table .
  • the fetal sheep serum exosomes described in Example 1 of the present invention are 1.2%, butanediol 5%, preservative 0.2% and 0.6% sodium chloride, water 93%, and weigh the appropriate amount of each raw material group. Minute.
  • the above-mentioned raw material components are mixed to obtain anti-aging skin care water.
  • Volunteers apply the anti-aging cosmetics of the present invention once a day in the morning and evening, and wash their face with clean water after 30 minutes of each application. The use period is 4 weeks, and no other cosmetics are allowed to be used during the test period. Then, the skin elasticity tester and the facial wrinkle imager were used to compare and analyze the improvement of the volunteers' skin elasticity and wrinkles by the anti-aging cosmetics of the present invention. The results are as follows:
  • Example 21 Examples of cosmetic preparations
  • compositions for topical application especially for cosmetics.
  • Nylon 6 0%-2% Polyacrylamide gel 1%-5% Vitamin B6 0%-1% spices 0%-1% Pure water QSP100%
  • Raw material/brand name % castor oil QSP 100% Oleyl alcohol 10%-20% Palm oil 10%-20% Polyglycerol-3-beeswax 10%-20% Candelilla wax 10%-20% Hectorite 10%-20% Titanium dioxide 0%-5% Fetal Sheep Serum Exosomes 0.01%-0.1% Seborin 0%-5% Vitamin E 0%-1%

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Abstract

Disclosed is the use of an exosome derived from a carcass in a skin regulation product. Experiments prove that the exosome realizes an anti-aging function by means of promoting the expression of cell collagen, etc.; the exosome realizes the pharmaceutical function of resisting acne by means of inhibiting inflammatory mediators in keratinocytes, etc.; the exosome realizes the dermatological uses of enhancing hair follicles and preventing hair loss by means of promoting ATP synthesis in hair follicle dermal papilla fibroblasts, etc.; and the exosome prevents skin problems caused by Asian dust by means of inhibiting the production of active oxygen, etc.

Description

胎体来源的外泌体在皮肤调节产品中的用途Use of exosomes derived from carcass in skin conditioning products 技术领域Technical field
本发明属于生物皮肤调节产品技术领域，具体涉及一种胎体来源的外泌体在皮肤调节产品中的用途以及包含该外泌体的皮肤调节产品。The invention belongs to the technical field of biological skin conditioning products, and in particular relates to the use of exosomes derived from carcass in skin conditioning products and skin conditioning products containing the exosomes.
背景技术Background technique
在化妆品领域，“羊胎素”的概念和生物医学效应是具有争议性的。较为早期的记录是1931年，瑞士保罗尼翰医生(Dr.Paul Niehans)首次将从小羊胎体中来源的甲状旁腺鲜活细胞注射应用，这就是胎羊素细胞活化疗法的最初起源，它的发现对人类在抗衰老领域的医学发展有着里程碑式的意义，因此Paul Niehans被世人誉称为“活细胞之父”、“胎羊素细胞活化治疗的创始人”，罗马教皇为了表示对Dr.Paul Niehans的敬意，授封Paul Niehans为梵蒂冈科学院院士。In the field of cosmetics, the concept and biomedical effects of "Sheep Placenta" are controversial. The earliest record is that in 1931, Dr. Paul Niehans from Switzerland injected fresh parathyroid cells from lamb fetus for the first time. This is the original origin of fetal sheep cell activation therapy. The discovery of Niehans has a milestone significance for the medical development of human beings in the field of anti-aging. Therefore, Paul Niehans is known as the "father of living cells" and "the founder of fetal sheep cell activation therapy". The Pope of Rome expressed his gratitude to Dr. . In the respect of Paul Niehans, Paul Niehans was awarded as a member of the Vatican Academy of Sciences.
记载描述性的文献并未对羊胎素的实际成分进行描述，目前市面上的羊胎素产品，多以物理化学手段打碎裂解羊胎，再进一步获取其中的蛋白质等分子。一方面，其生物学效应有待进一步观察，一般认为比较有争议。另一方面，和保罗尼翰所述的羊胎疗法已经区别很大。因此，羊胎素的活性成分未明确，一直是困扰领域内的科学问题。The descriptive literature does not describe the actual composition of sheep placenta. At present, most of the sheep placenta products on the market use physical and chemical means to break and crack the fetus, and then further obtain the protein and other molecules in it. On the one hand, its biological effects need to be further observed, which is generally considered to be more controversial. On the other hand, it is very different from the sheep fetal therapy described by Paul Nihan. Therefore, the active ingredients of sheep placenta are not clear, which has always been a scientific problem in the field.
值得关注的是，现代生命科学证实：活细胞能分泌大量的外泌体(exosome)，外泌体是胞内体逆出芽形成多囊泡胞内体，多囊泡胞内体与细胞膜融合，向细胞外释放形成外泌体。几乎所有的细胞都可以分泌外泌体。这些外泌体的特点是：直径在30-150nm之间；密度在1.13-1.19g/mL之间；表达特异性的蛋白，携带了亲本细胞的重要信号分子，包括蛋白质、脂质和RNA等；保持着与亲代细胞相似的生活学活性。特别的是，一旦细胞死亡，外泌体的分泌量则立刻降低，说明外泌体是活细胞分泌的一种活性效应物。It is worth noting that modern life sciences have confirmed that: living cells can secrete a large number of exosomes (exosome), exosomes are endosomes that reverse budding to form multivesicular endosomes, and multivesicular endosomes fuse with cell membranes. Released outside the cell to form exosomes. Almost all cells can secrete exosomes. The characteristics of these exosomes are: the diameter is between 30-150nm; the density is between 1.13-1.19g/mL; they express specific proteins and carry important signal molecules of the parent cell, including proteins, lipids and RNA, etc. ; Maintain the life activity similar to the parental cells. In particular, once the cell dies, the amount of exosomes secreted immediately decreases, indicating that exosomes are an active effector secreted by living cells.
哺乳动物的脐带、胎盘等是外泌体的重要来源，从哺乳动物的脐带或胎盘中提取外泌体并用于皮肤调理的研究不少，但未见以动物胎体作为该类外泌体来源的研究。在少数以动物胎体作为原材料进行物质提取的研究中，其提取物多集中在小分子量的水溶性分子，如专利文献CN 103462864B公开了从动物胎体内脏如分离分子量小于4000D的水溶性分子用于化妆品和食品生产的用途。然而公开的是分子量小于4000D的水溶性分 子，并不是外泌体；专利文献CN 103462865 B公开了从从动物胎体皮肤及肌肉中提取分子量为5000D以下分子量的水提物和用途，然而公开的是分子量小于5000D的水溶性分子，并不是外泌体。Mammalian umbilical cord, placenta, etc. are important sources of exosomes. There have been many studies on extracting exosomes from mammalian umbilical cord or placenta and using them for skin conditioning, but animal fetuses have not been seen as the source of such exosomes. Research. In a small number of studies using animal carcass as a raw material for material extraction, the extracts are mostly concentrated in small molecular weight water-soluble molecules. For example, the patent document CN 103462864B discloses the separation of water-soluble molecules with a molecular weight of less than 4000D from the internal organs of animals. Used in cosmetics and food production. However, what is disclosed are water-soluble molecules with a molecular weight of less than 4000D, not exosomes; patent document CN 103462865 B discloses the extraction and use of water extracts with molecular weights below 5000D from animal carcass skin and muscle. It is a water-soluble molecule with a molecular weight of less than 5000D, not an exosome.
因而，没有现有技术公开或提示胎体内脏、体液提取的外泌体用于化妆品和食品用途。Therefore, there is no prior art that discloses or suggests that exosomes extracted from fetal organs and body fluids are used in cosmetics and food.
发明内容Summary of the invention
本发明是为解决上述技术问题进行的，目的在于提供胎体来源的外泌体在皮肤调节产品中的用途、该外泌体的制备方法以及以该外泌体作为活性成分的皮肤调节产品。The present invention was made to solve the above-mentioned technical problems, and aims to provide the use of exosomes derived from carcass in skin conditioning products, a method for preparing the exosomes, and skin conditioning products using the exosomes as active ingredients.
本发明的第一方面在于提供了胎体来源的外泌体在皮肤调节产品中的用途。该外泌体是提取自非人胎生动物胎体、新生体内脏器或生理性流体内的外泌体。The first aspect of the present invention is to provide the use of carcass-derived exosomes in skin conditioning products. The exosomes are exosomes extracted from the carcass of non-human viviparous animals, new internal organs or physiological fluids.
所述的胎体指非人胎生动物母体内的幼体和新出生动物，不包括胎儿附属物。优选羊、牛、鹿和骆驼，即胎羊、胎牛、胎鹿和胎骆驼；动物幼体优选是至少2月龄的胎羊、至少4月龄的胎牛、至少3月龄的胎鹿、至少6月龄的胎骆驼；新生体指自然分娩后24小时内的新生动物。The fetus refers to larvae and new-born animals in the mothers of non-human viviparous animals, excluding fetal appendages. Preferably sheep, cattle, deer and camels, namely fetal sheep, fetal cattle, fetal deer and fetal camels; animal larvae are preferably fetal sheep at least 2 months old, fetal cattle at least 4 months old, fetal deer at least 3 months old, Fetal camels that are at least 6 months old; newborns refer to newborn animals within 24 hours of natural delivery.
所述脏器包括肺、乳房、胃、胰、前列腺、膀胱、骨、卵巢及附件、子宫、皮肤、肾、窦、结肠、直肠、食道、血液、大脑和其覆盖物、脊髓和其覆盖物、肌肉、结缔组织、肾上腺、副甲状腺、甲状腺、睾丸、垂体、生殖器、肝脏、胆囊、眼、耳、鼻、喉、扁桃腺、口、淋巴结和淋巴系统及其它器官；优选是肝脏，骨髓。The organs include lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and accessories, uterus, skin, kidney, sinus, colon, rectum, esophagus, blood, brain and its covering, spinal cord and its covering , Muscle, connective tissue, adrenal gland, parathyroid, thyroid, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymphatic system and other organs; preferably liver, bone marrow.
所述生理性流体包括以下的流体：鼻咽、口腔、食道、胃、胰腺、肝、胸膜、心包、腹膜、肠、前列腺、精液、阴道分泌物、眼泪、唾液、粘液、胆汁、血液、淋巴、血浆、血清、滑液、脑脊液、宫腔及附件、尿，以及间隙、细胞内和细胞外的流体；优选是血液、血浆和血清。The physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph , Plasma, serum, synovial fluid, cerebrospinal fluid, uterine cavity and appendages, urine, and interstitial, intracellular and extracellular fluids; preferably blood, plasma and serum.
发明人意外发现上述外泌体具有促进细胞增殖、提高细胞存活能力、促进细胞ATP合成、提高线粒体活性、减轻炎症反应和细胞衰老以及增加一些局部靶标的表达，该提取物在增加细胞活力、抗衰老方面是有效的。即在应用方面，发明人发现外泌体具有美容化妆和皮肤病学用途，美容化妆和皮肤病学用途包括抗衰老、增强皮肤弹性、对抗痤疮、增强毛囊、抗脱发等。The inventors unexpectedly discovered that the above-mentioned exosomes can promote cell proliferation, improve cell survival, promote cell ATP synthesis, increase mitochondrial activity, reduce inflammation and cell senescence, and increase the expression of some local targets. It is effective in aging. That is, in terms of application, the inventor found that exosomes have cosmetic and dermatological uses. The cosmetic and dermatological uses include anti-aging, enhancing skin elasticity, fighting acne, strengthening hair follicles, and anti-hair loss.
因此，本发明所述的皮肤调节产品包括直接涂抹式护肤产品、口服式皮肤调理产品或皮肤病学用途产品。Therefore, the skin conditioning products of the present invention include direct application skin care products, oral skin conditioning products, or dermatological use products.
优选的，直接涂抹式护肤产品为抗衰老或增强皮肤弹性的护肤产品；口服式皮肤调理产品包括含有该外泌体的乳制品、基于脂肪制品、饮料制品或谷物制品；皮肤病学用途产品为对抗痤疮或增强毛囊、抗脱发的皮肤病学用途产品。Preferably, the direct application type skin care product is an anti-aging or skin elasticity skin care product; the oral skin conditioning product includes dairy products containing the exosomes, fat-based products, beverage products or cereal products; products for dermatological purposes are Dermatological products for fighting acne or strengthening hair follicles and anti-hair loss.
更优选的，抗衰老或增强皮肤弹性的护肤产品为促进细胞胶原蛋白表达、增加细胞肌原纤蛋白合成、维持干细胞干性以及刺激干细胞增殖的护肤产品中的一种或多种组合；对抗痤疮的皮肤病学用途产品为抑制角质形成细胞中炎症介质或基质蛋白酶的基因表达的产品、以及抑制角质形成细胞中VEGF合成和释放产品中的一种或两种组合；增强毛囊、抗脱发的皮肤病学用途产品为促进毛囊乳头成纤维细胞中ATP合成或增强线粒体代谢活性的产品、增加微毛囊的细胞存活力或减轻微毛囊膜损伤产品中的任意一种或两种。More preferably, the anti-aging or skin elasticity skin care products are one or more combinations of skin care products that promote cell collagen expression, increase cell myofibril protein synthesis, maintain stem cell stemness, and stimulate stem cell proliferation; fight acne The dermatological use products of keratinocytes are products that inhibit the gene expression of inflammatory mediators or matrix proteases in keratinocytes, and one or a combination of products that inhibit VEGF synthesis and release in keratinocytes; enhance hair follicles and anti-hair loss skin The products for disease use are any one or two of products that promote ATP synthesis in hair follicle papillary fibroblasts or enhance mitochondrial metabolic activity, increase cell viability of microhair follicles, or reduce microhair follicle membrane damage.
根据实施例，本发明胎体来源的外泌体意图通过刺激增殖、维持干细胞活性的特异性作用抗衰老、促愈合。因而，下文提出的实例显示外泌体针对人间充质干细胞细胞具有以下几种作用：增加ALDH阳性细胞比率、Ki67比率的表达。According to the examples, the exosomes derived from the fetus of the present invention are intended to resist aging and promote healing by stimulating proliferation and maintaining the activity of stem cells. Therefore, the examples presented below show that exosomes have the following effects on human mesenchymal stem cells: increase the ratio of ALDH-positive cells and the expression of Ki67 ratio.
此外，还意图通过针对痤疮丙酸杆菌的特异性作用抑制炎症。外泌体特异地锁定在病变发展和加重中发挥核心作用的痤疮炎性过程。因而，下文提出的实例显示外泌体针对角质形成细胞具有以下几种作用：In addition, it is also intended to suppress inflammation through a specific action against P. acnes. Exosomes specifically target the inflammatory process of acne that plays a central role in the development and aggravation of the disease. Therefore, the examples presented below show that exosomes have the following effects on keratinocytes:
抗炎作用：抑制炎症早期介质(IL-1α，IL-1β)和晚期介质(IL-8)；对角质形成细胞诱导MMP(MMP-2和MMP-9)的抑制作用；对VEGF释放的抑制作用。Anti-inflammatory effect: Inhibition of early mediators of inflammation (IL-1α, IL-1β) and late mediators (IL-8); inhibition of keratinocyte-induced MMP (MMP-2 and MMP-9); inhibition of VEGF release effect.
外泌体也对痤疮丙酸杆菌具有以下作用：抑制痤疮丙酸杆菌诱导IL-8；抑制痤疮丙酸杆菌诱导MMP-9。Exosomes also have the following effects on P. acnes: inhibit P. acnes from inducing IL-8; inhibit P. acnes from inducing MMP-9.
因此，将该胎体来源的外泌体局部或口服施用用于维持和/或增加V型胶原蛋白和/或肌原纤蛋白-1表达和/或用于增加毛囊成纤维细胞增殖和/或用于增加细胞存活力和/或ATP合成和/或线粒体活性和/或减轻细胞损伤和/或减轻细胞衰老，用于增强毛囊，进而减少皮肤附属器损失，如脱发。Therefore, topical or oral administration of the fetus-derived exosomes is used to maintain and/or increase the expression of type V collagen and/or myofibrin-1 and/or to increase the proliferation of hair follicle fibroblasts and/or It is used to increase cell viability and/or ATP synthesis and/or mitochondrial activity and/or reduce cell damage and/or reduce cell aging, and is used to strengthen hair follicles, thereby reducing the loss of skin appendages, such as hair loss.
在本发明的一些具体的实施方案中，本发明所述胎体来源的外泌体局部或口服施用用于：通过增加抗氧化酶NQO1(Entrez Gene:1728)和GSTT1(Entrez Gene:2952)的表达实现清除和排除毒素；通过增加天然免疫因子的皮肤抗微生物肽hBD3(Entrez Gene:55894)的表达实现对皮肤的保护；以及通过增加抗氧化蛋白GPX1(Entrez Gene:2876)、过氧化氢酶(catalase,Entrez Gene:847)、TRX1(Entrez Gene:7295)和PRDX1(Entrez Gene:5052)的表达来实现抑制活性氧的产生。因此，组合物可以用作预防由亚洲沙尘(PM2.5)引起的皮肤问题、保护皮肤以及预防和治疗皮肤老化的药物组合物或美容组合 物。In some specific embodiments of the present invention, the topical or oral administration of exosomes derived from the carcass of the present invention is used to increase the antioxidant enzymes NQO1 (Entrez Gene: 1728) and GSTT1 (Entrez Gene: 2952). Expression to eliminate and eliminate toxins; increase the expression of the natural immune factor skin antimicrobial peptide hBD3 (Entrez Gene: 55894) to achieve skin protection; and increase the antioxidant protein GPX1 (Entrez Gene: 2876) and catalase (catalase, Entrez Gene: 847), TRX1 (Entrez Gene: 7295) and PRDX1 (Entrez Gene: 5052) expression to inhibit the production of reactive oxygen species. Therefore, the composition can be used as a pharmaceutical composition or a cosmetic composition for preventing skin problems caused by Asian sand dust (PM2.5), protecting the skin, and preventing and treating skin aging.
本发明的第二方面在于，提供了胎体来源外泌体的制备方法，由包括如下步骤的方法制备得到：The second aspect of the present invention is to provide a method for preparing carcass-derived exosomes, which are prepared by a method including the following steps:
(1)脏器细胞分离和培养液收集(1) Organ cell separation and culture fluid collection
取无菌脏器，经磷酸盐缓冲液(PBS)反复冲洗后，剪成直径约1-2mm的组织块；经2型胶原酶和胰酶消化依次后，将上清液离心，取细胞沉淀放入培养瓶，使用含10％胎牛血清DMEM/F12培养基，5％CO2、37℃饱和湿度培养；去除非贴壁细胞，在贴壁细胞80％融合后更换无血清培养基培养48h；收集培养上清液；0.45μm滤膜过滤得到富含外泌体的培养液。Take the sterile organs, wash them repeatedly with phosphate buffered saline (PBS), and cut them into tissue pieces with a diameter of about 1-2mm; after digestion with type 2 collagenase and trypsin, the supernatant is centrifuged and the cell pellet is taken Put it into a culture flask, use DMEM/F12 medium containing 10% fetal bovine serum, 5% CO2, 37°C saturated humidity; remove non-adherent cells, replace the serum-free medium for 48h after the adherent cells are 80% fused; The culture supernatant was collected; 0.45μm filter membrane was filtered to obtain a culture solution rich in exosomes.
(2)生理性流体内预处理(2) Pretreatment in physiological fluid
对于血液等生理性流体，首先利用离心、过滤等方法去除生理性流体内的细胞成分。0.45μm滤膜过滤得到富含外泌体的流体。For physiological fluids such as blood, first, centrifugation, filtration, and other methods are used to remove cellular components in the physiological fluid. A 0.45μm filter membrane filters to obtain a fluid rich in exosomes.
(2)外泌体分离纯化包括：将培养液或流体在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。(2) The separation and purification of exosomes includes: centrifuging the culture solution or fluid at 4°C, 1000g for 10 minutes, and collecting the supernatant; collecting the supernatant at 4°C and centrifuging at 2000g for 20 minutes to collect the supernatant; and collecting the supernatant at 4°C Centrifuge at 10,000g for 30min to collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer. Filtration with 0.45μm filter membrane to obtain exosomes.
根据上述方法制备得到的外泌体，具有如下特征：直径约为30-150nm，具有脂质膜，内包裹蛋白质和mRNA、microRNA等遗传物质。经蛋白分析含有CD63、CD81、Alix等蛋白分子。The exosomes prepared according to the above method have the following characteristics: a diameter of about 30-150 nm, a lipid membrane, and genetic materials such as protein, mRNA, and microRNA, etc. are encapsulated in the exosomes. Protein analysis contains CD63, CD81, Alix and other protein molecules.
本发明的第三方面，提供了含有外泌体为活性成分的组合物。该组合物包含至少一种可药用的赋形剂，特别是美容和皮肤病学可接受的赋形剂。如该赋形剂可以是适于体外局部施加的赋形剂，也可以是能够供人体应用的赋形剂。The third aspect of the present invention provides a composition containing exosomes as an active ingredient. The composition contains at least one pharmaceutically acceptable excipient, especially cosmetically and dermatologically acceptable excipients. For example, the excipient may be an excipient suitable for topical application in vitro, or an excipient that can be applied to the human body.
根据第一变例，多种制剂适于局部施加，并且包括乳膏剂、凝胶剂、乳化剂、乳液剂、香膏剂、洗剂、油剂、水溶液剂或氢化-醇或乙醇酸溶液剂、粉剂、贴剂、喷雾剂或用于外部施加的任何其他产品。在下文实例中提出此类制剂。According to the first variant, a variety of formulations are suitable for topical application, and include creams, gels, emulsifiers, emulsions, ointments, lotions, oils, aqueous solutions or hydrogen-alcohol or glycolic acid solutions, Powder, patch, spray or any other product for external application. Such formulations are presented in the examples below.
根据第二变例，多种制剂适于口服施用，其中可以在膳食补充剂或膳食组合物中包含所述外泌体。According to a second variant, various formulations are suitable for oral administration, wherein the exosomes can be included in a dietary supplement or dietary composition.
在本发明背景下，可以以硬质或软质明胶或植物胶囊剂的形式提供膳食补充剂。所述膳食补充剂因而可以含有以重量计1％至100％的外泌体。In the context of the present invention, dietary supplements can be provided in the form of hard or soft gelatin or vegetable capsules. The dietary supplement may thus contain 1% to 100% exosomes by weight.
在不限制的情况下，也可以在膳食组合物如食物、饮料和营养药中并入本发明的外泌体，所述膳食组合物包括以下制品：Without limitation, the exosomes of the present invention can also be incorporated into dietary compositions such as foods, beverages and nutraceuticals, and the dietary compositions include the following products:
1)乳制品如乳酪、黄油、乳和其他乳状饮料，含有乳产品、冰淇淋和酸乳的混合物和糊状食品；1) Dairy products such as cheese, butter, milk and other milky beverages, mixtures and pastes containing milk products, ice cream and yogurt;
2)基于脂肪的制品如人造黄油、糊状食品、蛋黄酱、烹调用脂肪、油炸用油和维纳格雷酸辣沙司(vinaigrette)；2) Fat-based products such as margarine, pasty food, mayonnaise, cooking fat, frying oil and vinaigrette;
3)由谷物组成的基于谷物的制品，如面包和面食制品，无论这些食品是否经过烹煮、焙烘或加工；3) Cereal-based products composed of grains, such as bread and pasta products, regardless of whether these foods have been cooked, baked or processed;
4)糖果如巧克力、糖食、口香糖、甜品、顶端配料(topping)、清凉果汁饮料(sorbet)、蛋糕糖衣(icing)和其他饰菜(garnishe)；4) Candies such as chocolate, confectionery, chewing gum, desserts, toppings, refreshing fruit drinks (sorbet), cake icing and other garnishes;
5)酒精或无酒精饮料，包括苏打水和其他软饮料、果汁、膳食补充物、饮料形式的正餐代用品如在品牌名称Boost TM和Ensure TM下销售的那些正餐代用品，以及； 5) Alcoholic or non-alcoholic beverages, including soda and other soft drinks, juices, dietary supplements, meal substitutes in the form of beverages such as those sold under the brand names Boost TM and Ensure TM, and;
6)多种制品如蛋类、加工食品如汤、即用型面食制品调味汁、准备好的菜肴和相同类型的其他制品。6) Various products such as eggs, processed foods such as soups, ready-to-use pasta products, sauces, prepared dishes and other products of the same type.
可以借助技术如混合、灌注、注射、掺混、吸附、揉捏和喷洒直接且无其他修饰下将外泌体并入食物中、营养药中、膳食产品，尤其高蛋白产品中或饮料中。The exosomes can be incorporated into food, nutraceuticals, dietary products, especially high-protein products or beverages directly with the help of techniques such as mixing, infusion, injection, blending, adsorption, kneading and spraying without other modifications.
可以根据建立适于患者的药物疗法、尤其皮肤病学疗法时通常所考虑的标准例如，该患者的年龄或体重、该患者整体状态的严重性、对所述疗法的耐受性、明显的副作用和皮肤类型，确定本发明化合物和组合物的施用模式及、给药方案和最佳植物制剂形式。It can be based on the criteria commonly considered when establishing drug therapy, especially dermatological therapy, suitable for the patient, for example, the age or weight of the patient, the severity of the patient's overall condition, the tolerance to the therapy, and obvious side effects. And skin type, determine the application mode and dosage regimen of the compound and composition of the present invention, and the optimal botanical preparation form.
本发明的第四方面还涉及美容治疗痤疮的方法，其特征在于将本发明的组合物施加至患病的皮肤区域或者患病的个体口服服用本发明的营养药组合物。The fourth aspect of the present invention also relates to a method for the cosmetic treatment of acne, which is characterized in that the composition of the present invention is applied to a diseased skin area or a diseased individual orally takes the nutraceutical composition of the present invention.
因此，本发明还可涉及将外泌体用于制备治疗或预防痤疮的化妆品、营养药或皮肤病学组合物的用途。Therefore, the present invention may also relate to the use of exosomes for the preparation of cosmetics, nutraceuticals or dermatological compositions for the treatment or prevention of acne.
在美容或皮肤病学用途的背景下，将会优选地以适于局部施加的制剂形式配制所述组合物。In the context of cosmetic or dermatological use, the composition will preferably be formulated in a formulation suitable for topical application.
在用于食物中的背景下，针对营养或美容目的(美容食物)，将会优选地以适于口服施用的制剂形式配制所述组合物。In the context of use in food, for nutritional or cosmetic purposes (cosmetic food), the composition will preferably be formulated in a formulation suitable for oral administration.
本发明的美容化妆用护理方法包括将本发明的外泌体或包含其的组合物局部应用至选自如下的身体的全部或部分：腿，足，腋，手，大腿，腹部，颈线，颈，胳膊，躯干，背部，面部，皮肤附属器，优选毛发，和/或头皮，更有利地为头皮。The cosmetic care method of the present invention includes topical application of the exosomes of the present invention or a composition containing the same to all or part of the body selected from the group consisting of legs, feet, armpits, hands, thighs, abdomen, neckline, The neck, arms, trunk, back, face, skin appendages, preferably hair, and/or scalp, more advantageously the scalp.
此外，本发明的组合物还可以包含本领域技术人员已知的至少一种药用佐剂，如增稠剂、防腐剂、香料、色料、化学或矿物遮光物、润湿剂、温泉水等。In addition, the composition of the present invention may also contain at least one pharmaceutical adjuvant known to those skilled in the art, such as thickeners, preservatives, fragrances, colorants, chemical or mineral sunscreens, wetting agents, and hot spring water. Wait.
本发明的组合物还可以包含选自皮脂调节剂、抗细菌剂、抗真菌剂、角质层分离剂、角质层调节剂、收敛剂、抗炎/抗刺激剂、抗氧化/自由基清除剂、结瘢剂、抗老化剂和/或润湿剂的至少一种抗衰老化合物。The composition of the present invention may also comprise a sebum regulator, antibacterial agent, antifungal agent, keratolytic agent, stratum corneum regulator, astringent, anti-inflammatory/anti-irritant, antioxidant/radical scavenger, At least one anti-aging compound of scarring agent, anti-aging agent and/or wetting agent.
术语“皮脂调节剂”例如指5-α-还原酶抑制剂，如活性物质
Figure PCTCN2020130931-appb-000001
锌及其葡萄糖酸盐、水杨酸盐和焦谷氨酸也具有皮脂抑制活性。也可以提到在12周应用后显著降低皮脂分泌速率的螺内酯，一种抗雄激素和醛固酮拮抗剂。其他的提取分子(例如来自西葫芦(Cucurbita pepo)的种子、南瓜种子油以及扇叶菜棕(palm cabbage))通过抑制5-α-还原酶转录和活性来限制皮脂产生。影响皮脂性质的其他脂源皮脂调节剂如亚油酸是也可以使用。 The term "sebum regulator" refers, for example, to 5-α-reductase inhibitors, such as active substances
Figure PCTCN2020130931-appb-000001
Zinc and its gluconate, salicylate and pyroglutamic acid also have sebum inhibitory activity. Mention can also be made of spironolactone, an antiandrogen and aldosterone antagonist, which significantly reduces the rate of sebum secretion after 12 weeks of application. Other extracted molecules (such as seeds from Cucurbita pepo, pumpkin seed oil, and palm cabbage) limit sebum production by inhibiting 5-α-reductase transcription and activity. Other lipid-derived sebum regulators that affect the properties of sebum, such as linoleic acid, can also be used.
术语“抗细菌剂”和“抗真菌剂”指限制致病微生物如某些细菌如痤疮丙酸杆菌或某些真菌(糠秕马拉色菌(Malassezia furfur)生长或摧毁这些致病微生物的分子。最传统的是化妆品或营养药中通常使用的防腐剂，即具有抗细菌活性的分子(假防腐剂)如辛酸衍生物(辛酰甘氨酸、辛酸甘油酯等)，如己二醇和乙酰丙酸钠、锌和铜衍生物(葡糖酸盐和PCA)、植物鞘氨醇及其衍生物、过氧苯甲酰、吡罗克酮乙醇胺、巯氧吡啶锌、二硫化硒、益康唑、酮康唑、或局部用抗生素如红霉素和克林霉素等。The terms "antibacterial agent" and "antifungal agent" refer to molecules that restrict the growth of or destroy pathogenic microorganisms such as certain bacteria such as Propionibacterium acnes or certain fungi (Malassezia furfur). The most traditional are the preservatives commonly used in cosmetics or nutraceuticals, that is, molecules with antibacterial activity (pseudopreservatives) such as caprylic acid derivatives (caprylylglycine, caprylic glyceride, etc.), such as hexylene glycol and sodium levulinate , Zinc and copper derivatives (gluconate and PCA), phytosphingosine and its derivatives, benzoyl peroxide, piroctone ethanolamine, zinc pyrithione, selenium disulfide, econazole, ketone Conazole, or topical antibiotics such as erythromycin and clindamycin.
术语“角质层调节剂”和“角质层分离剂”指调节或辅助表皮角质层死细胞消除的物质。常使用的角质层调节剂/角质层分离剂包括：最常使用的角质层调节剂/角质层分离剂包括：果实的α-羟酸(AHAS)(柠檬酸、羟乙酸、苹果酸、乳酸等)、AHA酯、AHAS与其他分子的组合如苹果酸和杏仁蛋白的组合
Figure PCTCN2020130931-appb-000002
羟乙酸或乳酸与精氨酸的组合或羟酸与脂质分子如
Figure PCTCN2020130931-appb-000003
(脂-羟酸)的组合、两性羟酸复合物(AHCare)、柳树皮(白柳树皮提取物)、壬二酸和其盐及酯、水杨酸及其衍生物如辛酰基水杨酸或与其他分子的组合如水杨酸和多糖(β-羟酸或BHA)的组合、他扎罗汀、阿达帕林、以及类视黄醇的分子如维甲酸、视黄醛、异维甲酸和视黄醇。 The terms "stratum corneum regulator" and "keratolytic agent" refer to substances that regulate or assist the elimination of dead cells in the epidermal stratum corneum. Commonly used stratum corneum regulators/stratum corneum separation agents include: The most commonly used stratum corneum regulators/stratum corneum separation agents include: fruit alpha-hydroxy acid (AHAS) (citric acid, glycolic acid, malic acid, lactic acid, etc.) ), the combination of AHA ester, AHAS and other molecules such as the combination of malic acid and marzipan
Figure PCTCN2020130931-appb-000002
The combination of glycolic acid or lactic acid and arginine or hydroxy acid and lipid molecules such as
Figure PCTCN2020130931-appb-000003
(Lipo-hydroxy acid) combination, amphoteric hydroxy acid complex (AHCare), willow bark (white willow bark extract), azelaic acid and its salts and esters, salicylic acid and its derivatives such as caprylyl salicylic acid Or in combination with other molecules such as salicylic acid and polysaccharides (β-hydroxy acid or BHA), tazarotene, adapalene, and retinoid molecules such as tretinoin, retinal, isotretinoin and Retinol.
术语“收敛剂”指帮助收窄毛孔的物质，最常使用的是多酚、锌衍生物和北美金缕梅。The term "astringent" refers to substances that help narrow pores. The most commonly used are polyphenols, zinc derivatives, and North American witch hazel.
术语“抗炎性/抗刺激剂”指限制由细胞因子或花生四烯酸代谢介质引起的炎症反应并且具有顺滑和抗刺激性质的物质。最常规的是甘草次酸(甘草衍生物)及其盐和酯、α-没药醇、银杏(Ginkgo biloba)、金盏花(Calendula)、硫辛酸、β-胡萝卜素、维生素B3(烟酰胺尼古酰胺)、维生素E、维生素C、维生素B12、类黄酮(绿茶、槲皮素等)、番茄红 素或叶黄素、鳄梨糖、鳄梨油精华素(avocado oleodistillate)、阿拉伯半乳聚糖、羽扇豆肽、羽扇豆总提取物、昆诺阿藜肽提取物、循环神经酰胺
Figure PCTCN2020130931-appb-000004
(噁唑啉衍生物)、抗糖化剂如肌肽、N-乙酰半胱氨酸、异黄酮例如染料木黄酮/染料木苷、黄豆苷原/黄豆苷、泉水或温泉水(雅漾(eau d′Avène)、理肤泉(eau de la Roche Posay)、圣泉薇(eau de SaintGervais)、依泉(eau d′Uriage)、珂玛德(eau de Gamarde)、枸杞(Lycium barbarum)提取物、植物氨基酸肽或复合物、局部用氨苯砜、或抗炎药。 The term "anti-inflammatory/anti-irritant" refers to a substance that limits the inflammatory response caused by cytokines or arachidonic acid metabolism mediators and has smooth and anti-irritant properties. The most common are glycyrrhetinic acid (glycyrrhizin derivatives) and its salts and esters, α-bisabolol, Ginkgo biloba, Calendula, lipoic acid, β-carotene, vitamin B3 (niacinamide) Nicolamide), vitamin E, vitamin C, vitamin B12, flavonoids (green tea, quercetin, etc.), lycopene or lutein, avocado sugar, avocado oleodistillate, arabic gal Glycan, lupin peptide, total lupin extract, quinoa peptide extract, circulating ceramide
Figure PCTCN2020130931-appb-000004
(Oxazoline derivatives), anti-glycation agents such as carnosine, N-acetylcysteine, isoflavones such as genistein/genistin, daidzein/daidzin, spring water or hot spring water (Eau d' Avène, eau de la Roche Posay, eau de Saint Gervais, eau d'Uriage, eau de Gamarde, Lycium barbarum extract, plant amino acid peptide Or complexes, topical dapsone, or anti-inflammatory drugs.
术语“抗氧化剂”指减少或防止其他化学物质氧化的分子。可以组合使用的抗氧化/自由基清除剂优选地选自由以下物质组成的组：硫醇和酚、甘草衍生物如甘草次酸和其盐及酯、α-没药醇、银杏提取物、金盏花(Calendula)提取物、循环神经酰胺
Figure PCTCN2020130931-appb-000005
(噁唑啉衍生物)、鳄梨肽、微量元素如铜、锌和硒、硫辛酸、维生素B12、维生素B3(烟酰胺、尼古酰胺)、维生素C、维生素E、辅酶Q10、磷虾油(krill)、谷胱甘肽、丁羟甲苯(BHT)、丁羟茴醚(BHA)、番茄红素或叶黄素、β-胡萝卜素、多酚家族如单宁、酚酸、花青素、类黄酮，例如绿茶、红浆果、可可、葡萄、粉色西番莲(Passiflora incarnata)或柑橘(Citrus)的提取物，或异黄酮，例如染料木黄酮/染料木苷和黄豆苷元/黄豆苷。抗氧化剂类群还包括抗糖化剂如肌肽或某些肽、N-乙酰半胱氨酸以及抗氧化或自由基清除酶，如超氧化物歧化酶(SOD)、过氧化氢酶、谷胱甘肽过氧化物酶、硫氧还蛋白还原酶及其拮抗剂。 The term "antioxidant" refers to molecules that reduce or prevent the oxidation of other chemicals. Antioxidant/radical scavengers that can be used in combination are preferably selected from the group consisting of mercaptans and phenols, licorice derivatives such as glycyrrhetinic acid and its salts and esters, α-bisabolol, ginkgo extract, marigold Flower (Calendula) extract, circulating ceramide
Figure PCTCN2020130931-appb-000005
(Oxazoline derivatives), avocado peptides, trace elements such as copper, zinc and selenium, lipoic acid, vitamin B12, vitamin B3 (niacinamide, nicotiamide), vitamin C, vitamin E, coenzyme Q10, krill oil (krill), glutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), lycopene or lutein, β-carotene, polyphenol family such as tannins, phenolic acids, anthocyanins , Flavonoids, such as extracts of green tea, red berries, cocoa, grapes, pink passionflower (Passiflora incarnata) or citrus (Citrus), or isoflavones, such as genistein/genistin and daidzein/daidzein . The antioxidant group also includes anti-glycation agents such as carnosine or certain peptides, N-acetylcysteine, and antioxidant or free radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, glutathione Peroxidase, thioredoxin reductase and its antagonists.
可以组合使用的生成瘢痕/修复屏障功能的物质优选地是维生素A、泛醇(维生素B5)、鳄梨呋鳄梨糖喃
Figure PCTCN2020130931-appb-000006
羽扇醇、玛卡肽提取物、昆诺阿藜肽提取物、阿拉伯半乳聚糖、氧化锌、镁、硅、羟基积雪草酸或积雪草酸、硫酸葡聚糖、辅酶Q10、葡糖胺及其衍生物、硫酸软骨素和总体糖胺聚糖(GAGs)、硫酸葡聚糖、神经酰胺、胆固醇、角鲨烷、磷脂、发酵或未发酵过的大豆肽、植物肽、海洋多糖、植物多糖或生物技术多糖，如藻类提取物或蕨类提取物、微量元素、富含单宁的植物的提取物如从没食子酸衍生的单宁，其称作没食性单宁或可水解单宁，最初在栎树瘿中找到，和从黄烷单元聚合产生的儿茶素单宁，其模型由儿茶(Acacia catechu)提供。可以使用的微量元素优选地选自由铜、镁、锰、铬、硒、硅、锌及其混合物组成。 The substance that can be used in combination to generate scars/repair barrier function is preferably vitamin A, panthenol (vitamin B5), avocado furan
Figure PCTCN2020130931-appb-000006
Lupin, Macatide Extract, Quinoa Peptide Extract, Arabinogalactan, Zinc Oxide, Magnesium, Silicon, Asiatic Acid or Asiatic Acid, Dextran Sulfate, Coenzyme Q10, Glucosamine And its derivatives, chondroitin sulfate and total glycosaminoglycans (GAGs), dextran sulfate, ceramide, cholesterol, squalane, phospholipids, fermented or unfermented soybean peptides, plant peptides, marine polysaccharides, plants Polysaccharides or biotechnological polysaccharides, such as algae extracts or fern extracts, trace elements, tannin-rich plant extracts such as tannins derived from gallic acid, which are called gallic tannins or hydrolyzable tannins, It was originally found in oak gall, and catechin tannins produced from the polymerization of flavan units, the model of which was provided by Acacia catechu. The trace elements that can be used are preferably selected from copper, magnesium, manganese, chromium, selenium, silicon, zinc and mixtures thereof.
可以组合使用以治疗成熟受试者中痤疮的抗老化剂是抗氧化剂，尤其是维生素C、维生素A、视黄醇、视黄醛、任何分子量的透明质酸、鳄梨呋喃
Figure PCTCN2020130931-appb-000007
羽扇豆肽和玛卡肽提取物。 Anti-aging agents that can be used in combination to treat acne in mature subjects are antioxidants, especially vitamin C, vitamin A, retinol, retinal, hyaluronic acid of any molecular weight, avocado furan
Figure PCTCN2020130931-appb-000007
Lupin peptide and maca peptide extract.
最常使用的润湿剂/润肤剂是甘油或其衍生物、脲、吡咯烷酮羧酸及其衍生物、任何 分子量的透明质酸、糖胺聚糖和海洋源、植物源或生物技术源的任何其他多糖，例如黄原胶、
Figure PCTCN2020130931-appb-000008
某些脂肪酸如月桂酸、肉豆蔻酸、单不饱和以及多不饱和ω-3、-6、-7和-9脂肪酸(亚油酸、棕榈油酸等)、向日葵油精华素(sunflower oleodistillate)、鳄梨肽和古朴阿苏果油(cupuacu butter)。 The most commonly used humectants/emollients are glycerin or its derivatives, urea, pyrrolidone carboxylic acid and its derivatives, hyaluronic acid of any molecular weight, glycosaminoglycans and those of marine, plant or biotechnological origin Any other polysaccharides, such as xanthan gum,
Figure PCTCN2020130931-appb-000008
Certain fatty acids such as lauric acid, myristic acid, monounsaturated and polyunsaturated omega-3, -6, -7 and -9 fatty acids (linoleic acid, palmitoleic acid, etc.), sunflower oleodistillate , Avocado peptides and cupuacu butter.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及植物和动物不可皂化物，例如，鳄梨和大豆不可皂化物和不可皂化的植物油或动物油浓缩物，例如，向日葵油或棕榈油浓缩物，或含有不可皂化物的植物油，例如，大豆油和菜籽油，以及不可皂化物的衍生物如鳄梨呋喃、甾醇酯和维生素衍生物。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and plant and animal unsaponifiables, for example, avocado and soybean unsaponifiables and unsaponifiable vegetable oils or animal oil concentrates For example, sunflower oil or palm oil concentrate, or vegetable oils containing unsaponifiables, such as soybean oil and rapeseed oil, and derivatives of unsaponifiables such as avocado furan, sterol esters, and vitamin derivatives.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及鳄梨糖(见申请WO2005/115421)。所述组合物特别适于治疗皮肤屏障修复和炎症。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and avocado sugar (see application WO2005/115421). The composition is particularly suitable for the treatment of skin barrier repair and inflammation.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体和鳄梨肽(见国际申请WO2005/105123)。所述组合物特别适于治疗刺激和炎症。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and avocado peptides (see International Application WO2005/105123). The composition is particularly suitable for treating irritation and inflammation.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及鳄梨油(见国际申请WO2004/012496、WO2004/012752、WO2004/016106、WO2007/057439)。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and avocado oil (see international applications WO2004/012496, WO2004/012752, WO2004/016106, WO2007/057439).
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及鳄梨呋喃
Figure PCTCN2020130931-appb-000009
(鳄梨呋喃，其可以通过国际申请WO01/21605中所述的方法获得)。所述组合物特别适于治疗炎症、促进瘢痕形成和适于其抗老化性质。 In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and avocado furan
Figure PCTCN2020130931-appb-000009
(Avocado furan, which can be obtained by the method described in the international application WO01/21605). The composition is particularly suitable for treating inflammation, promoting scar formation and suitable for its anti-aging properties.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及鳄梨和大豆不可皂化物。可以组合使用的鳄梨和大豆不可皂化物优选地分别是以大约1:3-2:3比例的鳄梨呋喃性不可皂化物和大豆不可皂化物的混合物。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and avocado and soy unsaponifiables. The avocado and soybean unsaponifiables that can be used in combination are preferably a mixture of avocado furanic unsaponifiables and soybean unsaponifiables in a ratio of about 1:3-2:3, respectively.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及羽扇醇(FR2822821，FR2857596)。所述组合物特别适于支持瘢痕形成。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and lupeol (FR2822821, FR2857596). The composition is particularly suitable for supporting scar formation.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及羽扇豆肽，如根据申请WO2005/102259中所述的方法获得的羽扇豆肽。所述组合物特别适于治疗炎症，并且因其抗老化性质而使用。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and lupin peptides, such as lupin peptides obtained according to the method described in application WO2005/102259. The composition is particularly suitable for treating inflammation and is used because of its anti-aging properties.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及总羽扇豆提取物(见国际申请WO2005/102259)。所述组合物特别适于治疗刺激。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and total lupin extract (see International Application WO2005/102259). The composition is particularly suitable for treating irritation.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及羽扇豆油，优选地是甜味白色羽扇豆油，如国际申请WO98/47479中所述的甜味白色 羽扇豆油。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and lupin oil, preferably sweet white lupin oil, as described in the international application WO98/47479 White lupin oil.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及玛卡肽提取物(见国际申请WO2004/112742)。所述组合物因其结瘢性质和抗老化性质而特别优选。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and a maca peptide extract (see International Application WO2004/112742). The composition is particularly preferred due to its scarring properties and anti-aging properties.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及稻肽(见国际申请2008/009709)。所述组合物因其与刺激黑素形成和与黑色素转移相关的性质而特别优选。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and rice peptides (see International Application 2008/009709). The composition is particularly preferred due to its properties related to the stimulation of melanin formation and the transfer of melanin.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及循环神经酰胺
Figure PCTCN2020130931-appb-000010
(噁唑啉衍生物)，如国际申请WO2004/050052、WO2004/050079和WO2004/112741中所述。所述组合物特别适于治疗炎症反应。 In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and circulating ceramide
Figure PCTCN2020130931-appb-000010
(Oxazoline derivatives), as described in international applications WO2004/050052, WO2004/050079 and WO2004/112741. The composition is particularly suitable for the treatment of inflammatory reactions.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及昆诺阿藜提取物，尤其肽提取物(见国际申请WO2008/080974)。所述组合物特别适于治疗炎性疾病和皮肤屏障修复。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and quinoa extract, especially peptide extract (see International Application WO2008/080974). The composition is particularly suitable for the treatment of inflammatory diseases and skin barrier repair.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及古朴阿苏果油(cupuacu butter)。所述组合物因其润湿性质而特别优选。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and cupuacu butter. The composition is particularly preferred because of its wetting properties.
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及油菜籽油精华素(rapeseed oleodistillate)。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and rapeseed oil essence (rapeseed oleodistillate).
在本发明的一些优选的实施例中，本发明组合包括组合物，该组合物包含外泌体以及玉米油精华素(corn oleodistillate)。In some preferred embodiments of the present invention, the combination of the present invention includes a composition comprising exosomes and corn oleodistillate.
除外泌体之外，全部这些组合包含至少一种其他活性化合物，并且可以包含如上所述的两种、三种、四种或更多种活性化合物。Except for exosomes, all of these combinations contain at least one other active compound, and may contain two, three, four or more active compounds as described above.
除这些活性物质之外，单独或与上文提及的活性物质组合，本发明的外泌体可以与本领域技术人员已知的防晒活性物质如UVB和/或UVA滤光物或遮光物或任何无机和/或有机遮光物或滤光物组合使用，所述技术人员将根据所寻求的保护程度调整它们的选择和其浓度。In addition to these active substances, alone or in combination with the above-mentioned active substances, the exosomes of the present invention can be combined with sunscreen active substances known to those skilled in the art such as UVB and/or UVA filters or shades or Any inorganic and/or organic light-shielding material or filter is used in combination, and the skilled person will adjust their selection and concentration according to the degree of protection sought.
作为防晒活性物质的实例，可以具体提到二氧化钛、氧化锌、亚甲基双-苯并三唑基四甲基丁基酚(品牌名称：TINOSORB M)和双-乙基己氧苯酚甲氧苯基三嗪(品牌名称：TINOSORB S)、辛-水杨酸、丁基甲氧基二苯甲酰基甲烷、对苯二亚甲基二樟脑磺酸、4-甲基亚苄基樟脑、二苯酮、甲氧基肉桂酸乙基己酯、二甲基PABA乙基己酯和二乙基己基丁酰胺基三嗪酮。As examples of sunscreen active substances, titanium dioxide, zinc oxide, methylene bis-benzotriazole tetramethyl butyl phenol (brand name: TIINOORB M) and bis-ethylhexyloxyphenol methoxybenzene can be specifically mentioned. Triazine (brand name: TINOSORB S), octyl-salicylic acid, butylmethoxydibenzoylmethane, terephthalmethylene dicamphorsulfonic acid, 4-methylbenzylidene camphor, benzophenone, Ethylhexyl methoxycinnamate, ethylhexyl dimethyl PABA and diethylhexyl butamido triazone.
在本发明的背景下术语“衰老”以及术语“皮肤衰老”用于描述皮肤外观上出现的可见变化以及那些通过触摸可察觉的可见变化，例如像，在非限制意义上的皱纹、细纹、粗糙、表情纹、妊娠纹、不连续性、深皱纹、松弛、皮肤下垂(如脸颊下垂、眼袋、双下巴)、毛孔大小的增加、失去弹性、失去回弹性、失去紧致性、弹性组织变性、异常分化、角化过度、角化病、皮肤颜色变化(如斑点、发红或眼袋)、色素沉着过度的区域的形成(如老年斑、黄褐斑、或雀斑)、失去柔滑性、橘皮样皮肤、失去胶原蛋白结构以及角质层、真皮、表皮、血管系统(例如蜘蛛静脉或毛细血管扩张的形成)、或靠近皮肤的这些组织的其他组织学变化。皮肤衰老是一个具有两个主要部分的过程，这两个主要部分为：时序衰老，这归因于时间的流逝；和光诱导的衰老，这归因于暴露于紫外线辐射的水平并且被称作光老化。若干环境因素如暴露于烟草烟雾、暴露于污染、以及气候条件(如寒冷和/或风)的总和也促成了皮肤衰老。在本发明的背景中，“皮肤抗衰老治疗”为一种用于防止、延迟、和/或减轻人类皮肤衰老的治疗。In the context of the present invention, the term "aging" and the term "skin aging" are used to describe visible changes in the appearance of the skin and those visible changes that can be detected by touch, such as wrinkles, fine lines, and wrinkles in a non-limiting sense. Roughness, expression lines, stretch marks, discontinuities, deep wrinkles, sagging, sagging skin (such as sagging cheeks, eye bags, double chin), increase in pore size, loss of elasticity, loss of resilience, loss of firmness, elastic tissue degeneration , Abnormal differentiation, hyperkeratosis, keratosis, skin color changes (such as spots, redness, or bags under the eyes), formation of hyperpigmented areas (such as age spots, melasma, or freckles), loss of smoothness, orange peel Skin-like skin, loss of collagen structure and other histological changes in the stratum corneum, dermis, epidermis, vascular system (such as the formation of spider veins or telangiectasia), or these tissues near the skin. Skin aging is a process with two main parts. The two main parts are: temporal aging, which is attributed to the passage of time; and light-induced aging, which is attributed to the level of exposure to ultraviolet radiation and is called light Ageing. The sum of several environmental factors such as exposure to tobacco smoke, exposure to pollution, and climatic conditions (such as cold and/or wind) also contribute to skin aging. In the context of the present invention, "skin anti-aging treatment" is a treatment for preventing, delaying, and/or reducing the aging of human skin.
相比现有技术，本发明的技术效果如下：Compared with the prior art, the technical effects of the present invention are as follows:
本发明提供了胎体来源的外泌体在皮肤调节产品中的用途，通过实验证实，该外泌体能够通过促进细胞胶原蛋白表达、增加细胞肌原纤蛋白合成、维持干细胞干性以及刺激干细胞增殖实现抗衰老功能；能够通过抑制角质形成细胞中炎症介质或基质蛋白酶的基因表达的产品、以及抑制角质形成细胞中VEGF合成和释放实现对抗痤疮的药学功能；能够通过促进毛囊乳头成纤维细胞中ATP合成或增强线粒体代谢活性的产品、增加微毛囊的细胞存活力或减轻微毛囊膜损伤，实现增强毛囊、抗脱发的皮肤病学用途；能够通过增加抗氧化酶NQO1和GSTT1的表达实现清除和排除毒素；通过增加天然免疫因子的皮肤抗微生物肽hBD3的表达实现对皮肤的保护；以及通过增加抗氧化蛋白GPX1、过氧化氢酶、TRX1和PRDX1的表达来实现抑制活性氧的产生，进而可以用作预防由亚洲沙尘(PM2.5)引起的皮肤问题、保护皮肤以及预防和治疗皮肤老化的药物组合物或美容组合物。因此，本发明为抗衰老皮肤护理、痤疮对抗、防脱发、防亚洲沙尘影响等皮肤护理提供了一种新的途径。The present invention provides the use of carcass-derived exosomes in skin conditioning products. Experiments have confirmed that the exosomes can promote cell collagen expression, increase cell myofibrillar protein synthesis, maintain stem cell stemness, and stimulate stem cells. Proliferation to achieve anti-aging function; products that can inhibit inflammatory mediators or matrix protease gene expression in keratinocytes, and inhibit the synthesis and release of VEGF in keratinocytes to achieve pharmaceutical functions against acne; can promote hair follicle papillary fibroblasts ATP synthesis or products that enhance the metabolic activity of mitochondria, increase the cell viability of the microhair follicles or reduce the damage of the microhair follicle membrane, realize the dermatological use of enhancing hair follicles and anti-hair loss; it can achieve clearance and elimination by increasing the expression of antioxidant enzymes NQO1 and GSTT1 Eliminate toxins; increase the expression of the skin antimicrobial peptide hBD3, which is a natural immune factor, to protect the skin; and increase the expression of antioxidant proteins GPX1, catalase, TRX1 and PRDX1 to inhibit the production of reactive oxygen species. It is used as a pharmaceutical or cosmetic composition to prevent skin problems caused by Asian sand dust (PM2.5), protect the skin, and prevent and treat skin aging. Therefore, the present invention provides a new approach for skin care such as anti-aging skin care, acne fighting, anti-hair loss, and anti-Asian dust effects.
附图说明Description of the drawings
图1为本发明胎体来源外泌体的电镜检测结果；Figure 1 is the electron microscope detection result of the exosomes derived from the carcass of the present invention;
图2为NTA检测的外泌体粒径分布结果。Figure 2 shows the results of the particle size distribution of exosomes detected by NTA.
具体实施方式Detailed ways
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制，同时实施例不包括对传统方法的详细描述。在下述实施例中，多组间的比较统计学以方差分析及组间检验计算P值，仅有两组时以非参数T检验计算P值，多个率的比较以卡方检验计算P指。The following examples and experimental examples further illustrate the present invention, and should not be construed as limiting the present invention. At the same time, the examples do not include detailed descriptions of traditional methods. In the following examples, the comparison statistics between multiple groups uses analysis of variance and between-group tests to calculate the P value. When there are only two groups, the non-parametric T test is used to calculate the P value. The comparison of multiple rates uses the chi-square test to calculate the P value. .
实施例1、胎羊血清外泌体的制备Example 1. Preparation of Fetal Sheep Serum Exosomes
将4月龄高山黑绵羊胎羊血清用0.45μm滤膜过滤，在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。用Bradford法检测外泌体总蛋白量(Bio-Rad Protein Assay Reagent)。外泌体冻干后储存于-80℃。成年羊血清用同样的方法提取外泌体，作为对照外泌体。The 4-month-old Alpine black sheep fetal serum was filtered with a 0.45μm filter, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant Centrifuge the supernatant at 10,000g for 30min at 4℃, and collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet in phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend in a small amount of phosphate buffer. Precipitate, filter with 0.45μm filter membrane to obtain exosomes. The Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80°C. Adult goat serum was used to extract exosomes in the same way as control exosomes.
实施例2、胎骆驼血清外泌体的制备Example 2. Preparation of Fetal Camel Serum Exosomes
将11月龄澳洲羊驼血清用0.45μm滤膜过滤，在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。用Bradford法检测外泌体总蛋白量(Bio-Rad Protein Assay Reagent)。外泌体冻干后储存于-80℃。成年羊驼血清用同样的方法提取外泌体，作为对照外泌体。The 11-month-old Australian alpaca serum was filtered with a 0.45μm filter, centrifuged at 4°C, 1000g for 10min, and collected the supernatant; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant was 4°C Centrifuge at 10,000g for 30min to collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer. Filtration with 0.45μm filter membrane to obtain exosomes. The Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80°C. Adult alpaca serum was used to extract exosomes in the same way as control exosomes.
实施例3、胎牛血清外泌体的制备Example 3. Preparation of Fetal Bovine Serum Exosomes
将8月龄澳洲胎牛血清用0.22μm无菌膜过滤，在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤 膜过滤，得到外泌体。用Bradford法检测外泌体总蛋白量(Bio-Rad Protein Assay Reagent)。外泌体冻干后储存于-80℃。The 8-month-old Australian fetal bovine serum was filtered with a 0.22μm sterile membrane, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant 4 Centrifuge the supernatant at 10,000g for 30min at ℃, and collect the supernatant; centrifuge the collected supernatant at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer , 0.45μm filter membrane to obtain exosomes. The Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80°C.
实施例4、胎羊肝脏外泌体的制备Example 4. Preparation of Fetal Sheep Liver Exosomes
将4月龄高山黑绵羊胎羊肝脏经磷酸盐缓冲液(PBS)反复冲洗后，剪成直径约1-2mm的组织块；经2型胶原酶和胰酶消化依次后，将上清液离心，取细胞沉淀放入培养瓶，使用含10％胎牛血清DMEM/F12培养基，5％CO 2、37℃饱和湿度培养；去除非贴壁细胞，在贴壁细胞80％融合后更换无血清培养基培养48h；收集培养上清液；0.45μm滤膜过滤得到富含外泌体的培养液。 The liver of 4-month-old alpine black sheep fetal sheep was washed repeatedly with phosphate buffered saline (PBS) and cut into tissue pieces with a diameter of about 1-2 mm; after digestion with type 2 collagenase and trypsin, the supernatant was centrifuged Put the cell pellet into a culture flask, and use DMEM/F12 medium containing 10% fetal bovine serum, 5% CO 2 , 37°C saturated humidity; remove non-adherent cells, and replace with no serum after 80% of adherent cells are fused The culture medium was cultured for 48 hours; the culture supernatant was collected; the culture solution rich in exosomes was obtained by filtering with a 0.45 μm filter membrane.
将培养液在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。Centrifuge the culture broth at 4°C and 1000g for 10 minutes to collect the supernatant; the collected supernatant was centrifuged at 4°C and 2000g for 20 minutes to collect the supernatant; the collected supernatant was centrifuged at 4°C and 10000g for 30 minutes to collect the supernatant; the collected supernatant Centrifuge the clear solution at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, resuspend the pellet with a small amount of phosphate buffer, and filter with a 0.45μm filter to obtain exosomes.
实施例5、外泌体生化表征检测Example 5 Biochemical characterization and detection of exosomes
照以下步骤进行外泌体电镜检测：将实施例1中得到的胎羊血清来源的外泌体解冻后用缓冲液重悬得到样品；将样品滴于带有支持膜的铜网上；将染液与样品进行混匀得到混匀液，待铜网上的悬液稍微干燥后将混匀液滴于铜网上，负染2分钟后自然风干；在透射电镜下观察。观察到符合外泌体30-150nm形态特征的双层膜囊泡(图1)。经ELISA检测，外泌体表达CD63，CD81和Alix。Perform the electron microscopy detection of exosomes according to the following steps: Thaw the exosomes derived from fetal goat serum obtained in Example 1 and resuspend them in a buffer to obtain a sample; drop the sample on a copper mesh with a supporting film; transfer the dye solution Mix with the sample to obtain a mixed solution. After the suspension on the copper mesh is slightly dried, the mixed solution is dropped on the copper mesh, negatively dyed for 2 minutes and then air-dried naturally; observe under a transmission electron microscope. A bilayer membrane vesicle conforming to the morphological characteristics of 30-150 nm of exosomes was observed (Figure 1). The ELISA test showed that the exosomes expressed CD63, CD81 and Alix.
利用NanoFCM Flow NanoAnalyzer(纳米粒径分析仪)对实施例1中得到的胎羊血清来源的外泌体进行粒径检测方法，具体包括以下步骤：The particle size detection method of exosomes derived from fetal sheep serum obtained in Example 1 by NanoFCM Flow NanoAnalyzer (nano particle size analyzer) includes the following steps:
粒径检测：粒径标准品、空白对照和待测样品(子宫腔液)均需要在相同检测条件下(激光功率和散射通道衰减系数完全相同)采样；所有样品均在较低进样压力下(取样不大于1.0kPa)检测；在合适的条件下检测粒径标准品(粒径最小的硅球的散射光信号与背景完全分开且最大的硅球信号未饱和，检测器饱和值为3.6k)；检测重悬待测样品用的缓冲液，用于扣除背景颗粒，作为空白对照；检测待测样品，若待测样品中20％以上的颗粒在散射通道的信号强度达到饱和，则表明当前的粒径标准品不是该样品粒径表征的最优选择；经上述步骤检测到样品中的粒径大部分集中在85nm左右，符合外泌体直径30-150nm范围，说明检测到的样品为外泌体，如图2。Particle size detection: The particle size standard, blank control and sample (uterine cavity fluid) need to be sampled under the same detection conditions (laser power and attenuation coefficient of the scattering channel are exactly the same); all samples are under lower injection pressure (Sampling is not more than 1.0kPa) detection; under appropriate conditions, detect the particle size standard (the scattered light signal of the smallest particle size silicon ball is completely separated from the background and the largest silicon ball signal is not saturated, and the detector saturation value is 3.6k ); Detection of the buffer used to resuspend the sample to be tested, used to deduct background particles, as a blank control; to detect the sample to be tested, if the signal intensity of more than 20% of the particles in the sample to be tested reaches saturation in the scattering channel, it indicates that the current The particle size standard product is not the best choice for the particle size characterization of the sample; after the above steps, it is detected that most of the particle size in the sample is around 85nm, which is in the range of 30-150nm in exosomal diameter, indicating that the detected sample is external Exosomes, as shown in Figure 2.
实施例6、外泌体增强1型胶原蛋白Example 6. Exosomes enhance type 1 collagen
将实施例1所述胎羊血清外泌体(0.02％质量体积比)加入到人成纤维细胞的培养液(DMEM培养液，1％抗生素)中，以证实外泌体在细胞水平上促进1型胶原蛋白合成。合成的胶原蛋白的测定是使用PICP试剂盒(前胶原蛋白I型C肽酶免疫测定试剂盒)而进行定量。使用人成纤维细胞培养液将人源成纤维细胞(human fibroblast)以2×10 5个细胞/孔等分到6孔版中。确认细胞粘附后，将作为有效成分的胎羊血清外泌体溶解于培养液，对照组利用浓度一致的成年羊血清外泌体，空白组不添加任何外泌体，仅培养液。在37℃、5％CO2条件的培养箱中培养24小时后，测定细胞裂解物中I型胶原蛋白的含量，其结果如表1所示，结果显示胎羊血清外泌体可以促进胶原蛋白表达。 The fetal goat serum exosomes (0.02% mass-volume ratio) described in Example 1 were added to the culture medium (DMEM medium, 1% antibiotics) of human fibroblasts to confirm that the exosomes promote 1 at the cellular level. Type collagen synthesis. The synthetic collagen was measured using the PICP kit (Procollagen Type I C Peptidase Immunoassay Kit) for quantification. Using human fibroblast culture medium, human fibroblasts (human fibroblast) were divided into 6-well plates at 2×10 5 cells/well. After confirming cell adhesion, fetal goat serum exosomes as the active ingredient were dissolved in the culture medium. The control group used adult goat serum exosomes with the same concentration. The blank group did not add any exosomes, only the culture medium. After culturing for 24 hours in an incubator at 37°C and 5% CO2, the content of type I collagen in the cell lysate was determined. The results are shown in Table 1. The results show that fetal goat serum exosomes can promote collagen expression .
表1.胶原蛋白浓度Table 1. Collagen concentration
 To 处理组Treatment group 对照组Control group 空白组Blank group
1型胶原蛋白浓度μg/mlConcentration of type 1 collagen μg/ml 0.0180.018 0.0010.001 0.0010.001
增加率(％，较对照组)Increase rate (%, compared to the control group) 18001800 --  To
实施例7.外泌体对人造皮肤影响Example 7. The effect of exosomes on artificial skin
利用实施例2所述胎骆驼血清外泌体、对照外泌体进行本实施例。This example was performed using the fetal camel serum exosomes and control exosomes described in Example 2.
通过紫外线照射进行了光衰老的皮肤的情况下，表皮-真皮结合部位的胶原纤维网络受损。因此，胶原纤维的量和形态用作皮肤光衰老的指标。利用实施例2所述胎骆驼血清外泌体进行本实施例。以含有0.02％来自成年骆驼血清外泌体作为对照(对照)。空白组不添加任何外泌体，仅培养液。涂布后，将接受紫外线照射的无毛小鼠的皮肤组织用马松三色法(Masson's Trichrome)染色，用光学显微镜观察检测染色面积，相对于对照组的染色面积为标准，结果如表2。In the case of skin that has undergone photoaging by ultraviolet irradiation, the collagen fiber network at the epidermis-dermis junction is damaged. Therefore, the amount and morphology of collagen fibers are used as an indicator of skin photoaging. This example was carried out using the fetal camel serum exosomes described in example 2. Exosomes containing 0.02% serum from adult camels were used as a control (control). No exosomes were added to the blank group, only culture medium. After coating, the skin tissue of hairless mice irradiated with ultraviolet rays was stained with Masson's Trichrome, and the stained area was observed with an optical microscope. The stained area relative to the control group was the standard. The results are shown in Table 2. .
表2相对染色面积Table 2 Relative staining area
 To 平均值average value SDSD p值p value
对照[0.02％(w/v培养基)成年骆驼血清外泌体]Control [0.02% (w/v medium) adult camel serum exosomes] 100100 10.510.5  To
空白组[不添加任何外泌体]Blank group [do not add any exosomes] 98.6198.61 6.76.7 P＞0.05P>0.05
实施例1所述胎骆驼血清外泌体0.02％(w/v培养基)Fetal camel serum exosomes described in Example 1 0.02% (w/v medium) 155.1155.1 18.518.5 p＜0.05p＜0.05
进一步的，购买人造皮肤(来自MatTek，Ashland，USA的EpiDermFT  TM)后，在专用培养基(EFT-400)中稳定18小时。在人造皮肤的表面上，涂布浓度为0.02％(质量体积比)的100μl的实施例1所述胎羊血清外泌体作为有效成分。以含有1％来自成年羊血 清外泌体作为对照(对照)。空白组不添加任何外泌体，仅培养液。24小时后，将人造皮肤组织用10％甲醛固定，并用石蜡包埋，用马松三色法(Masson's Trichrome：M&T)染色并切成5μm的组织，从而观察计算胶原纤维(collagen fibers)的染色面积，结果如表3。 Further, after purchasing artificial skin (EpiDermFT ™ from MatTek, Ashland, USA), it was stabilized in a special medium (EFT-400) for 18 hours. On the surface of the artificial skin, 100 μl of the fetal sheep serum exosomes described in Example 1 at a concentration of 0.02% (mass volume ratio) was applied as an active ingredient. Exosomes containing 1% serum from adult goats were used as a control (control). No exosomes were added to the blank group, only culture medium. After 24 hours, the artificial skin tissue was fixed with 10% formaldehyde, embedded in paraffin, stained with Masson's Trichrome (M&T) and cut into 5μm tissues to observe and calculate the staining of collagen fibers (collagen fibers) Area, the results are shown in Table 3.
表3相对染色面积Table 3 Relative staining area
 To 平均值average value SDSD p值p value
对照[0.02％(w/v培养基)成年羊血清外泌体]Control [0.02% (w/v medium) adult goat serum exosomes] 100100 12.512.5  To
空白组[不添加任何外泌体]Blank group [do not add any exosomes] 102.42102.42 7.57.5 P＞0.05P>0.05
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 148.61148.61 16.116.1 p＜0.05p＜0.05
这些结果显示，根据本发明的所述的外泌体对具有抗光衰老作用。These results show that the exosomes according to the present invention have anti-photoaging effects.
实施例8.外泌体增强间充质干细胞ALDH表达。Example 8. Exosomes enhance the expression of ALDH in mesenchymal stem cells.
利用实施例1所述胎羊血清外泌体、对照外泌体进行本实施例。This example was performed using the fetal goat serum exosomes and control exosomes described in Example 1.
将人骨髓来源的间充质干细胞(MSC)，以2×10 5个细胞/孔等分到6孔版中。确认细胞粘附后，将作为有效成分的胎羊血清外泌体溶解于培养液，对照组利用成年羊血清外泌体，各孔外泌体均为200μg/ml的浓度。空白组不添加任何外泌体，仅培养液。在37℃、5％CO 2条件的培养箱中培养24小时后，利用流式细胞术检测MSC细胞的ALDH阳性率和Ki67阳性率，其结果如表4。 The mesenchymal stem cells (MSC) derived from human bone marrow were divided into 6-well plates at 2×10 5 cells/well. After confirming cell adhesion, fetal goat serum exosomes as the active ingredient were dissolved in culture medium. Adult goat serum exosomes were used in the control group, and the concentration of exosomes in each well was 200 μg/ml. No exosomes were added to the blank group, only culture medium. After culturing in an incubator at 37°C and 5% CO 2 for 24 hours, flow cytometry was used to detect the ALDH positive rate and Ki67 positive rate of MSC cells. The results are shown in Table 4.
表4.ALDH阳性率和Ki67阳性率Table 4. ALDH positive rate and Ki67 positive rate
Figure PCTCN2020130931-appb-000011
Figure PCTCN2020130931-appb-000011
结果显示胎羊血清外泌体具有非常强的维持干细胞干性和刺激干细胞增殖的作用。The results show that fetal goat serum exosomes have a very strong role in maintaining stem cell stemness and stimulating stem cell proliferation.
实施例9.外泌体对预防和治疗痤疮的作用Example 9. The effect of exosomes on the prevention and treatment of acne
利用实施例1所述胎羊血清外泌体、对照外泌体进行本实施例。空白组不添加任何外泌体，仅培养基。角质形成细胞单层模型：L-1α、IL-1β、IL-8、MMP-2和MMP-9表达的研究、角质形成细胞/痤疮丙酸杆菌共培养模型：MMP-9表达的研究、角质形成细胞单层模型：对IL-1α和VEGF的作用研究、角质形成细胞/痤疮丙酸杆菌共培养模型： 对IL-8产生的作用研究的模型建立、实验方法、实时RT-PCR方法和结果计算参考专利(CN102481245B)。结果如下：This example was performed using the fetal goat serum exosomes and control exosomes described in Example 1. No exosomes were added to the blank group, only medium. Keratinocyte monolayer model: study of L-1α, IL-1β, IL-8, MMP-2 and MMP-9 expression, keratinocyte/P. acnes co-culture model: study of MMP-9 expression, keratin Formation cell monolayer model: study on the effects of IL-1α and VEGF, keratinocyte/P. acnes co-culture model: model establishment, experimental methods, real-time RT-PCR methods and results for the study on the effects of IL-8 Calculation reference patent (CN102481245B). The results are as follows:
A.外泌体对初始炎症介质：IL-1α和IL-1β的影响。分析编码IL-1α和IL-1β的基因的表达。A. The influence of exosomes on initial inflammatory mediators: IL-1α and IL-1β. The expression of genes encoding IL-1α and IL-1β was analyzed.
在48小时处理后，测试的0.02％和0.05％外泌体显著抑制角质形成细胞中的IL-1α基因表达(表5)和IL-1β基因表达(表6)。After 48 hours of treatment, 0.02% and 0.05% of the tested exosomes significantly inhibited IL-1α gene expression (Table 5) and IL-1β gene expression (Table 6) in keratinocytes.
表5：角质形成细胞中的IL-1α基因表达Table 5: IL-1α gene expression in keratinocytes
IL-1α分析IL-1α analysis RQRQ 抑制％inhibition% p值p value
对照Control 11  To  To
空白blank 0.980.98  To  To
0.02％外泌体0.02% exosomes 0.410.41 -59-59 p＜0.05p＜0.05
0.05％外泌体0.05% exosomes 0.160.16 -84-84 p＜0.05p＜0.05
表6：角质形成细胞中的IL-1β基因表达Table 6: IL-1β gene expression in keratinocytes
IL-1β分析IL-1β analysis RQRQ 抑制％inhibition% p值p value
对照Control 11  To  To
空白blank 1.051.05  To  To
0.02％外泌体0.02% exosomes 0.250.25 -75-75 p＜0.05p＜0.05
0.05％外泌体0.05% exosomes 0.070.07 -93-93 p＜0.01p＜0.01
B.外泌体对IL-8表达的影响：次级炎症介质B. The effect of exosomes on IL-8 expression: secondary inflammatory mediators
1.分析编码IL-8的基因的表达1. Analyze the expression of the gene encoding IL-8
在48小时处理后，测试的0.02％和0.05％外泌体显著抑制角质形成细胞中的IL-8基因表达(表7)。After 48 hours of treatment, 0.02% and 0.05% of exosomes tested significantly inhibited IL-8 gene expression in keratinocytes (Table 7).
表7：角质形成细胞中的IL-8基因表达Table 7: IL-8 gene expression in keratinocytes
IL-8分析IL-8 analysis RQRQ 抑制％inhibition% p值p value
对照Control 11  To  To
空白blank 1.021.02  To  To
0.02％外泌体0.02% exosomes 0.260.26 -74-74 p＜0.05p＜0.05
0.05％外泌体0.05% exosomes 0.150.15 -85-85 p＜0.01p＜0.01
2.对受到痤疮丙酸杆菌刺激的角质形成细胞中IL-8产生的影响。2. The effect on IL-8 production in keratinocytes stimulated by P. acnes.
用痤疮丙酸杆菌悬液处理角质形成细胞极强烈地刺激IL-8释放，一种炎症次生标记(表8)。用外泌体预处理60分钟抑制了角质形成细胞释放IL-8。IL-8生产的抑制在0.02％开始并且在0.1％达到完全。Treatment of keratinocytes with a suspension of P. acnes extremely strongly stimulated the release of IL-8, a secondary marker of inflammation (Table 8). Pretreatment with exosomes for 60 minutes inhibited the release of IL-8 by keratinocytes. The inhibition of IL-8 production started at 0.02% and reached completion at 0.1%.
表8：分析角质形成细胞/痤疮丙酸杆菌共培养物中的IL-8Table 8: Analysis of IL-8 in keratinocytes/P. acnes co-culture
Figure PCTCN2020130931-appb-000012
Figure PCTCN2020130931-appb-000012
C.外泌体对基质蛋白酶MMP-2和MMP-9的影响C. The effect of exosomes on matrix protease MMP-2 and MMP-9
1.MMP-2表达的分析1. Analysis of MMP-2 expression
用0.05％外泌体处理角质形成细胞48小时导致MMP-2基因表达的显著抑制(-75％抑制)(表9)。Treatment of keratinocytes with 0.05% exosomes for 48 hours resulted in a significant inhibition of MMP-2 gene expression (-75% inhibition) (Table 9).
表9：角质形成细胞中的MMP-2基因表达Table 9: MMP-2 gene expression in keratinocytes
MMP-2MMP-2 RQRQ 抑制％inhibition% p值p value
对照Control 11  To  To
空白blank 0.980.98  To  To
0.05％外泌体0.05% exosomes 0.1670.167 -83-83 p＜0.01p＜0.01
2.MMP-9表达的分析2. Analysis of MMP-9 expression
在参与痤疮病理的基质蛋白酶当中，MMP-9发挥特别重要的作用。实际上，这种蛋 白酶受促炎性细胞因子正调节，还受痤疮丙酸杆菌正调节。Among the matrix proteases involved in the pathology of acne, MMP-9 plays a particularly important role. In fact, this protease is positively regulated by pro-inflammatory cytokines and also positively regulated by P. acnes.
如表10中所示，Q-PCR结果的分析显示用外泌体处理48小时后抑制角质形成细胞中的MMP-9基因表达(在0.02％时-49％抑制和在0.05％时-72％抑制)。As shown in Table 10, the analysis of the Q-PCR results showed that the MMP-9 gene expression in keratinocytes was inhibited after 48 hours of treatment with exosomes (inhibition of -49% at 0.02% and -72% at 0.05% inhibition).
表10：角质形成细胞中的MMP-9基因表达Table 10: MMP-9 gene expression in keratinocytes
MMP-9MMP-9 RQRQ 抑制％inhibition% p值p value
对照Control 11  To  To
空白blank 1.021.02  To  To
0.02％外泌体0.02% exosomes 0.510.51 -49-49 p＜0.01p＜0.01
0.05％外泌体0.05% exosomes 0.280.28 -72-72 p＜0.01p＜0.01
类似地，还在角质形成细胞/痤疮丙酸杆菌共培养模型中评价对MMP-9表达的调节(表11)。Similarly, the regulation of MMP-9 expression was also evaluated in a keratinocyte/P. acnes co-culture model (Table 11).
使用这个模型，展示了痤疮丙酸杆菌细菌悬液对MMP-9基因表达的刺激性影响(+67％)。Using this model, the irritant effect of P. acnes bacterial suspension on the expression of MMP-9 gene was demonstrated (+67%).
相反，用外泌体预处理角质形成细胞对抗衰老丙酸杆菌的这种刺激性作用并且还抑制这种刺激性作用(相对于用细菌悬液处理的角质形成细胞，-57％和-71％)。In contrast, pretreatment of keratinocytes with exosomes counteracts this irritating effect of Propionibacterium senescens and also inhibits this irritating effect (compared to keratinocytes treated with bacterial suspension, -57% and -71% ).
表11：角质形成细胞/痤疮丙酸杆菌共培养物中的MMP-9基因表达Table 11: MMP-9 gene expression in keratinocyte/P. acnes co-culture
Figure PCTCN2020130931-appb-000013
Figure PCTCN2020130931-appb-000013
D.外泌体对血管发生(VEGF)的影响D. The effect of exosomes on angiogenesis (VEGF)
最后，研究了外泌体对角质形成细胞合成和释放VEGF的潜在抑制性影响。结果呈现于表12中。Finally, the potential inhibitory effects of exosomes on the synthesis and release of VEGF by keratinocytes were studied. The results are presented in Table 12.
因而，PMA刺激角质形成细胞24小时诱导VEGF的大量和明显释放(+226％)。相反，用外泌体预处理角质形成细胞24小时对这种作用并且显著地抑制这种作用。Therefore, PMA stimulates keratinocytes for 24 hours to induce a large and significant release of VEGF (+226%). In contrast, pretreatment of keratinocytes with exosomes for 24 hours had this effect and significantly inhibited this effect.
表12：分析角质形成细胞中的VEGFTable 12: Analysis of VEGF in keratinocytes
Figure PCTCN2020130931-appb-000014
Figure PCTCN2020130931-appb-000014
实施例10.胎猪胃来源外泌体的制备Example 10. Preparation of fetal pig stomach-derived exosomes
将3月龄胎猪胃经磷酸盐缓冲液(PBS)反复冲洗后，剪成直径约1-2mm的组织块；经2型胶原酶和胰酶消化依次后，将上清液离心，取细胞沉淀放入培养瓶，使用含10％胎牛血清DMEM/F12培养基，5％CO 2、37℃饱和湿度培养；去除非贴壁细胞，在贴壁细胞80％融合后更换无血清培养基培养48h；收集培养上清液；0.45μm滤膜过滤得到富含外泌体的培养液。 The stomach of a 3-month-old fetal pig was washed repeatedly with phosphate buffered saline (PBS) and cut into tissue pieces with a diameter of about 1-2mm; after digestion with type 2 collagenase and trypsin, the supernatant was centrifuged and the cells were collected Put the precipitate in a culture flask, use DMEM/F12 medium containing 10% fetal bovine serum, 5% CO 2 , 37°C saturated humidity; remove non-adherent cells, replace the serum-free medium after the adherent cells are 80% fused 48h; collect the culture supernatant; filter with a 0.45μm filter membrane to obtain a culture solution rich in exosomes.
将培养液在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。Centrifuge the culture broth at 4°C and 1000g for 10 minutes to collect the supernatant; the collected supernatant was centrifuged at 4°C and 2000g for 20 minutes to collect the supernatant; the collected supernatant was centrifuged at 4°C and 10000g for 30 minutes to collect the supernatant; the collected supernatant Centrifuge the clear solution at 110,000g for 90min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 90min, discard the supernatant, resuspend the pellet with a small amount of phosphate buffer, and filter with a 0.45μm filter to obtain exosomes.
实施例11.新生羊胃液来源外泌体的制备Example 11. Preparation of Exosomes from Newborn Sheep Gastric Juice
将新生黑绵羊胎胃液用0.45μm滤膜过滤，在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心90min，弃上清，使用磷酸盐缓冲液 重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。用Bradford法检测外泌体总蛋白量(Bio-Rad Protein Assay Reagent)。外泌体冻干后储存于-80℃。The fetal gastric juice of newborn black sheep was filtered with a 0.45μm filter, centrifuged at 4°C, 1000g for 10min, and the supernatant was collected; the collected supernatant was centrifuged at 4°C, 2000g for 20min, and the supernatant was collected; the collected supernatant was 4°C, 10000g Centrifuge for 30 min, collect the supernatant; centrifuge the collected supernatant at 110,000 g for 90 min, discard the supernatant, and resuspend the pellet in phosphate buffer; centrifuge again at 110,000 g for 90 min, discard the supernatant, and resuspend the pellet in a small amount of phosphate buffer, 0.45 μm Filter membrane to obtain exosomes. The Bradford method was used to detect the total protein content of exosomes (Bio-Rad Protein Assay Reagent). The exosomes were lyophilized and stored at -80°C.
实施例12：外泌体增强V型胶原蛋白表达Example 12: Exosomes enhance the expression of type V collagen
利用实施例1所述胎羊血清外泌体、实施例4所述胎羊肝脏外泌体进行本实施例。The fetal sheep serum exosomes described in Example 1 and the fetal sheep liver exosomes described in Example 4 were used for this example.
正常人成纤维细胞在添加不同终浓度的各种外泌体的FGM培养基中培养48小时，然后除去细胞培养基。添加0.05％来自成年羊血清外泌体的相同培养基用作对照(对照)。空白组不添加任何外泌体，仅培养液。将细胞层用氢氧化铵溶液裂解，然后用在缓冲溶液(PBS)中稀释至1/4000的抗-胶原蛋白V抗体测定V型胶原蛋白。60分钟后，应用稀释至1/25000的二级抗体达60分钟。在洗涤后，加入显示溶液，测定荧光(ENVision,PerkinElmer)。将荧光结果相对于对照培养基得到的荧光(对照)进行标准化，使其与在每个条件下得到的胶原蛋白量相关。所示结果为3次测定的平均值(n＝3)(SD：标准偏差)。结果如表13。Normal human fibroblasts were cultured in FGM medium supplemented with various exosomes at different final concentrations for 48 hours, and then the cell culture medium was removed. The same medium supplemented with 0.05% of adult goat serum exosomes was used as a control (control). No exosomes were added to the blank group, only culture medium. The cell layer was lysed with ammonium hydroxide solution, and then type V collagen was measured with an anti-collagen V antibody diluted to 1/4000 in a buffer solution (PBS). After 60 minutes, apply the secondary antibody diluted to 1/25000 for 60 minutes. After washing, display solution was added, and fluorescence was measured (ENVision, PerkinElmer). The fluorescence results were normalized to the fluorescence obtained from the control medium (control) to correlate with the amount of collagen obtained under each condition. The results shown are the average of 3 measurements (n=3) (SD: standard deviation). The results are shown in Table 13.
表13 V型胶原蛋白表达情况Table 13 Expression of Type V Collagen
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.54100.54 8.48.4  To
空白blank 96.8196.81 7.517.51 P＞0.05P>0.05
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 122.66122.66 16.6416.64 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 195.55195.55 24.3224.32 p＜0.01p＜0.01
实施例4所述胎羊肝脏外泌体0.02％(w/v培养基)Fetal sheep liver exosomes described in Example 4 0.02% (w/v medium) 135.15135.15 123.6123.6 p＜0.05p＜0.05
实施例4所述胎羊肝脏外泌体0.05％(w/v培养基)The fetal sheep liver exosomes described in Example 4 0.05% (w/v medium) 175.68175.68 29.829.8 p＜0.05p＜0.05
结论：在所分析的成纤维细胞中，所述外泌体使V型胶原蛋白表达增加至少22％和35％，显示了外泌体在增强胶原蛋白表达的性质，可以用于增强毛囊和减少脱发方面的性质。Conclusion: In the analyzed fibroblasts, the exosomes increased the expression of type V collagen by at least 22% and 35%, showing the properties of exosomes in enhancing the expression of collagen, which can be used to strengthen hair follicles and reduce The nature of hair loss.
实施例13：外泌体增加肌原纤蛋白-1合成Example 13: Exosomes increase myofibrillin-1 synthesis
将正常人成纤维细胞在FGM培养基中培养48小时，然后用实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞，最终重量浓度相对于培养基的最终体积为0.02％或0.05％。采集细胞，然后用裂解缓冲液裂解以进行免疫印迹法(Western Blots)。通过BCA方法测定了蛋白质浓度。将所有样品相对于蛋白质总量进行标准化(n＝3)。使用抗-肌原纤蛋白-1抗体(LS-BIO)，因为是过氧化物酶-偶联的二级抗体。化学发光底物使得能够说明信号并对其进行定量(Protein Simple)，结果如表14。Normal human fibroblasts were cultured in FGM medium for 48 hours, and then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2, and the final weight concentration was relative to the medium The final volume is 0.02% or 0.05%. The cells were collected and then lysed with lysis buffer for Western Blots. The protein concentration was determined by the BCA method. All samples were normalized to total protein (n=3). Anti-myofibrillin-1 antibody (LS-BIO) was used because it is a peroxidase-conjugated secondary antibody. The chemiluminescent substrate enables the interpretation and quantification of the signal (Protein Simple). The results are shown in Table 14.
表14肌原纤蛋白-1表达Table 14 Myofibrillin-1 expression
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.31100.31 6.456.45  To
空白blank 105.25105.25 10.1510.15 P＞0.05P>0.05
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 184.65184.65 17.6117.61 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 266.14266.14 20.1220.12 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 159.68159.68 19.8519.85 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 233.12233.12 29.6329.63 p＜0.05p＜0.05
结论：与对照相比和，本发明所述外泌体显示出在成纤维细胞中的肌原纤蛋白-1合成增加。Conclusion: Compared with the control, the exosomes of the present invention show an increase in the synthesis of myofibrin-1 in fibroblasts.
实施例14：外泌体促进毛囊乳头成纤维细胞中ATP合成增加Example 14: Exosomes promote increased ATP synthesis in hair follicle papilla fibroblasts
将来自毛囊乳头的非病理性人成纤维细胞在DMEM培养基上培养24小时，然后用实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞处理6天，终浓度/体积相对于培养基总体积而言为0.02％或0.05％体积。在6天后通过酶促法(荧光素/荧光素酶络合物；ATP Bioluminescence kit Roche Diagnostics)测量ATP含量(表15)。Non-pathological human fibroblasts from hair follicle papilla were cultured on DMEM medium for 24 hours, and then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 6 On days, the final concentration/volume is 0.02% or 0.05% by volume relative to the total volume of the culture medium. After 6 days, the ATP content was measured by enzymatic method (luciferin/luciferase complex; ATP Bioluminescence kit Roche Diagnostics) (Table 15).
表15 ATP含量Table 15 ATP content
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.64100.64 11.3111.31  To
空白blank 94.2594.25 4.654.65  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 145145 16.6516.65 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 167167 19.1819.18 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 135135 15.9515.95 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 157157 20.3320.33 p＜0.01p＜0.01
结论：外泌体与对照相比显示出ATP合成增加，证实其用于增强毛囊的活性的用途。Conclusion: The exosomes showed increased ATP synthesis compared with the control, confirming their use for enhancing the activity of hair follicles.
实施例15：外泌体增强毛乳头成纤维细胞的线粒体代谢活性Example 15: Exosomes enhance the mitochondrial metabolic activity of dermal papilla fibroblasts
将毛囊乳头的非病理性人成纤维细胞培养24小时，实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞处理6天，终浓度/体积相对于培养基总体积而言为0.02％和0.05％。在琥珀酸脱氢酶的存在下、通过MTT(3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑鎓溴化物)的还原测量了成纤维细胞的线粒体活性。将所得沉淀物用DMSO提取，然后于540nm测量DMSO溶液的光密度(表16)。The non-pathological human fibroblasts from the hair follicle papilla were cultured for 24 hours, and the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 were treated for 6 days. The total volume of the medium is 0.02% and 0.05%. Measurement of fibroblasts by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in the presence of succinate dehydrogenase Mitochondrial activity. The resulting precipitate was extracted with DMSO, and then the optical density of the DMSO solution was measured at 540 nm (Table 16).
表16相对线粒体活性Table 16 Relative mitochondrial activity
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100100 1010  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 115115 1212 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 154154 1414 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 126126 1414 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 171171 1616 p＜0.05p＜0.05
结论：本发明所述外泌体与对照相比显示出毛囊乳头成纤维细胞的线粒体活性增加。该线粒体活性参与毛囊的增强。Conclusion: Compared with the control, the exosomes of the present invention show increased mitochondrial activity of hair follicle papilla fibroblasts. This mitochondrial activity participates in the enhancement of hair follicles.
实施例16：外泌体对微毛囊的作用Example 16: The effect of exosomes on microhair follicles
微毛囊重建方法：微毛囊模型包括将乳头成纤维细胞、来自毛囊外鞘的角质形成细胞和黑素细胞在三维上共培养。由于能够整合各种细胞类型之间的神经-表皮-间质相互作用，该细胞模型构成了最接近毛囊的重建器官模型。Micro hair follicle reconstruction method: The micro hair follicle model includes papillary fibroblasts, keratinocytes from the outer sheath of hair follicles and melanocytes co-cultured in three dimensions. Due to the ability to integrate the neuro-epidermal-mesenchymal interactions between various cell types, this cell model constitutes the closest reconstructed organ model to hair follicles.
将非病理性乳头成纤维细胞培养3天。然后，将黑素细胞和毛囊外鞘的角质形成细胞加入到新型乳头中以形成微毛囊。培养24小时后，加入实施例1所述胎羊血清外泌体，终浓度相对于培养基总体积而言为0.05％体积。将相同的培养基在成年羊血清外泌体的情况下培养(对照)。处理48小时后，对培养基和微毛囊采样用于分析。Non-pathological papillary fibroblasts were cultured for 3 days. Then, melanocytes and keratinocytes of the outer sheath of hair follicles are added to the new type of papilla to form micro hair follicles. After culturing for 24 hours, the fetal goat serum exosomes described in Example 1 were added, and the final concentration was 0.05% by volume relative to the total volume of the culture medium. The same medium was cultured in the case of adult goat serum exosomes (control). After 48 hours of treatment, the culture medium and microfollicles were sampled for analysis.
通过PrestoBlue方法(Thermo Fisher Scientific)测量了微毛囊的细胞存活力。处理48 小时后进行测定。值表示为对照进行标准化后的平均值％(表17)。The cell viability of microhair follicles was measured by the PrestoBlue method (Thermo Fisher Scientific). The measurement is carried out after 48 hours of treatment. The value is expressed as the mean% of the normalized control (Table 17).
表17相对细胞活力Table 17 Relative cell viability
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.65100.65 7.817.81  To
空白blank 101.11101.11 3.513.51 P＞0.05P>0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 156156 20.120.1 p＜0.05p＜0.05
结论：本发明所述外泌体具有增加微毛囊的细胞存活力的能力，使其成为对增强毛囊具有活性的外泌体。Conclusion: The exosomes of the present invention have the ability to increase the cell viability of microhair follicles, making them active exosomes for enhancing hair follicles.
进一步评估微毛囊水平上的膜损伤的减轻To further evaluate the reduction of membrane damage at the microfollicle level
利用乳酸脱氢酶检测法，通过比色法测量细胞损伤，使得能够基于培养基中受损细胞中乳酸脱氢酶活性的测量来定量细胞损伤。细胞膜损伤的增加和细胞溶解导致乳酸脱氢酶活性增加，其与裂解细胞的数量成比例。处理48小时后，在培养基中进行了乳酸脱氢酶活性测量(表18)。Using the lactate dehydrogenase detection method, cell damage is measured by a colorimetric method, so that cell damage can be quantified based on the measurement of lactate dehydrogenase activity in damaged cells in the culture medium. Increased cell membrane damage and cell lysis lead to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells. After 48 hours of treatment, lactate dehydrogenase activity was measured in the culture medium (Table 18).
表18相对细胞损伤Table 18 Relative cell damage
 To 平均值average value SDSD p值p value
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.33100.33 10.610.6  To
空白blank 89.9189.91 9.919.91 P＞0.05P>0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 4747 6.76.7 p＜0.05p＜0.05
结论：本发明的外泌体具有减轻膜损伤的能力，使其成为对增强毛囊具有活性的本发明外泌体，增强毛囊促进了脱发的减少。Conclusion: The exosomes of the present invention have the ability to reduce membrane damage, making them the exosomes of the present invention that are active in enhancing hair follicles, and enhancing hair follicles promotes the reduction of hair loss.
实施例17：外泌体应对亚洲沙尘引起的人角质形成细胞中的活性氧产生Example 17: Exosomes respond to the production of reactive oxygen species in human keratinocytes caused by Asian dust
亚洲沙尘是起源于我国、蒙古、中亚和俄罗斯等国沙漠的一种气候现象。在这些地方，沙子或黄色沙尘随风刮向东并落到地上。几乎每年春季北京等北方地区都会受到亚洲沙尘的影响。亚洲沙尘包含有机和无机物质，并且其物理性质和组成是不同的。其甚至包含可能具有生物效应的金属。亚洲沙尘中包含的大颗粒保留在原地或其周围，而直径为10μm或更小的微粒(颗粒物10；PM10)，近年来持续爆发的PM2.5也属于这类气候污染。Asian dust is a climatic phenomenon that originated in the deserts of my country, Mongolia, Central Asia and Russia. In these places, sand or yellow dust blows eastward with the wind and falls to the ground. Almost every spring, Beijing and other northern regions are affected by Asian dust. Asian dust contains organic and inorganic substances, and their physical properties and composition are different. It even contains metals that may have biological effects. The large particles contained in the Asian dust remain in or around the place, while the particles with a diameter of 10 μm or less (particulate matter 10; PM10), the continuous outbreak of PM2.5 in recent years also belong to this type of climate pollution.
据报道，微粒在被吸入时可以深入到达亚段支气管和肺的气体交换区域并可能损伤呼吸系统(Yanagisawa et al,.Exp Biol Med232：1109-1118，2007；Kim et al,.J Biosci28(1)：77-81，2003)。亚洲沙尘对人呼吸疾病方面的健康影响作已经有大量报道。在小鼠实验性研究中，在被灌注亚洲沙尘的小鼠肺中发现氧化应激相关指数增大(Naota et al.,Toxicol Pathol.2010Sep30。临床上，如果皮肤直接暴露于亚洲沙尘，它可能通过过敏反应导致接触性皮炎。此外，已知干燥和强烈的亚洲沙尘暴通过使皮肤丧失水分导致皮肤干燥、角质化、瘙痒、痤疮、特应性等。近来，关于亚洲沙尘对皮肤的有害作用也有报道，如研究显示亚洲沙尘导致角质形成细胞中包括细胞因子白介素6和白介素8的促炎性因子的增加(Choi et al,.Toxicol Lett.15；200(1-2)：92-9.2010)。According to reports, when inhaled, particles can reach the gas exchange area of subsegmental bronchi and lungs and may damage the respiratory system (Yanagisawa et al, Exp Biol Med232: 1109-1118, 2007; Kim et al, J Biosci28(1 ): 77-81, 2003). There have been numerous reports on the health effects of Asian dust on human respiratory diseases. In experimental studies in mice, it was found that the oxidative stress-related index increased in the lungs of mice infused with Asian dust (Naota et al., Toxicol Pathol. 2010 Sep 30. Clinically, if the skin is directly exposed to Asian dust, It may cause contact dermatitis through allergic reactions. In addition, dry and strong Asian dust storms are known to cause dry skin, keratinization, itching, acne, atopy, etc. by depriving the skin of moisture. Recently, there has been a discussion about the effects of Asian dust on the skin. Harmful effects have also been reported. For example, studies have shown that Asian sand dust causes an increase in keratinocytes including the cytokines interleukin 6 and interleukin 8 pro-inflammatory factors (Choi et al,. Toxicol Lett. 15; 200(1-2): 92 -9.2010).
人表皮新生角质形成细胞细胞培养24小时，培养条件同Lonza公司说明书所述。然后用实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞处理12小时，终浓度/体积相对于培养基总体积而言为0.02％或0.05％体积，然后加入，25μg/mL亚洲沙尘样品，并将每组细胞继续孵育24小时。亚洲沙尘样品是在亚洲沙尘季节期间在国贸三期A座大楼(北京市)的阳台屋顶上收集的。将细胞用10mL磷酸缓冲盐(phosphate buffered saline，PBS)洗涤两次，并使用Glomax20/20发光检测仪在加入1mM鲁米诺和20单位/mL辣根过氧化物酶(HRP)1分钟后在2mL PBS中测量化学发光。结果如表19Human epidermal neonatal keratinocyte cells were cultured for 24 hours under the same culture conditions as those described in the Lonza company's instructions. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% or 0.05% volume relative to the total volume of the culture medium. , And then add 25μg/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season. Wash the cells twice with 10mL phosphate buffered saline (PBS), and use the Glomax 20/20 luminescence detector to add 1 mM luminol and 20 units/mL horseradish peroxidase (HRP) for 1 minute after adding 1 mM luminol and 20 units/mL horseradish peroxidase (HRP). Chemiluminescence was measured in 2mL PBS. The results are shown in Table 19
表19活性氧相对水平Table 19 Relative levels of active oxygen
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土空白对照No Asian dust blank control 14.0914.09 1.111.11  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.01100.01 5.455.45  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 34.4334.43 4.304.30 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 21.8321.83 1.861.86 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 59.8059.80 7.887.88 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 15.6215.62 2.332.33 p＜0.05p＜0.05
结果显示，本发明所述的胎体来源外泌体可以抑制由亚洲沙尘介导的正常人角质形成细胞中活性氧的产生。The results show that the carcass-derived exosomes of the present invention can inhibit the production of reactive oxygen species in normal human keratinocytes mediated by Asian sand dust.
实施例18：外泌体对毒素去毒和排除因子、抗微生物肽的表达作用Example 18: Expression of Exosomes on Toxin Detoxification and Elimination Factors and Antimicrobial Peptides
同实施例17，人表皮新生角质形成细胞细胞培养24小时，培养条件同Lonza公司说明书所述。然后用实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞处理12小时，终浓度/体积相对于培养基总体积而言为0.02％和0.05％体积，然后加入，25μg/mL亚洲沙尘样品，并将每组细胞继续孵育24小时。亚洲沙尘样品是在亚洲沙尘季节期间在国贸三期A座大楼(北京市)的阳台屋顶上收集的。实验各组细胞用10mL磷酸盐缓冲液洗涤两次,并使用TRIzol(Invitrogen)从细胞中分离出总RNA(total RNA)。再次使用RNA试剂盒(Qiagen)对分离出的RNA进行纯化并使用Bioanalyzer2100(Agilent)分析RNA的质量(quality)。使用Superscript逆转录酶(RT)II试剂盒(Superscriptreverse transcriptase(RT)II kit)由分离出的RNA合成cDNA,并通过实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,qPCR)对cDNA进行定量分析。使用TaqMan基因表达分析试剂盒(TaqMan gene expression assay kit,A pplied Biosys tems)对GSTT1(Hs00184475_m1)、NQO1(Hs01045994_m1)和hBD3(hs00218678_m1)的表达水平进行检测，结果如表20-22。As in Example 17, the human epidermal keratinocyte cells were cultured for 24 hours, and the culture conditions were the same as those described in the instructions of Lonza Company. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% and 0.05% volume relative to the total volume of the culture medium. , And then add 25μg/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season. The cells in each group were washed twice with 10mL phosphate buffer, and TRIzol (Invitrogen) was used to separate total RNA from the cells. The RNA kit (Qiagen) was used to purify the isolated RNA and the quality of RNA was analyzed using Bioanalyzer 2100 (Agilent). Use Superscript reverse transcriptase (RT) II kit (Superscript reverse transcriptase (RT) II kit) to synthesize cDNA from the isolated RNA, and use real-time reverse transcription polymerase chain reaction (qPCR) Quantitative analysis of cDNA. The expression levels of GSTT1 (Hs00184475_m1), NQO1 (Hs01045994_m1) and hBD3 (hs00218678_m1) were detected using the TaqMan gene expression assay kit (TaqMan gene expression assay kit, A pplied Biosystems tems), and the results are shown in Table 20-22.
表20 NQO1相对表达水平Table 20 Relative expression level of NQO1
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 125.54125.54 28.6028.60  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 8.308.30  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 212.14212.14 14.9514.95 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 435.40435.40 33.2933.29 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 245.63245.63 14.1714.17 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 268.04268.04 26.7826.78 p＜0.05p＜0.05
表21 GSTT1相对表达水平Table 21 Relative expression level of GSTT1
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土空白对照No Asian dust blank control 393.75393.75 64.0864.08  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 12.5812.58  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 333.11333.11 18.7918.79 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 626.60626.60 57.1457.14 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 331.97331.97 33.2933.29 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 743.04743.04 97.0097.00 p＜0.05p＜0.05
表22 hBD3相对表达水平Table 22 Relative expression level of hBD3
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 214.41214.41 12.0612.06  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 9.239.23  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 231.58231.58 11.7111.71 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 558.07558.07 36.4036.40 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 355.62355.62 58.8358.83 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 602.69602.69 93.6893.68 p＜0.05p＜0.05
结果所示，本发明所述胎体外泌体可以增加去毒素蛋白NQO1、天然免疫因子的皮肤抗微生物肽hBD3和排除毒素的酶的GSTT1在正常人角质形成细胞中的表达。The results show that the extrafetal body of the present invention can increase the expression of detoxified protein NQO1, skin antimicrobial peptide hBD3 of natural immune factors, and GSTT1 of detoxifying enzyme in normal human keratinocytes.
实施例19：外泌体对抗氧化蛋白基因的表达作用Example 19: Expression of Antioxidant Protein Gene by Exosomes
同实施例17，人表皮新生角质形成细胞细胞培养24小时，培养条件同Lonza公司说明书所述。然后用实施例1所述胎羊血清外泌体、实施例2所述胎骆驼血清外泌体处理细胞处理12小时，终浓度/体积相对于培养基总体积而言为0.02％和0.05％体积，然后加入，25μg/mL亚洲沙尘样品，并将每组细胞继续孵育24小时。亚洲沙尘样品是在亚洲沙尘季节期间在国贸三期A座大楼(北京市)的阳台屋顶上收集的。实验各组细胞用10mL磷酸盐缓冲液洗涤两次,并使用TRIzol(Invitrogen)从细胞中分离出总RNA(total RNA)。再次使用RNA试剂盒(Qiagen)对分离出的RNA进行纯化并使用Bioanalyzer2100(Agilent)分析RNA的质量(quality)。使用Superscript逆转录酶(RT)II试剂盒(Superscriptreverse transcriptase(RT)II kit)由分离出的RNA合成cDNA,并通过实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,qPCR)对cDNA进行定量分析。使用TaqMan基因表达分析试剂盒(TaqMan gene expression assay kit,A pplied Biosystems)对GPX1(Hs00829989_gH)、过氧化氢酶(Hs00156308_m1)、TRX1(Hs01555212_g1)和PRDX1(Hs00602020_mH)的表达水平进行检测，结果如表。As in Example 17, the human epidermal keratinocyte cells were cultured for 24 hours, and the culture conditions were the same as those described in the instructions of Lonza Company. The cells were then treated with the fetal goat serum exosomes described in Example 1 and the fetal camel serum exosomes described in Example 2 for 12 hours, and the final concentration/volume was 0.02% and 0.05% volume relative to the total volume of the culture medium. , And then add 25μg/mL Asian sand dust sample, and incubate each group of cells for 24 hours. The Asian dust samples were collected on the balcony roof of Tower A of China World Trade Center Phase III (Beijing) during the Asian dust season. The cells in each group were washed twice with 10mL phosphate buffer, and TRIzol (Invitrogen) was used to separate total RNA from the cells. The RNA kit (Qiagen) was used to purify the isolated RNA and the quality of RNA was analyzed using Bioanalyzer 2100 (Agilent). Use Superscript reverse transcriptase (RT) II kit (Superscript reverse transcriptase (RT) II kit) to synthesize cDNA from the isolated RNA, and use real-time reverse transcription polymerase chain reaction (qPCR) Quantitative analysis of cDNA. Use TaqMan gene expression assay kit (TaqMan gene expression assay kit, A plied Biosystems) to detect the expression levels of GPX1 (Hs00829989_gH), catalase (Hs00156308_m1), TRX1 (Hs01555212_g1) and PRDX1 (Hs00602020_mH), and the results are shown in the table .
表23 GPX1相对表达水平Table 23 Relative expression level of GPX1
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 158.42158.42 23.2323.23  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 5.015.01  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 187.28187.28 12.7712.77 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 353.73353.73 49.8249.82 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 223.01223.01 35.5535.55 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 503.20503.20 29.0029.00 p＜0.05p＜0.05
表24过氧化氢酶相对表达水平Table 24 Relative expression levels of catalase
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 223.01223.01 35.5535.55  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 13.7413.74  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 187.03187.03 21.2621.26 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 426.89426.89 71.0671.06 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 248.91248.91 17.2017.20 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 533.55533.55 88.8288.82 p＜0.05p＜0.05
表25 TRX1相对表达水平Table 25 Relative expression level of TRX1
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 153.48153.48 20.9620.96  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 15.1815.18  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 196.10196.10 14.9314.93 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 276.73276.73 41.7441.74 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 155.23155.23 20.5320.53 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 377.66377.66 41.6741.67 p＜0.05p＜0.05
表26 PRDX1相对表达水平Table 26 Relative expression level of PRDX1
 To 平均值(％)average value(%) SDSD p值p value
未加入亚洲尘土对照No Asian dust control added 276.73276.73 41.7441.74  To
对照[0.05％(w/v培养基)成年羊血清外泌体]Control [0.05% (w/v medium) adult goat serum exosomes] 100.00100.00 11.4811.48  To
实施例1所述胎羊血清外泌体0.02％(w/v培养基)Fetal goat serum exosomes described in Example 1 0.02% (w/v medium) 229.40229.40 37.7337.73 p＜0.05p＜0.05
实施例1所述胎羊血清外泌体0.05％(w/v培养基)The fetal goat serum exosomes described in Example 1 0.05% (w/v medium) 454.47454.47 74.9874.98 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.02％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.02% (w/v medium) 183.80183.80 29.5229.52 p＜0.05p＜0.05
实施例2所述胎骆驼血清外泌体0.05％(w/v培养基)The fetal camel serum exosomes described in Example 2 0.05% (w/v medium) 300.65300.65 65.1265.12 p＜0.05p＜0.05
结果所示，本发明所述胎体外泌体可以增加清除活性氧的抗氧化酶GPX1、过氧化氢酶、TRX1和PRDX1的相对表达水平。有效对抗亚洲尘土介导的氧化应激反应。The results show that the extrafetal body of the present invention can increase the relative expression levels of antioxidant enzymes GPX1, catalase, TRX1 and PRDX1 that scavenges reactive oxygen species. Effective against the oxidative stress response mediated by Asian dust
实施例20：抗衰老实例Example 20: Anti-aging example
按照质量百分数，本发明实施例1所述的述胎羊血清外泌体1.2％，丁二醇5％，防腐剂0.2％以及0.6％氯化钠，水93％，分别称取适量各原料组分。将上述原料组分混合，即得抗衰老护肤水。According to the mass percentage, the fetal sheep serum exosomes described in Example 1 of the present invention are 1.2%, butanediol 5%, preservative 0.2% and 0.6% sodium chloride, water 93%, and weigh the appropriate amount of each raw material group. Minute. The above-mentioned raw material components are mixed to obtain anti-aging skin care water.
选择志愿者20名，性别随机。将志愿者随机分为2组，并分别随机在每位志愿者的左脸颊或右脸颊中的一侧脸颊，以及左眼角和右眼角中的一侧眼角使用本发明抗衰护肤水，并作为实验组；志愿者未使用抗衰护肤水的一侧脸颊和眼角作为对照组。Twenty volunteers were selected, with random gender. The volunteers were randomly divided into 2 groups, and the anti-aging skin care lotion of the present invention was used randomly on each of the volunteers’ left cheek or right cheek, and one of the left and right corners of the eye. Experimental group: Volunteers did not use the anti-aging skin care lotion on one cheek and the corner of the eye as the control group.
志愿者者于每日早晚各涂一次本发明抗衰老化妆品，每次涂用30分钟后以清水洗面。使用周期为4周，在测试周期内不得使用其他化妆品。然后，采用皮肤弹性测试仪和脸部皱纹成像仪，对比分析本发明抗衰老化妆品对志愿者皮肤弹性和皱纹改善情况，结果如下：Volunteers apply the anti-aging cosmetics of the present invention once a day in the morning and evening, and wash their face with clean water after 30 minutes of each application. The use period is 4 weeks, and no other cosmetics are allowed to be used during the test period. Then, the skin elasticity tester and the facial wrinkle imager were used to compare and analyze the improvement of the volunteers' skin elasticity and wrinkles by the anti-aging cosmetics of the present invention. The results are as follows:
Figure PCTCN2020130931-appb-000015
Figure PCTCN2020130931-appb-000015
实施例21：化妆品制剂的实例Example 21: Examples of cosmetic preparations
本发明人在下文提供几种用于局部施加、尤其适用于美容的组合物。In the following, the inventors provide several compositions for topical application, especially for cosmetics.
(1)清洁霜(1) Cleansing cream
原料/品牌名称Raw material/brand name ％%
纯水Pure water QSP 100％QSP 100%
阿拉同恩(Arlatone)Arlatone 10％-30％10%-30%
椰油基葡糖苷Coco Glucoside 5％-20％5%-20%
羟丙基瓜尔胶Hydroxypropyl guar 1％-5％1%-5%
辛酰甘氨酸Caprylylglycine 0％-2％0%-2%
防腐剂preservative 0％-2％0%-2%
香料spices 0％-1％0%-1%
柠檬酸 Citric acid 0％-1％0%-1%
锌PCAZinc PCA 0％-1％0%-1%
胎羊血清外泌体Fetal Sheep Serum Exosomes 0.01％-0.1％0.01%-0.1%
(2)调节皮脂的抗衰老乳剂(2) Anti-aging emulsion that regulates sebum
原料/品牌名称Raw material/brand name ％%
PEG40硬脂酸酯PEG40 stearate 1％-5％1%-5%
PEG5硬脂酸甘油PEG5 Stearic Acid Glycerin 1％-5％1%-5%
地蜡Ozokerite 1％-5％1%-5%
单硬脂酸甘油酯Glyceryl Monostearate 1％-5％1%-5%
失水山梨醇硬脂酸酯Sorbitan Stearate 0％-2％0%-2%
鲸蜡醇Cetyl alcohol 0％-2％0%-2%
苹果酸氢酯醇Malate Alcohol 5％-20％5%-20%
维生素EVitamin E 0％-1％0%-1%
5-αAvocuta5-αAvocuta 1％-5％1%-5%
胎羊血清外泌体Fetal Sheep Serum Exosomes 0.01％-0.1％0.01%-0.1%
丁二醇Butanediol 1％-5％1%-5%
吡罗克酮乙醇胺(Piroctolamine)Piroctolamine 0％-1％0%-1%
防腐剂preservative 0％-1％0%-1%
甘油glycerin 1％-10％1%-10%
黄原胶Xanthan gum 0％-1％0%-1%
锌PCAZinc PCA 0％-2％0%-2%
米淀粉Rice starch 1％-5％1%-5%
尼龙6Nylon 6 0％-2％0%-2%
聚丙烯酰胺凝胶Polyacrylamide gel 1％-5％1%-5%
维生素B6Vitamin B6 0％-1％0%-1%
香料spices 0％-1％0%-1%
纯水Pure water QSP100％QSP100%
(3)抗炎性促愈合乳剂(3) Anti-inflammatory promoting healing emulsion
原料/品牌名称Raw material/brand name ％%
PEG40硬脂酸酯PEG40 stearate 1％-5％1%-5%
PEG5硬脂酸甘油PEG5 Stearic Acid Glycerin 1％-5％1%-5%
地蜡Ozokerite 1％-5％1%-5%
单硬脂酸甘油酯Glyceryl Monostearate 1％-5％1%-5%
失水山梨醇硬脂酸酯Sorbitan Stearate 0％-2％0%-2%
鲸蜡醇Cetyl alcohol 0％-2％0%-2%
苹果酸氢酯醇Malate Alcohol 5％-20％5%-20%
维生素EVitamin E 0％-1％0%-1%
维生素B3Vitamin B3 1％-5％1%-5%
玛卡肽提取物Macatide Extract 1％-5％1%-5%
胎骆驼肝脏外泌体Fetal camel liver exosomes 0.01％-0.1％0.01%-0.1%
丁二醇Butanediol 1％-5％1%-5%
吡罗克酮乙醇胺Piroctone ethanolamine 0％-1％0%-1%
防腐剂preservative 0％-1％0%-1%
甘油glycerin 1％-10％1%-10%
黄原胶Xanthan gum 0％-1％0%-1%
锌PCAZinc PCA 0％-2％0%-2%
米淀粉Rice starch 1％-5％1%-5%
尼龙6Nylon 6 0％-2％0%-2%
聚丙烯酰胺凝胶Polyacrylamide gel 1％-5％1%-5%
维生素B6Vitamin B6 0％-1％0%-1%
香料spices 0％-1％0%-1%
纯水Pure water QSP100％QSP100%
(4)修复性抗衰老乳剂(4) Restorative anti-aging emulsion
原料/品牌名称Raw material/brand name ％%
PEG40硬脂酸酯PEG40 stearate 1％-5％1%-5%
PEG5硬脂酸甘油PEG5 Stearic Acid Glycerin 1％-5％1%-5%
地蜡Ozokerite 1％-5％1%-5%
单硬脂酸甘油酯Glyceryl Monostearate 1％-5％1%-5%
失水山梨醇硬脂酸酯Sorbitan Stearate 0％-2％0%-2%
鲸蜡醇Cetyl alcohol 0％-2％0%-2%
苹果酸氢酯醇Malate Alcohol 5％-20％5%-20%
维生素EVitamin E 0％-1％0%-1%
泛醇Panthenol 0％-5％0%-5%
玛卡肽提取物Macatide Extract 1％-5％1%-5%
胎骆驼血清外泌体Fetal Camel Serum Exosomes 0.01％-0.1％0.01%-0.1%
丁二醇Butanediol 1％-5％1%-5%
吡罗克酮乙醇胺Piroctone ethanolamine 0％-1％0%-1%
防腐剂preservative 0％-1％0%-1%
甘油glycerin 1％-10％1%-10%
黄原胶Xanthan gum 0％-1％0%-1%
锌PCAZinc PCA 0％-2％0%-2%
米淀粉Rice starch 1％-5％1%-5%
尼龙6Nylon 6 0％-2％0%-2%
聚丙烯酰胺凝胶Polyacrylamide gel 1％-5％1%-5%
维生素B6Vitamin B6 0％-1％0%-1%
香料spices 0％-1％0%-1%
纯水Pure water QSP100％QSP100%
(5)调节角质层的抗衰老乳剂(5) Anti-aging emulsion that regulates the stratum corneum
原料/品牌名称Raw material/brand name ％%
异壬酸异壬酯Isononyl isononanoate 1％-10％1%-10%
异鲸蜡醇硬脂酸酯Isocetyl Stearate 1％-10％1%-10%
PEG40硬脂酸酯PEG40 stearate 1％-5％1%-5%
PEG5硬脂酸甘油PEG5 Stearic Acid Glycerin 1％-5％1%-5%
防腐剂preservative 0％-1％0%-1%
C16-C18鲸蜡醇C16-C18 cetyl alcohol 0％-2％0%-2%
PPG/SMDI聚合物PPG/SMDI polymer 0％-1％0%-1%
水杨酸 Salicylic acid 0％-2％0%-2%
角鲨烷凝胶 Squalane Gel 0％-2％0%-2%
二辛醚Dioctyl Ether 1％-10％1%-10%
苹果酸氢酯醇Malate Alcohol 1％-10％1%-10%
向日葵提取物Sunflower extract 1％-10％1%-10%
氨丁三醇Tromethamine 1％-5％1%-5%
丁二醇Butanediol 1％-10％1%-10%
柠檬酸三钠Trisodium Citrate 0％-1％0%-1%
小核菌胶Sclerotium 0％-1％0%-1%
聚丙烯酰胺凝胶Polyacrylamide gel 0％-1％0%-1%
维生素CVitamin C 0％-2％0%-2%
甘氨酸Glycine 0％-2％0%-2%
香料spices 0％-1％0%-1%
维生素EVitamin E 0％-2％0%-2%
柠檬酸 Citric acid 0％-1％0%-1%
胎鹿骨髓外泌体Fetal Deer Bone Marrow Exosomes 0.01％-0.1％0.01%-0.1%
纯水Pure water QSP 100％QSP 100%
(6)抗老化抗皱乳剂(6) Anti-aging and anti-wrinkle emulsion
原料/品牌名称Raw material/brand name ％%
异壬酸异壬酯Isononyl isononanoate 1％-10％1%-10%
异鲸蜡醇硬脂酸酯Isocetyl Stearate 1％-10％1%-10%
PEG40硬脂酸酯PEG40 stearate 1％-5％1%-5%
PEG5硬脂酸甘油PEG5 Stearic Acid Glycerin 1％-5％1%-5%
防腐剂preservative 0％-1％0%-1%
C16-C18鲸蜡醇C16-C18 cetyl alcohol 0％-2％0%-2%
PPG/SMDI聚合物PPG/SMDI polymer 0％-1％0%-1%
水杨酸 Salicylic acid 0％-2％0%-2%
角鲨烷凝胶Squalane Gel 0％-2％0%-2%
二辛醚Dioctyl Ether 1％-10％1%-10%
苹果酸氢酯醇Malate Alcohol 1％-10％1%-10%
向日葵提取物Sunflower extract 1％-10％1%-10%
氨丁三醇Tromethamine 1％-5％1%-5%
丁二醇Butanediol 1％-10％1%-10%
柠檬酸三钠Trisodium Citrate 0％-1％0%-1%
小核菌胶 Sclerotium 0％-1％0%-1%
米淀粉Rice starch 1％-5％1%-5%
聚丙烯酰胺凝胶Polyacrylamide gel 0％-1％0%-1%
维生素CVitamin C 0％-2％0%-2%
甘氨酸Glycine 0％-2％0%-2%
香料spices 0％-1％0%-1%
维生素EVitamin E 0％-2％0%-2%
柠檬酸 Citric acid 0％-1％0%-1%
视黄醇Retinol 0％-5％0%-5%
玛卡肽提取物Macatide Extract 0.1％-5％0.1%-5%
胎羊肝脏外泌体Fetal Sheep Liver Exosomes 0.01％-0.1％0.01%-0.1%
纯水Pure water QSP 100％QSP 100%
(7)抗细菌抗衰老滚搽棒(stick roll-on)(7) Anti-bacterial and anti-aging stick roll-on
原料/品牌名称Raw material/brand name ％%
蓖麻油castor oil QSP 100％QSP 100%
油醇Oleyl alcohol 10％-20％10%-20%
棕榈油Palm oil 10％-20％10%-20%
聚甘油-3-蜂蜡Polyglycerol-3-beeswax 10％-20％10%-20%
小烛树蜡Candelilla wax 10％-20％10%-20%
锂蒙脱石Hectorite 10％-20％10%-20%
二氧化钛Titanium dioxide 0％-5％0%-5%
胎羊血清外泌体Fetal Sheep Serum Exosomes 0.01％-0.1％0.01%-0.1%
雪亚脂Seborin 0％-5％0%-5%
维生素EVitamin E 0％-1％0%-1%
(8)SPF 50+抗衰老防晒霜(8)SPF 50+ anti-aging sunscreen
原料/品牌名称Raw material/brand name ％%
B4纯水B4 pure water QSP100％QSP100%
氧化钛Titanium Oxide 10％-20％10%-20%
环五硅氧烷Cyclopentasiloxane 5％-15％5%-15%
棕榈酸辛酯Octyl palmitate 5％-15％5%-15%
C12-C15烷基苯甲酸酯C12-C15 alkyl benzoate 5％-10％5%-10%
戊酸癸酯Decyl valerate 5％-10％5%-10%
氧化锌Zinc oxide 5％-10％5%-10%
甘油glycerin 1％-5％1%-5%
PEG-45/十二烷甘醇共聚物PEG-45/Dodecyl Glycol Copolymer 1％-5％1%-5%
胎羊血清外泌体Fetal Sheep Serum Exosomes 0.01％-0.1％0.01%-0.1%
氯化钠Sodium chloride 1％-5％1%-5%
糊精棕榈酸酯Dextrin palmitate 1％-2％1%-2%
维生素EVitamin E 0％-2％0%-2%
防腐剂preservative 0％-2％0%-2%
羟丙基瓜尔胶Hydroxypropyl guar 0％-1％0%-1%
芦荟(Aloe vera)Aloe vera 0％-1％0%-1%
苏打碱液Soda lye 0％-1％0%-1%
EDTA2-NaEDTA2-Na 0％-1％0%-1%
葡萄糖酸锌Zinc gluconate 0％-1％0%-1%
(9)SPF 50+抗衰老防晒喷雾剂(9) SPF 50+ anti-aging sunscreen spray
Figure PCTCN2020130931-appb-000016
Figure PCTCN2020130931-appb-000016
Figure PCTCN2020130931-appb-000017
Figure PCTCN2020130931-appb-000017
(10)擦剂(10) Liniment
原料/品牌名称Raw material/brand name ％%
Arlatone duo(阿拉同恩多)Arlatone duo 5％-20％5%-20%
表皮剥脱剂Epidermal exfoliant 1％-10％1%-10%
小核菌胶Sclerotium 1％-10％1%-10%
防腐剂preservative 0％-1％0%-1%
辛酰甘氨酸Caprylylglycine 0％-1％0%-1%
苏打soda 0％-1％0%-1%
胎牛血清外泌体Fetal Bovine Serum Exosomes 0.01％-0.1％0.01%-0.1%
多价螯合剂Sequestrant 0％-1％0%-1%
柠檬酸 Citric acid 0％-1％0%-1%
纯水Pure water QSP 100％QSP 100%
香料spices 0％-1％0%-1%
本发明中涉及的未说明部分与现有技术相同或采用现有技术加以实现。申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The unspecified parts involved in the present invention are the same as the prior art or implemented by using the prior art. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, which does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims (8)

  1. 胎体来源的外泌体在皮肤调节产品中的用途，其特征在于，所述胎体指的是非人胎体。The use of carcass-derived exosomes in skin conditioning products is characterized in that the carcass refers to a non-human carcass.
  2. 根据权利要求1所述的胎体来源的外泌体在制备皮肤调节产品中的用途，其特征在于：The use of carcass-derived exosomes in the preparation of skin conditioning products according to claim 1, characterized in that:
    其中，所述外泌体提取自非人胎生动物胎体、非人新生体内脏器或生理性流体内。Wherein, the exosomes are extracted from the carcass of non-human viviparous animals, non-human neonatal internal organs or physiological fluids.
  3. 根据权利要求2所述的胎体来源的外泌体在制备皮肤调节产品中的用途，其特征在于：The use of carcass-derived exosomes in the preparation of skin conditioning products according to claim 2, characterized in that:
    其中，所述非人胎生动物胎体为非人胎生动物母体内的幼体和新出生动物；所述非人新生体内脏器为自然分娩后24小时内的新生动物的肝脏或骨髓；所述生理性流体包括非人新生体的血液、血浆或血清。Wherein, the fetus of the non-human viviparous animal is the larvae and the newborn animal in the mother of the non-human viviparous animal; the internal organ of the non-human viviparous animal is the liver or bone marrow of the newborn animal within 24 hours after natural delivery; the physiology Sexual fluids include blood, plasma or serum of non-human newborns.
  4. 根据权利要求1所述的胎体来源的外泌体在制备皮肤调节产品中的用途，其特征在于：The use of carcass-derived exosomes in the preparation of skin conditioning products according to claim 1, characterized in that:
    其中，所述皮肤调节产品包括抗衰老或增强皮肤弹性的护肤产品、皮肤病学用途产品以及预防由亚洲沙尘引起的皮肤问题的护肤产品；Wherein, the skin conditioning products include anti-aging or skin elasticity skin care products, dermatological products, and skin care products that prevent skin problems caused by Asian sand and dust;
    所述皮肤调节产品的形态包括直接涂抹式护肤产品或口服式皮肤调理产品。The form of the skin conditioning product includes a direct application type skin care product or an oral skin conditioning product.
  5. 根据权利要求4所述的胎体来源的外泌体在制备皮肤调节产品中的用途，其特征在于：The use of carcass-derived exosomes in the preparation of skin conditioning products according to claim 4, characterized in that:
    其中，所述皮肤病学用途产品为对抗痤疮或增强毛囊、抗脱发的皮肤病学用途产品；Wherein, the dermatological use product is a dermatological use product for fighting acne or enhancing hair follicles and preventing hair loss;
    所述预防由亚洲沙尘引起的皮肤问题的护肤产品为清除和排除皮肤毒素的产品、增强皮肤免疫性的产品或抑制活性氧的产生的产品；The skin care products for preventing skin problems caused by Asian sand dust are products that remove and eliminate skin toxins, products that enhance skin immunity, or products that inhibit the production of active oxygen;
    所述口服式皮肤调理产品包括含有该外泌体的乳制品、基于脂肪制品、饮料制品或谷物制品。The oral skin conditioning products include dairy products, fat-based products, beverage products or cereal products containing the exosomes.
  6. 根据权利要求5所述的胎体来源的外泌体在制备皮肤调节产品中的用途，其特征在于：The use of carcass-derived exosomes in the preparation of skin conditioning products according to claim 5, characterized in that:
    其中，所述抗衰老或增强皮肤弹性的护肤产品为促进细胞胶原蛋白表达、增加细胞肌原纤蛋白合成、维持干细胞干性以及刺激干细胞增殖的护肤产品中的一种或多种组合；Wherein, the anti-aging or skin elasticity-enhancing skin care product is one or more combinations of skin care products that promote cell collagen expression, increase cell myofibrillar protein synthesis, maintain stem cell stemness, and stimulate stem cell proliferation;
    所述对抗痤疮的皮肤病学用途产品为抑制角质形成细胞中炎症介质或基质蛋白酶的基因表达的产品、以及抑制角质形成细胞中VEGF合成和释放产品中的一种或两种组合；The anti-acne dermatological product is one or a combination of a product that inhibits inflammatory mediators or matrix protease gene expression in keratinocytes, and a product that inhibits VEGF synthesis and release in keratinocytes;
    所述增强毛囊、抗脱发的皮肤病学用途产品为促进毛囊乳头成纤维细胞中ATP合成 或增强线粒体代谢活性的产品、增加微毛囊的细胞存活力或减轻微毛囊膜损伤产品中的任意一种或两种；The dermatological product for enhancing hair follicles and anti-hair loss is any one of products that promote ATP synthesis in hair follicle papillary fibroblasts or enhance mitochondrial metabolic activity, increase cell viability of microhair follicles, or reduce microhair follicle membrane damage Or two;
    所述清除和排除皮肤毒素的产品为增强抗氧化酶NQO1和GSTT1表达的产品；The product for removing and eliminating skin toxins is a product that enhances the expression of antioxidant enzymes NQO1 and GSTT1;
    所述增强皮肤免疫性的产品为增加天然免疫因子皮肤抗微生物肽hBD3表达的产品；The product that enhances skin immunity is a product that increases the expression of the natural immune factor skin antimicrobial peptide hBD3;
    所述抑制活性氧产生的产品为增加抗氧化蛋白GPX1、过氧化氢酶TRX1和PRDX1表达的产品。The product that inhibits the production of reactive oxygen species is a product that increases the expression of antioxidant proteins GPX1, catalase TRX1 and PRDX1.
  7. 根据权利要求1所述的胎体来源的外泌体在皮肤调节产品中的用途，其特征在于，该外泌体的制备方法如下：The use of carcass-derived exosomes in skin conditioning products according to claim 1, wherein the preparation method of the exosomes is as follows:
    A、原料处理：通过下述(1)或(2)中的处理方式进行A. Raw material processing: through the following processing methods (1) or (2)
    (1)胎体脏器细胞分离和培养液收集(1) Separation of fetal organ cells and collection of culture fluid
    取无菌脏器，反复冲洗后，剪成直径为1-2mm的组织块；依次经2型胶原酶和胰酶消化后，将上清液离心，取细胞沉淀放入培养瓶，使用含10％胎牛血清DMEM/F12培养基，5％CO 2、37℃饱和湿度培养；去除非贴壁细胞，在贴壁细胞80％融合后更换无血清培养基培养48h，而后收集培养上清液，滤膜过滤得到富含外泌体的培养液； Take the sterile organs, wash them repeatedly, and cut them into tissue pieces with a diameter of 1-2mm; after digestion with type 2 collagenase and trypsin, the supernatant is centrifuged, and the cell pellet is placed in a culture flask. % Fetal calf serum DMEM/F12 medium, 5% CO 2 , 37°C saturated humidity culture; remove non-adherent cells, replace the serum-free medium after 80% fusion of adherent cells, culture for 48 hours, and then collect the culture supernatant. Filter membrane to obtain culture solution rich in exosomes;
    (2)生理性流体预处理(2) Physiological fluid pretreatment
    利用离心或过滤方法去除生理性流体内的细胞成分，而后滤膜过滤得到富含外泌体的流体；Use centrifugation or filtration to remove cellular components in physiological fluids, and then filter membranes to obtain fluids rich in exosomes;
    B、外泌体分离纯化B. Separation and purification of exosomes
    将(1)中的培养液或(2)中的流体依次经4℃，1000g离心10min；4℃，2000g离心20min；4℃，10000g离心30min；110000g离心90min后，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。Centrifuge the culture solution in (1) or the fluid in (2) at 4°C and 1000g for 10 minutes; 4°C and 2000g for 20 minutes; 4°C and 10000g for 30 minutes; after centrifugation at 110000g for 90 minutes, discard the supernatant and use phosphate Resuspend the pellet in the buffer; centrifuge again at 110000g for 90 min, discard the supernatant, resuspend the pellet in a small amount of phosphate buffer, and filter with a 0.45μm filter membrane to obtain exosomes.
  8. 含有权利要求1～7任一项所述的胎体来源外泌体的皮肤调节产品，其特征在于，以所述外泌体为活性成分，还包括美容和皮肤病学可接受的赋形剂。A skin conditioning product containing exosomes derived from carcass according to any one of claims 1 to 7, characterized in that the exosomes are used as active ingredients and also include cosmetic and dermatologically acceptable excipients .
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