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WO2024186031A1 - Composition comprising mast cell-conditioned medium for wound healing - Google Patents

Composition comprising mast cell-conditioned medium for wound healing Download PDF

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Publication number
WO2024186031A1
WO2024186031A1 PCT/KR2024/002453 KR2024002453W WO2024186031A1 WO 2024186031 A1 WO2024186031 A1 WO 2024186031A1 KR 2024002453 W KR2024002453 W KR 2024002453W WO 2024186031 A1 WO2024186031 A1 WO 2024186031A1
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WIPO (PCT)
Prior art keywords
mast cells
skin
damage
conditioned medium
ulcers
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PCT/KR2024/002453
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French (fr)
Korean (ko)
Inventor
우소연
조경아
권지윤
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Ewha Womans University
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Ewha Womans University
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Priority claimed from KR1020230165797A external-priority patent/KR20240135344A/en
Application filed by Ewha Womans University filed Critical Ewha Womans University
Publication of WO2024186031A1 publication Critical patent/WO2024186031A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • 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

Definitions

  • the present invention relates to a composition for preventing, improving or treating skin wounds, comprising a regulated medium of mast cells.
  • Mast cells are cells involved in inflammatory responses.
  • IgE antibodies When IgE antibodies are activated and crosslinked by other external antigens or allergens, inflammatory substances such as histamine, ⁇ -hexosaminidase, and leukotrienes that were trapped in the granules of mast cells are released into the plasma membrane through various signaling pathways. These released inflammatory substances activate receptors and cytokines that induce inflammation, thereby further aggravating the inflammation.
  • allergic diseases are generally known to be caused by genetic/immunological IgE antibodies and acquired factors, such as environmental and psychological factors, that are sensitized by contact with and exposure to antigens.
  • immunological factors are currently the target of treatment, and it can be said that it is possible to apply allergic disease treatments by controlling the release of these allergic substances to the cell outer membrane from an immunological perspective.
  • a composition for treating skin wounds including a conditioned medium of mast cells has excellent wound healing ability in a wound mouse model, and in particular, a conditioned medium of mast cells treated with toll-like receptor 2 and 6 agonists further improves wound healing ability.
  • the present invention seeks to provide a pharmaceutical composition for preventing or treating skin wounds, which comprises a conditioned medium of mast cells.
  • the present invention seeks to provide a composition for external application to the skin containing a conditioned medium of mast cells.
  • the present invention aims to provide a food composition for preventing or improving skin wounds, which contains a conditioned medium for mast cells.
  • the present invention seeks to provide a method for preventing or treating skin wounds, comprising a step of administering a conditioned medium of mast cells to a subject in need thereof.
  • the present invention seeks to provide a use of a conditioned medium of mast cells in the manufacture of a medicament for preventing or treating skin wounds.
  • the present invention seeks to provide a composition comprising a conditioned medium of mast cells for use in the prevention or treatment of skin wounds.
  • the present invention seeks to provide a use of a conditioned medium of mast cells for the prevention or treatment of skin wounds.
  • the present invention provides a pharmaceutical composition for preventing or treating skin wounds, which contains a conditioned medium of mast cells.
  • the “conditioned medium for mast cells” means a culture medium obtained by culturing mast cells from which mast cells have been removed, or a supernatant thereof, and can be used interchangeably with “mast cell culture supernatant,” “mast cell conditioned culture medium,” or “mast cell culture medium.”
  • prevention refers to any act of inhibiting or delaying the onset of skin wounds by administering the mast cell-modified medium according to the present invention.
  • treatment refers to any act of improving or beneficially changing the symptoms of skin wounds by administering the mast cell-modified medium according to the present invention.
  • improvement refers to any act of improving the bad condition of skin wounds by administering or ingesting the composition of the present invention to a subject.
  • the skin wound may be specifically any one selected from the group consisting of: temperature damage such as burns, thermal burns, thermal burn ulcers, and frostbite; trauma such as lacerations, abrasions, incisions, cuts, puncture wounds, and strangulations; blood vessel and lymphatic vessel damage such as Buerger's disease, lymphedema, and leg ulcers; post-surgical wounds such as chaffing and suture wounds; stomas, bedsores, pressure ulcers, diabetic ulcers/gangrenes, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, chemical damage, and other skin wounds.
  • temperature damage such as burns, thermal burns, thermal burn ulcers, and frostbite
  • trauma such as lacerations, abrasions, incisions, cuts, puncture wounds, and strangulations
  • blood vessel and lymphatic vessel damage such as Buerger's disease, lymphedema, and leg ulcers
  • the mast cell may be a human-derived mast cell.
  • the mast cell-conditioned medium of the present invention can promote cell migration of skin cells, preferably skin keratinocytes.
  • the conditioned medium for mast cells includes a conditioned medium for cells isolated from an individual, cultured, and manufactured through special manipulation, and can be used as a medicine for the purposes of treatment, diagnosis, and prevention, and can be used on skin wounds.
  • the mast cell culture medium may be a culture medium obtained by culturing mast cells from which mast cells have been removed, a culture supernatant thereof, a concentrate thereof, or a lyophilized product thereof.
  • the mast cell culture solution can be obtained by subculturing mast cells in a serum medium and then culturing them in a serum-free medium.
  • Mast cell culture fluid can be obtained by subculturing mast cells in serum-free medium.
  • Mast cells can be conventionally cultured using a cell culture medium.
  • Mast cell culture fluid is obtained by subculturing mast cells in a serum medium and then subculturing them in a serum-free medium, and the supernatant obtained after removing mast cells and macromolecules by centrifugation or filtration using a filter can be used as is.
  • the obtained supernatant can be used as is or used as a concentrate obtained by concentrating it.
  • the above serum medium is a medium suitable for maintaining and storing a cell type identical to mast cells, and may be an IMDM medium supplemented with serum.
  • the serum may be, but is not limited to, fetal bovine serum (FBS), and may be 1 to 20 wt% based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents, and mycoplasma inhibitors may be included, and these may be 1 to 5 wt% based on the total weight of the medium.
  • FBS fetal bovine serum
  • antibiotics, antifungal agents, and mycoplasma inhibitors may be included, and these may be 1 to 5 wt% based on the total weight of the medium.
  • Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include, but are not limited to, gentamicin, ciprofloxacin, and tylosin.
  • Culturing in the above serum-free medium can be performed after removing the culture medium in the serum medium and washing the cells with a phosphate buffer solution.
  • the serum medium may be IMDM medium supplemented with 10% fetal bovine serum and 1% antibiotics.
  • the serum-free medium may be IMDM medium.
  • the mast cell culture solution can be obtained by a step of removing cells by centrifuging or filtering the mast cell culture solution.
  • the mast cell may be a mast cell stimulated with a toll-like receptor 2 and 6 agonist.
  • the above toll-like receptor 2 and 6 agonists are agonists that activate toll-like receptor 2 and 6 of mast cells and promote degranulation, and may be at least one selected from the group consisting of FSL-1, MALP-2, CBLB613, deacylated lipoprotein, and deacylated lipopeptide.
  • the mast cells stimulated with the above toll-like receptor 2 and 6 agonists may be mast cells pretreated with the toll-like receptor 2 and 6 agonists for 1 to 100 hours, preferably 24 to 72 hours. Additionally, the mast cells may be pretreated with the toll-like receptor 2 and 6 agonists at a concentration of 10 to 200 ng/ml.
  • the mast cell conditioning medium may contain tryptase.
  • the conditioned medium of mast cells stimulated with the toll-like receptor 2 and 6 agonists may contain tryptase.
  • the above tryptase is secreted from mast cells or mast cells stimulated by toll-like receptor 2 and 6 agonists and is a major factor in promoting cell migration of human skin keratinocytes.
  • the pharmaceutical composition of the present invention can be formulated into various dosage forms, such as a solution, suspension, emulsion, lotion, ointment, and lyophilisate, according to conventional methods.
  • the pharmaceutical composition of the present invention can be formulated into a unit dosage form pharmaceutical preparation suitable for administration into a patient's body according to a conventional method in the pharmaceutical field and administered, and the preparation contains an effective dosage amount by one or more administrations.
  • Preferred dosage forms for this purpose are parenteral administration preparations such as injections and infusions.
  • the pharmaceutical composition for preventing or treating skin wounds can contain a conventional inert carrier and diluent that are pharmaceutically acceptable.
  • Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, excipients such as starches, sugars, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, and the like, binders such as gelatin, alginates, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol, disintegrants such as povidone, crospovidone, and surfactants such as polysorbates, cetyl alcohol, and glycerol.
  • excipients such as starches, sugars, and mannitol
  • fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, and the like
  • binders such as gelatin, alginates, and polyvinyl pyrrolidone
  • the pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the conditioned medium of mast cells and the recipient to be transplanted therewith.
  • Diluents may include, but are not limited to, saline, an aqueous buffer, a solvent, and/or a dispersion media.
  • preservatives, analgesics, solubilizers or stabilizers may be additionally included, and in the case of topical administration preparations, bases, excipients, lubricants or preservatives may be additionally included.
  • the compositions of the present invention may be used unfrozen or frozen for later use. If frozen, standard cryopreservatives (e.g., DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologics)) may be added to the cell population prior to freezing.
  • standard cryopreservatives e.g., DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologic
  • the administration can be various administration methods such as oral administration, non-surgical administration using a catheter, and injection or transplantation after incision of the disease site.
  • the dosage may vary depending on the degree of concentration, but can be administered once or in several divided doses at 10 ⁇ l/kg to 1 ml/kg.
  • the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage does not limit the scope of the present invention in any way.
  • the present invention also provides a composition for external application to the skin comprising a regulated medium of mast cells.
  • the external skin composition of the present invention is not particularly limited as long as it is a formulation that can directly administer an active ingredient to the local surface of the skin, and for example, it can be used as a preparation such as an ointment, an ointment patch, a liquid (suspension, emulsion, lotion, etc.), a cataplasm, a tape, an external powder, and an aerosol.
  • a preparation such as an ointment, an ointment patch, a liquid (suspension, emulsion, lotion, etc.), a cataplasm, a tape, an external powder, and an aerosol.
  • any compounding ingredients used in a typical external skin composition can be used.
  • bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin, and squalene; solvents and solubilizers such as oleic acid, isopropyl myristate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylated hydroxyanisole; preservatives such as p-hydroxybenzoate; moisturizers such as glycerin, propylene glycol
  • tackifiers such as polyacrylic acid and polyacrylic acid copolymers, crosslinkers such as aluminum sulfate, potassium aluminum sulfate, aluminum chloride, magnesium aluminometasilicate and dihydroxy aluminum acetate, thickeners such as sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose and hydroxypropyl methylcellulose, polyhydric alcohols such as glycerin, polyethylene glycol (macrogol), propylene glycol and 1,3-butanediol, surfactants such as polyoxyethylene derivatives, fragrances such as 1-menthol, preservatives such as p-hydroxybenzoate, purified water and other suitable additives can be blended.
  • crosslinkers such as aluminum sulfate, potassium aluminum sulfate, aluminum chloride, magnesium aluminometasilicate and dihydroxy aluminum
  • adhesives such as styrene-isoprene-styrene block copolymers and acrylic resins, tackifiers such as alicyclic saturated-hydrocarbon resins, rosin resins, and terpene resins, softeners such as liquid rubber and liquid paraffin, antioxidants such as dibutylhydroxytoluene, polyhydric alcohols such as propylene glycol, absorption accelerators such as oleic acid, surfactants such as polyoxyethylene derivatives, and other appropriate additives can be blended.
  • a polymer capable of containing water such as sodium polyacrylate or polyvinyl alcohol and a small amount of purified water can be added to produce an aqueous tape.
  • excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, and other appropriate additives may be blended.
  • solvents and solubilizers such as oleic acid, isopropyl myristate, diisopropyl adipate, diisopropyl sebacate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, vegetable oils, etc.; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole; preservatives such as p-hydroxy benzoate; glycerin, propylene glycol, Humectants such as sodium hyaluronate, polyoxyethylene derivatives, glycerol esters of fatty acids, sucrose esters of fatty acids, etc.
  • solvents and solubilizers such as oleic acid, isopropyl myristate, diisopropyl
  • the external skin composition of the present invention may be formulated for topical application to the skin by containing a cosmetically or dermatologically acceptable medium or base.
  • a cosmetically or dermatologically acceptable medium or base may be any formulation suitable for topical application, for example, in the form of a solution, gel, solid, anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule, or an ionic (liposome) and nonionic vesicle dispersion, or in the form of a cream, a skin, a lotion, a powder, an ointment, a spray, or a concealer stick. It may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.
  • the skin external composition of the present invention may contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics.
  • the adjuvants are introduced in an amount commonly used in the fields of cosmetology or dermatology.
  • the present invention also provides a food composition for preventing or improving skin wounds, which comprises a regulated medium of mast cells.
  • the present invention can be generally used as a commonly used food.
  • the food composition of the present invention can be used as a health functional food.
  • the above "health functional food” means a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to the Health Functional Food Act, and “functionality” means that it is consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.
  • the food composition of the present invention may contain conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
  • Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.
  • the food composition of the present invention may contain a conditioned medium for mast cells in an amount of 0.01 to 95 wt%, preferably 5 to 90 wt%, based on the total weight of the composition.
  • the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc., for the purpose of preventing and/or improving skin wounds.
  • hard capsules can be manufactured by filling a mixture of the conditioned medium of mast cells according to the present invention and additives such as excipients into a conventional hard capsule
  • soft capsules can be manufactured by filling a mixture of the food composition according to the present invention and additives such as excipients into a capsule base such as gelatin.
  • the soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.
  • the present invention provides a method for preventing or treating skin wounds, comprising a step of administering a conditioned medium of mast cells to a subject in need thereof.
  • the terms “adjusted medium for mast cells,” “skin wound,” “administration,” etc. are the same as those described above.
  • the above subject refers to an animal, and may typically be a mammal that can show a beneficial effect by treatment using the mast cell-modified medium of the present invention.
  • Preferred examples of such subjects may include primates such as humans.
  • such subjects may include all subjects having symptoms of skin wounds or at risk of having such symptoms.
  • the present invention also provides the use of a conditioned medium of mast cells in the manufacture of a medicament for the prevention or treatment of skin wounds.
  • the present invention also provides a composition comprising a conditioned medium of mast cells for use in the prevention or treatment of skin wounds.
  • the present invention also provides the use of a mast cell-conditioned medium for the prevention or treatment of skin wounds.
  • composition for preventing, improving or treating skin wounds of the present invention comprises a mast cell-regulated medium, promotes cell migration of human skin keratinocytes and has an excellent effect of improving inflammation, especially during the wound healing process, and thus has high value as a wound treatment agent.
  • Figure 1 shows the results of a scratch assay image confirming the effect of human-derived mast cell-conditioned medium on human skin keratinocyte migration.
  • Figure 2 shows the results of confirming the effect of human-derived mast cell-conditioned medium on human skin keratinocyte migration through scratch assay quantification. (**p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001)
  • Figure 3 shows the results of a Transwell migration assay image confirming the effect of conditioned medium containing human-derived mast cells on the migration of human skin keratinocytes.
  • Figure 4 shows the results of confirming the effect of conditioned medium of human-derived mast cells on the migration of human skin keratinocytes through Transwell migration assay quantification. (**p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001)
  • Figure 5 shows the results of a scratch assay image showing the effect of conditioned media of human mast cells treated with toll-like receptor 2 and 6 agonists on the migration of human skin keratinocytes.
  • Figure 6 shows the results of the Scratch assay quantification to determine the effect of conditioned media of human mast cells treated with toll-like receptor 2 and 6 agonists on human skin keratinocyte migration. (**p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001)
  • Figure 7 shows the results of comparing the wound healing effect through images of the healing process of three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM).
  • Figure 8 shows the results of comparing the wound healing effect through quantification of the degree of healing in three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM). (**p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001)
  • Figure 9 shows the results of H&E staining on skin tissues of three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM).
  • Figure 10 shows the results of ELISA assay for tryptase content in conditioned medium derived from mast cells (HMC-1 CM), conditioned medium of mast cells stimulated with TLR2/6 agonist (T2/6 HMC-1 CM), conditioned medium derived from skin keratinocyte cell line (HaCaT CM), conditioned medium derived from monocyte cell line (THP-1 CM), and IMDM medium (IMDM) as a complete negative control. (****p ⁇ 0.0001)
  • Figure 11 shows the results of a scratch assay image confirming the effect of tryptase on human skin keratinocyte migration.
  • Figure 12 shows the results of the Scratch assay quantification to confirm the effect of tryptase on human skin keratinocyte migration. (****p ⁇ 0.0001)
  • conditioned media from human mast cells human mast cells were cultured in IMDM containing 10% serum and 1% penicillin/streptomycin. When the confluency reached 80% in a 100 mm cell culture plate, the cells were harvested and serum-free IMDM medium was added to 1 x 10 7 cells again. After 48 hours, the medium was harvested, centrifuged at 5300 xg for 1 hour and 30 minutes, and the supernatant was filtered. The filtered medium was concentrated to 1/5 using a centrifugal filter (cut-off of 3K, Millipore) and used in the experiment or stored at -80°C. In this way, mast cell conditioned media was prepared.
  • TLR2/6 agonist treatment 1x107 cells were treated with TLR2/6 agonist at a concentration of 50 ng/ml and cultured in serum IMDM medium for 24 hours. The supernatant was then removed by centrifugation, and 1x107 cells were cultured in serum-free IMDM medium for 48 hours, the medium was removed, centrifuged at 5300 xg for 1 hour 30 minutes, and the supernatant was filtered. The filtered medium was concentrated to 1/5 using a centrifugal filter (cut-off of 3K, Millipore) and used in the experiment or stored at -80°C. Through this, the conditioned medium of mast cells pretreated with TLR2/6 agonist was prepared.
  • Scratch assay was performed to confirm the cell migration promoting effect of human-derived mast cell-conditioned medium.
  • human skin keratinocytes were cultured in a 12-well cell culture dish at 37°C and 5% CO2 in a cell incubator until 90% confluency.
  • a scratch wound to which cells did not attach was created by scratching the bottom of the culture dish using a sterilized 200 ⁇ l micropipette tip.
  • 100 ⁇ l of the human-derived mast cell-conditioned medium obtained in Example 1 was added.
  • 100 ⁇ l of serum-free IMDM medium was added instead of the mast cell-conditioned medium.
  • Wound images were obtained at 0, 24, and 48 hours after the addition of the medium, and the results are shown in Fig. 1.
  • the wound opening gap was measured in each image, and the results are shown in Fig. 2.
  • a Transwell migration assay was performed using a Transwell plate with a porous membrane with a diameter of 0.4 um.
  • Scratch assay was performed to confirm the cell migration-promoting effect of human mast cell-conditioned medium treated with toll-like receptor 2 and 6 agonists.
  • the experimental method was the same as in Example 2, but 100 ⁇ l of human mast cell-conditioned medium treated with toll-like receptor 2 and 6 agonists was added to the scratch wound.
  • 100 ⁇ l of serum-free IMDM medium was added for the control group. Wound images were obtained 0 and 48 hours after the medium addition, and the results are shown in Fig. 5. In addition, the wound opening gap was measured in each image, and the results are shown in Fig. 6.
  • mice were divided into three groups as shown in Table 1 below and the experiment was conducted.
  • the corresponding substances were additionally injected into each group, and the body weights and wound healing processes of the mice were observed for a total of 12 days.
  • the wound images of the mice by group during the experimental period are shown in Fig. 7.
  • the wound opening gap was measured in each image, and this is shown in Fig. 8.
  • H&E staining was performed after the end of the animal experiment.
  • the mast cell-conditioned medium had an excellent effect on the treatment of skin wounds by promoting cell migration of keratinocytes and improving inflammation. In addition, it showed improved efficacy by treatment with the therapeutic agent in terms of epithelialization and granulation tissue formation.
  • ELISA assays were performed using conditioned medium derived from mast cells (HMC-1 CM), conditioned medium from mast cells stimulated with a TLR2/6 agonist (T2/6 HMC-1 CM), conditioned medium derived from a skin keratinocyte cell line (HaCaT CM), conditioned medium derived from a monocyte cell line (THP-1 CM), and IMDM medium (IMDM) as a complete negative control.
  • HMC-1 CM conditioned medium derived from mast cells
  • T2/6 HMC-1 CM conditioned medium from mast cells stimulated with a TLR2/6 agonist
  • HaCaT CM conditioned medium derived from a skin keratinocyte cell line
  • TTP-1 CM conditioned medium derived from a monocyte cell line
  • IMDM IMDM medium
  • the conditioned medium of each cell was diluted 1/50, and the tryptase concentration in the sample was measured using a Human Tryptase Elisa kit (Innovative Research, Inc.), and the results are shown in Figure 10.
  • the Scratch assay was performed in the same manner as in Example 2, but the experimental groups were composed of a control group, a mast cell-conditioned medium (HMC-1 CM) group, a mast cell-conditioned medium stimulated with toll-like receptor 2 and 6 agonists (T2/6 HMC-1 CM) group, and groups treated with 20 ug/ml of tryptase inhibitor (APC 366, trifluoroacetate salt, Sigma-Aldrich, SML2490) in each group (control+Inhibitor; HMC-1 CM+Inhibitor; and T2/6 HMC-1 CM+Inhibitor).
  • HMC-1 CM mast cell-conditioned medium
  • T2/6 HMC-1 CM mast cell-conditioned medium stimulated with toll-like receptor 2 and 6 agonists
  • APC 366 trifluoroacetate salt, Sigma-Aldrich, SML2490
  • the HMC-1 group and the T2/6 HMC-1 CM group showed that the gap between the wounds quickly closed over time, while the groups treated with tryptase inhibitor (HMC-1 CM+Inhibitor; and T2/6 HMC-1 CM+Inhibitor) showed a relatively slow rate of closing the gap between the wounds. In other words, it appeared that the effect of the conditioned medium on promoting HaCaT cell migration was limited when treated with tryptase inhibitor.
  • the mast cell-conditioned medium of the present invention promotes the migration of skin epithelial cells in a tryptase action-dependent manner, and it was confirmed that tryptase acts as a major factor in promoting cell migration.

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Abstract

The present invention relates to a composition for skin wound healing, comprising a mast cell-conditioned medium as an active ingredient. A mast cell-conditioned medium or a conditioned medium of mast cells stimulated with toll-like receptor 2 and 6 agonists, of the present invention, has the effect of promoting cell migration of human dermal keratinocytes and exhibits an excellent treatment effect during wound healing, especially in the inflammatory phase.

Description

비만세포의 조정 배지를 포함하는 창상 치료용 조성물Composition for wound healing comprising a regulated medium of mast cells

본 발명은 비만세포의 조정 배지를 포함하는 피부 창상 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating skin wounds, comprising a regulated medium of mast cells.

비만세포(mast cell)는 염증 반응에 관여하는 세포로서, IgE 항체가 활성화되고 외부의 다른 항원이나 알레르겐(allergen)에 의해서 교차(crosslink)되면, 여러 가지 다양한 신호기작(signalling pathway)에 의해 비만세포의 과립 안에 포집되어 있던 히스타민, 베타-헥소사미니다아제(β-hexosaminidase), 류코트리엔과 같은 염증유발 물질 등을 세포 외막(plasma membrane)으로 방출한다. 이렇게 방출된 염증유발 물질들은 염증을 유발하는 수용체와 사이토카인 등을 활성화시킴으로써 그 염증을 더욱 심화시키는 작용을 한다.Mast cells are cells involved in inflammatory responses. When IgE antibodies are activated and crosslinked by other external antigens or allergens, inflammatory substances such as histamine, β-hexosaminidase, and leukotrienes that were trapped in the granules of mast cells are released into the plasma membrane through various signaling pathways. These released inflammatory substances activate receptors and cytokines that induce inflammation, thereby further aggravating the inflammation.

특히, 비만세포에서 이러한 물질들의 방출은 알레르기성 질환 등과 밀접한 연관이 있으며, 알레르기성 질환은 보편적으로 유전학적/면역학적으로 IgE 항체가 관련되고 후천적 요소 즉 환경적, 정신적 요인 인자들이 항원에 접촉, 노출에 의해 감작되어 발병되는 것으로 알려져 있다. 이 중 면역학적 요인은 현재 치료의 타겟이 되고 있으며, 면역학적 측면에서 이러한 알레르기성 물질들의 세포 외막으로의 방출을 제어함으로써 알레르기성 질환 치료제로 적용 가능하다고 할 것이다.In particular, the release of these substances from mast cells is closely related to allergic diseases, etc., and allergic diseases are generally known to be caused by genetic/immunological IgE antibodies and acquired factors, such as environmental and psychological factors, that are sensitized by contact with and exposure to antigens. Among these, immunological factors are currently the target of treatment, and it can be said that it is possible to apply allergic disease treatments by controlling the release of these allergic substances to the cell outer membrane from an immunological perspective.

그러나 현재까지 이들 비만세포의 방출 물질들이 사람 피부 각질 세포의 세포 이동을 촉진시킴으로써 피부 창상 치료에 우수한 효과가 있다는 것은 알려지지 않았다.However, it has not been known until now that these substances released by mast cells are effective in treating skin wounds by promoting cell migration of human skin keratinocytes.

이에 본 발명자들은 비만세포의 조정배지를 포함하는 피부 창상 치료용 조성물이 창상 마우스 모델에서 우수한 상처 회복 능력을 가지며, 특히 톨 유사 수용체 2와 6 작용제를 처리한 비만세포의 조정배지는 창상 치유 능력이 더욱 향상되는 것을 확인함으로써 본 발명을 완성하였다.Accordingly, the inventors of the present invention have completed the present invention by confirming that a composition for treating skin wounds including a conditioned medium of mast cells has excellent wound healing ability in a wound mouse model, and in particular, a conditioned medium of mast cells treated with toll-like receptor 2 and 6 agonists further improves wound healing ability.

본 발명은 비만세포의 조정배지를 포함하는 피부 창상 예방 또는 치료용 약학 조성물을 제공하고자 한다. The present invention seeks to provide a pharmaceutical composition for preventing or treating skin wounds, which comprises a conditioned medium of mast cells.

본 발명은 비만세포의 조정배지를 포함하는 피부 외용제 조성물을 제공하고자 한다. The present invention seeks to provide a composition for external application to the skin containing a conditioned medium of mast cells.

본 발명은 비만세포의 조정배지를 포함하는 피부 창상 예방 또는 개선용 식품 조성물을 제공하고자 한다. The present invention aims to provide a food composition for preventing or improving skin wounds, which contains a conditioned medium for mast cells.

본 발명은 비만세포의 조정배지를 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 피부 창상의 예방 또는 치료 방법을 제공하고자 한다.The present invention seeks to provide a method for preventing or treating skin wounds, comprising a step of administering a conditioned medium of mast cells to a subject in need thereof.

본 발명은 피부 창상의 예방 또는 치료를 위한 약제의 제조에서 비만세포의 조정배지의 용도를 제공하고자 한다.The present invention seeks to provide a use of a conditioned medium of mast cells in the manufacture of a medicament for preventing or treating skin wounds.

본 발명은 피부 창상의 예방 또는 치료에 사용하기 위한 비만세포의 조정배지를 포함하는 조성물을 제공하고자 한다.The present invention seeks to provide a composition comprising a conditioned medium of mast cells for use in the prevention or treatment of skin wounds.

본 발명은 피부 창상의 예방 또는 치료를 위한 비만세포의 조정배지의 용도를 제공하고자 한다.The present invention seeks to provide a use of a conditioned medium of mast cells for the prevention or treatment of skin wounds.

본 발명은 비만세포의 조정배지를 포함하는 피부 창상 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating skin wounds, which contains a conditioned medium of mast cells.

본 발명에 있어서, "비만세포의 조정배지"는 비만세포를 배양하여 수득된 배양액으로부터 비만세포가 제거된 배양액 또는 이의 상층액을 의미하며, "비만세포 배양 상층액", "비만세포 조건 배양액" 또는 "비만세포 배양 배지"와 호환적으로 사용될 수 있다.In the present invention, the “conditioned medium for mast cells” means a culture medium obtained by culturing mast cells from which mast cells have been removed, or a supernatant thereof, and can be used interchangeably with “mast cell culture supernatant,” “mast cell conditioned culture medium,” or “mast cell culture medium.”

본 발명에서 용어 “예방”이란 본 발명에 따른 비만세포 조정배지의 투여로 피부 창상의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 비만세포 조정배지의 투여로 피부 창상의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명에서 용어 "개선"이란 본 발명의 조성물을 개체에 투여하거나 섭취시켜 피부 창상의 나쁜 상태를 좋게 하는 모든 행위를 의미한다.In the present invention, the term “prevention” refers to any act of inhibiting or delaying the onset of skin wounds by administering the mast cell-modified medium according to the present invention. In the present invention, the term “treatment” refers to any act of improving or beneficially changing the symptoms of skin wounds by administering the mast cell-modified medium according to the present invention. In the present invention, the term “improvement” refers to any act of improving the bad condition of skin wounds by administering or ingesting the composition of the present invention to a subject.

본 발명에 있어서, 피부 창상은 구체적으로, 화상, 열화상, 열화상성 궤양, 동상 등의 온도 손상, 열상(Laceration), 찰과상, 절창, 자상, 좌창(挫創), 교창(咬創) 등의 외상, 버거병, 림프부종, 하퇴궤양 등의 혈관 및 림프관 손상, 채피창(採皮創), 봉합창 등의 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 화학적 손상, 및 기타의 피부창상으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the present invention, the skin wound may be specifically any one selected from the group consisting of: temperature damage such as burns, thermal burns, thermal burn ulcers, and frostbite; trauma such as lacerations, abrasions, incisions, cuts, puncture wounds, and strangulations; blood vessel and lymphatic vessel damage such as Buerger's disease, lymphedema, and leg ulcers; post-surgical wounds such as chaffing and suture wounds; stomas, bedsores, pressure ulcers, diabetic ulcers/gangrenes, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, chemical damage, and other skin wounds.

본 발명의 일 실시양태에 따르면, 상기 비만세포는 사람 유래 비만세포일 수 있다.According to one embodiment of the present invention, the mast cell may be a human-derived mast cell.

본 발명의 비만세포의 조정배지는 피부 세포, 바람직하게 피부 각질 세포의 세포 이동을 촉진시킬 수 있다.The mast cell-conditioned medium of the present invention can promote cell migration of skin cells, preferably skin keratinocytes.

본 발명에 있어서, 비만세포의 조정배지는 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포의 조정 배지를 포함하며, 치료, 진단 및 예방의 목적으로 사용되는 의약품으로 사용되어 피부 창상에 사용될 수 있다. In the present invention, the conditioned medium for mast cells includes a conditioned medium for cells isolated from an individual, cultured, and manufactured through special manipulation, and can be used as a medicine for the purposes of treatment, diagnosis, and prevention, and can be used on skin wounds.

비만세포 배양액은 비만세포를 배양하여 수득된 배양액으로부터 비만세포가 제거된 배양액 또는 이의 배양 상층액 또는 이의 농축물이거나 이의 동결건조물일 수 있다.The mast cell culture medium may be a culture medium obtained by culturing mast cells from which mast cells have been removed, a culture supernatant thereof, a concentrate thereof, or a lyophilized product thereof.

본 발명에 있어서, 비만세포 배양액은 비만세포를 혈청 배지에서 계대 배양한 후, 무혈청 배지에서 배양하여 수득될 수 있다.In the present invention, the mast cell culture solution can be obtained by subculturing mast cells in a serum medium and then culturing them in a serum-free medium.

비만세포 배양액은 비만세포를 무혈청 배지에서 계대 배양하여 수득될 수 있다.Mast cell culture fluid can be obtained by subculturing mast cells in serum-free medium.

비만세포는 세포 배양용 배지를 사용하여 통상적으로 배양될 수 있다. 비만세포 배양액은 비만세포를 혈청 배지에서 계대배양한 후, 무혈청 배지에서 계대 배양하여 수득되고, 이를 그대로 사용하거나 또는 원심분리나 필터를 이용한 여과에 의해 비만세포 및 거대분자를 제거한 후에 수득된 상층액을 사용할 수 있다. 또한, 수득된 상층액은 그대로 사용하거나 또는 농축하여 수득된 농축물로 사용할 수 있다.Mast cells can be conventionally cultured using a cell culture medium. Mast cell culture fluid is obtained by subculturing mast cells in a serum medium and then subculturing them in a serum-free medium, and the supernatant obtained after removing mast cells and macromolecules by centrifugation or filtration using a filter can be used as is. In addition, the obtained supernatant can be used as is or used as a concentrate obtained by concentrating it.

비만세포의 배양을 위한 배양용 배지 및 배양 조건은 본 발명이 속하는 기술분야에서 잘 알려져 있으며, 통상의 지식을 가진 자가 적절하게 선택하거나 변형하여 이용할 수 있다.Culture media and culture conditions for culturing mast cells are well known in the technical field to which the present invention belongs, and can be appropriately selected or modified and used by a person skilled in the art.

상기 혈청 배지는 비만세포와 동일한 세포형을 유지하고 보관하는데 적합한 배지로서, 혈청이 보충된 IMDM 배지일 수 있다. 혈청은 우태아혈청(FBS)일 수 있으나, 이에 한정되지 않으며, 혈청 배지의 총 중량에 대해 1 내지 20 중량%일 수 있다. 필요에 따라, 항생제, 항진균제 및 마이코플라스마 억제제 등을 포함할 수 있으며, 이들은 배지의 총 중량에 대해 1 내지 5 중량%일 수 있다. 항생제는 페니실린-스트렙토마이신 등 세포 배양에서 통상적으로 사용되는 항생제를 포함하고, 항진균제는 암포테리신-B(fungizone) 등을 포함하며, 마이코플라스마 억제제는 겐타마이신, 시프로플록사신, 타일로신 등을 포함하나, 이에 한정되지 않는다.The above serum medium is a medium suitable for maintaining and storing a cell type identical to mast cells, and may be an IMDM medium supplemented with serum. The serum may be, but is not limited to, fetal bovine serum (FBS), and may be 1 to 20 wt% based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents, and mycoplasma inhibitors may be included, and these may be 1 to 5 wt% based on the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and mycoplasma inhibitors include, but are not limited to, gentamicin, ciprofloxacin, and tylosin.

상기 무혈청 배지 중에서의 배양은 혈청 배지에서의 배양액을 제거하고, 세포를 인산염 완충용액으로 세척한 후에 수행될 수 있다.Culturing in the above serum-free medium can be performed after removing the culture medium in the serum medium and washing the cells with a phosphate buffer solution.

바람직하게, 상기 혈청 배지는 10% 우태아혈청 및 1%의 항생제가 보충된 IMDM 배지일 수 있다.Preferably, the serum medium may be IMDM medium supplemented with 10% fetal bovine serum and 1% antibiotics.

바람직하게, 상기 무혈청 배지는 IMDM 배지일 수 있다.Preferably, the serum-free medium may be IMDM medium.

본 발명의 일 실시양태에 따르면, 비만세포 배양액은 비만세포 배양액을 원심분리나 여과시켜 세포를 제거하는 단계에 의해 수득될 수 있다.According to one embodiment of the present invention, the mast cell culture solution can be obtained by a step of removing cells by centrifuging or filtering the mast cell culture solution.

본 발명의 일 실시양태에 따르면, 비만세포는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포일 수 있다. According to one embodiment of the present invention, the mast cell may be a mast cell stimulated with a toll-like receptor 2 and 6 agonist.

상기 톨 유사 수용체 2와 6 작용제는 비만세포의 톨 유사 수용체 2와 6을 활성화시키고 탈과립을 촉진하는 작용제(agonist)로써, FSL-1, MALP-2, CBLB613, 디아실화된 지질단백질 및 디아실화된 리포펩티드로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The above toll-like receptor 2 and 6 agonists are agonists that activate toll-like receptor 2 and 6 of mast cells and promote degranulation, and may be at least one selected from the group consisting of FSL-1, MALP-2, CBLB613, deacylated lipoprotein, and deacylated lipopeptide.

상기 톨 유사 수용체 2와 6 작용제로 자극된 비만세포는 비만 세포를 1 내지 100 시간 동안, 바람직하게 24시간 내지 72시간 동안 톨 유사 수용체 2와 6 작용제로 전처리한 것일 수 있다. 또한, 톨 유사 수용체 2와 6 작용제를 10 내지 200 ng/ml 농도로 전처리한 것일 수 있다.The mast cells stimulated with the above toll-like receptor 2 and 6 agonists may be mast cells pretreated with the toll-like receptor 2 and 6 agonists for 1 to 100 hours, preferably 24 to 72 hours. Additionally, the mast cells may be pretreated with the toll-like receptor 2 and 6 agonists at a concentration of 10 to 200 ng/ml.

본 발명의 일 실시양태에 따르면, 상기 비만세포 조정배지는 트립타제(tryptase)를 포함할 수 있다.According to one embodiment of the present invention, the mast cell conditioning medium may contain tryptase.

본 발명의 일 실시양태에 따르면, 상기 톨 유사 수용체 2와 6 작용제로 자극된 비만세포의 조정배지는 트립타제(tryptase)를 포함할 수 있다.According to one embodiment of the present invention, the conditioned medium of mast cells stimulated with the toll-like receptor 2 and 6 agonists may contain tryptase.

상기 트립타제(tryptase)는 비만세포 또는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포에서 분비되며 사람 피부 각질세포의 세포이동 촉진의 주요 인자이다.The above tryptase is secreted from mast cells or mast cells stimulated by toll-like receptor 2 and 6 agonists and is a major factor in promoting cell migration of human skin keratinocytes.

본 발명의 약학 조성물은 통상의 방법에 따라 액제, 현탁액, 에멀젼, 로션, 연고, 동결건조제 등 다양한 제형으로 제제화될 수 있다.The pharmaceutical composition of the present invention can be formulated into various dosage forms, such as a solution, suspension, emulsion, lotion, ointment, and lyophilisate, according to conventional methods.

본 발명의 약학 조성물은 약학적 분야의 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 약학적 제제로 제형화시켜 투여할 수 있으며, 상기 제제는 1회 또는 수회 투여에 의해 효과적인 투여량을 포함한다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제, 주입제 등이 바람직하다. 또한, 상기 피부 창상 예방 또는 치료용 약학 조성물은 약학적으로 허용가능한 통상의 불활성 담체 및 희석제를 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 약학적으로 허용가능한 담체 및 희석제는 전분, 당, 및 만니톨과 같은 부형제, 칼슘 포스페이트 등과 같은 충전제 및 증량제, 카르복시메틸셀룰로오스, 히드록시프로필셀룰로오스 등과 같은 셀룰로오스 유도체, 젤라틴, 알긴산염, 및 폴리비닐 피롤리돈 등과 같은 결합제, 활석, 스테아린산 칼슘, 수소화 피마자유 및 폴리에틸렌 글리콜과 같은 윤활제, 포비돈, 크로스포비돈과 같은 붕해제, 폴리소르베이트, 세틸알코올, 및 글리세롤 등과 같은 계면활성제를 포함하나, 이에 한정되지 않는다. 상기 약학적으로 허용되는 담체 및 희석제는 비만세포의 조정 배지 및 이를 이식받을 수혜자에 대해 생물학적 및 생리학적으로 친화적인 것일 수 있다. 희석제로는 이에 한정되지 않으나, 염수, 수용성 완충액, 용매 및/또는 분산제(dispersion media)를 들 수 있다. 이외에도, 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소투여용 제제의 경우에는 기제(base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다. 본 발명의 조성물은 동결되지 않은 채 사용되거나 차후 사용을 위해 동결될 수 있다. 동결되어야 할 경우, 표준 냉동보존제 (예를 들어 DMSO, 글리세롤, 에피라이프 (Epilife®) 세포 동결 배지 (Cascade Biologics))가 동결 전 세포 집단에 첨가될 수 있다.The pharmaceutical composition of the present invention can be formulated into a unit dosage form pharmaceutical preparation suitable for administration into a patient's body according to a conventional method in the pharmaceutical field and administered, and the preparation contains an effective dosage amount by one or more administrations. Preferred dosage forms for this purpose are parenteral administration preparations such as injections and infusions. In addition, the pharmaceutical composition for preventing or treating skin wounds can contain a conventional inert carrier and diluent that are pharmaceutically acceptable. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, excipients such as starches, sugars, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, and the like, binders such as gelatin, alginates, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol, disintegrants such as povidone, crospovidone, and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the conditioned medium of mast cells and the recipient to be transplanted therewith. Diluents may include, but are not limited to, saline, an aqueous buffer, a solvent, and/or a dispersion media. In addition, for example, in the case of injectables, preservatives, analgesics, solubilizers or stabilizers may be additionally included, and in the case of topical administration preparations, bases, excipients, lubricants or preservatives may be additionally included. The compositions of the present invention may be used unfrozen or frozen for later use. If frozen, standard cryopreservatives (e.g., DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologics)) may be added to the cell population prior to freezing.

또한, 당업계에서 통상적으로 사용하는 투여방법을 이용하여 이식 및/또는 투여될 수 있으며, 바람직하게는 치료가 필요한 환자의 질환 부위에 직접 생착 또는 이식이 가능하나 이에 한정되지는 않는다. 또한, 상기 투여는 경구 투여, 카테터를 이용한 비외과적 투여, 및 질환부위 절개 후 주입 또는 이식 등 다양한 투여 방법이 이용될 수 있다. 투여량은 농축 정도에 따라 그 투여량이 달라질 수 있으나 10 μl/kg 내지 1 ml/kg을 1회 또는 수회로 나누어 투여할 수 있다. In addition, it can be transplanted and/or administered using a commonly used administration method in the art, and preferably, direct engraftment or transplantation is possible at the disease site of a patient requiring treatment, but is not limited thereto. In addition, the administration can be various administration methods such as oral administration, non-surgical administration using a catheter, and injection or transplantation after incision of the disease site. The dosage may vary depending on the degree of concentration, but can be administered once or in several divided doses at 10 μl/kg to 1 ml/kg.

그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage does not limit the scope of the present invention in any way.

본 발명은 또한 비만세포의 조정 배지를 포함하는 피부 외용제 조성물을 제공한다. The present invention also provides a composition for external application to the skin comprising a regulated medium of mast cells.

본 발명의 피부 외용제 조성물은, 피부의 국소 표면에 유효성분을 직접 투여할 수 있는 제형이면 특히 한정되지 않는데, 예를 들면 연고제, 연고첩부제, 액제(현탁제, 유제, 로션제 등), 습포제(cataplasms), 테이프제, 외용 분말 및, 에어로졸제 등의 제제로서 이용할 수가 있다. 본 발명의 외용제 조성물에 사용되는 배합성분으로는, 통상의 외용제 조성물에 사용되는 배합성분이면 어느 것이라도 사용할 수 있다.The external skin composition of the present invention is not particularly limited as long as it is a formulation that can directly administer an active ingredient to the local surface of the skin, and for example, it can be used as a preparation such as an ointment, an ointment patch, a liquid (suspension, emulsion, lotion, etc.), a cataplasm, a tape, an external powder, and an aerosol. As the compounding ingredients used in the external skin composition of the present invention, any compounding ingredients used in a typical external skin composition can be used.

연고제·크림제·겔제·로션제의 경우, 백색 바셀린, 황색 바셀린, 라놀린, 표백밀랍, 세틸 알콜, 스테아릴 알코올, 스테아린산, 경화유(hydrogenated oil), 탄화수소 겔, 폴리에틸렌글리콜, 액상 파라핀, 스쿠알렌 등의 기제, 올레산, 이소프로필 미리스테이트, 글리세릴 트리이소옥타노에이트, 크로타미톤, 디에틸 세바케이트, 디이소프로필 아디페이트, 헥실 라우레이트, 지방산, 지방산 에스테르, 지방족 알콜, 식물성 유지 등의 용매 및 용해보조제, 토코페롤 유도체, L-아스코르브산, 디부틸히드록시톨루엔, 부틸화 히드록시 아니솔 등의 산화방지제, p-히드록시벤조에이트 등의 방부제, 글리세린, 프로필렌 글리콜, 히알루로네이트소듐 등의 보습제, 폴리옥시에틸렌 유도체, 지방산의 글리세롤 에스테르, 지방산의 슈크로스 에스테르, 지방산의 소르비탄 에스테르, 지방산의 프로필렌 글리콜 에스테르, 레시틴 등의 계면활성제, 카르복시 비닐폴리머, 크산탄 검, 카르복시메틸 셀룰로오스, 카르복시메틸 셀룰로오스 소듐, 히드록시프로필셀룰로오스, 히드록시프로필 메틸셀룰로오스 등의 증점제(增粘劑), 액화 석유가스, 액화 이산화탄소, 디메틸 에테르, 질소, 등유, 이산화탄소 등의 분사제, 안정제, 보존제, 흡수촉진제 등과 기타 적당한 첨가제가 배합될 수 있다.In the case of ointments, creams, gels, and lotions, bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin, and squalene; solvents and solubilizers such as oleic acid, isopropyl myristate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylated hydroxyanisole; preservatives such as p-hydroxybenzoate; moisturizers such as glycerin, propylene glycol, and sodium hyaluronate; polyoxyethylene derivatives, and glycerol esters of fatty acids; Surfactants such as sucrose esters of fatty acids, sorbitan esters of fatty acids, propylene glycol esters of fatty acids, and lecithin; thickeners such as carboxy vinyl polymer, xanthan gum, carboxymethyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose; propellants such as liquefied petroleum gas, liquefied carbon dioxide, dimethyl ether, nitrogen, kerosene, and carbon dioxide; stabilizers, preservatives, absorption accelerators, and other appropriate additives can be blended.

습포제의 경우, 폴리아크릴산, 폴리아크릴산 코폴리머 등의 점착부여제, 황산알루미늄, 황산칼륨알루미늄, 염화 알루미늄, 마그네슘 알루미노메타실리케이트, 디히드록시 알루미늄 아세테이트 등의 가교제, 폴리아크릴산 나트륨, 폴리비닐 알콜, 폴리비닐피롤리돈, 젤라틴, 알긴산 나트륨, 카르복시메틸 셀룰로오스, 카르복시메틸 셀룰로오스 소듐, 히드록시프로필 셀룰로오스, 히드록시프로필 메틸셀룰로오스 등의 증점제, 글리세린, 폴리에틸렌 글리콜(마크로골), 프로필렌 글리콜, 1, 3-부탄디올 등의 다가 알콜류, 폴리옥시에틸렌 유도체 등의 계면활성제, 1-멘톨 등의 향료, p-히드록시벤조에이트 등의 방부제, 정제수, 및 기타 적당한 첨가제가 배합될 수 있다.In the case of wet compresses, tackifiers such as polyacrylic acid and polyacrylic acid copolymers, crosslinkers such as aluminum sulfate, potassium aluminum sulfate, aluminum chloride, magnesium aluminometasilicate and dihydroxy aluminum acetate, thickeners such as sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose and hydroxypropyl methylcellulose, polyhydric alcohols such as glycerin, polyethylene glycol (macrogol), propylene glycol and 1,3-butanediol, surfactants such as polyoxyethylene derivatives, fragrances such as 1-menthol, preservatives such as p-hydroxybenzoate, purified water and other suitable additives can be blended.

테이프제의 경우, 스티렌-이소프렌-스티렌 블록 코폴리머 및 아크릴수지 등의 점착제, 지환족(脂環族) 포화-탄화수소계 수지, 로진계 수지, 테르펜계 수지 등의 점착부여제, 액상고무, 액상 파라핀 등의 연화제, 디부틸히드록시톨루엔 등의 산화방지제, 프로필렌 글리콜 등의 다가알콜, 올레산 등의 흡수촉진제, 폴리옥시에틸렌 유도체 등의 계면활성제, 및 기타 적당한 첨가제가 배합될 수 있다. 또한, 폴리아크릴산 나트륨 또는 폴리비닐알콜과 같은 물을 함유할 수 있는 폴리머와 소량의 정제수를 가해 수성 테이프제로 제조될 수도 있다.In the case of tapes, adhesives such as styrene-isoprene-styrene block copolymers and acrylic resins, tackifiers such as alicyclic saturated-hydrocarbon resins, rosin resins, and terpene resins, softeners such as liquid rubber and liquid paraffin, antioxidants such as dibutylhydroxytoluene, polyhydric alcohols such as propylene glycol, absorption accelerators such as oleic acid, surfactants such as polyoxyethylene derivatives, and other appropriate additives can be blended. In addition, a polymer capable of containing water such as sodium polyacrylate or polyvinyl alcohol and a small amount of purified water can be added to produce an aqueous tape.

외용 분말의 경우, 감자 전분, 쌀 전분, 옥수수 전분, 탈크, 산화아연 등의 부형제(賦型劑) 및 기타 적당한 첨가제가 배합될 수 있다.In the case of external powder, excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, and other appropriate additives may be blended.

에어로졸제의 경우, 연고제·크림제·겔제·현탁제·유제·액제·로션제·외용 분말 등에 쓰이는, 백색 바셀린, 황색 바셀린, 라놀린, 표백밀랍, 세틸 알콜, 스테아릴 알코올, 스테아린산, 경화유, 탄화수소 겔, 폴리에틸렌글리콜, 액상 파라핀, 스쿠알렌 등의 기제, 올레산, 이소프로필 미리스테이트, 디이소프로필 아디페이트, 디이소프로필 세바케이트, 글리세릴 트리이소옥타노에이트, 크로타미톤, 디에틸 세바케이트, 헥실라우레이트, 지방산, 지방산 에스테르, 지방족 알콜, 식물성 유지 등의 용매 및 용해보조제, 토코페롤 유도체, L-아스코르브산, 디부틸히드록시톨루엔, 부틸화 히드록시아니솔 등의 산화방지제, p-히드록시 벤조에이트 등의 방부제, 글리세린, 프로필렌 글리콜, 히알루로네이트 소듐 등의 보습제, 폴리옥시에틸렌 유도체, 지방산의 글리세롤 에스테르, 지방산의 슈크로스 에스테르, 지방산의 소르비탄 에스테르, 지방산의 프로필렌 글리콜, 레시틴 등의 계면활성제, 카르복시 비닐 폴리머, 크산탄 검, 카르복시메틸 셀룰로오스, 카르복시메틸셀룰로오스 소듐, 히드록시프로필셀룰로오스, 히드록시프로필 메틸셀룰로오스 등의 증점제(增粘劑), 감자 전분, 쌀 전분, 옥수수 전분, 탈크, 산화아연 등의 부형제, 액화 석유가스, 액화 이산화탄소, 디메틸 에테르, 질소, 등유, 이산화탄소 등의 분사제, 완충제, 교미제(correctives), 현탁화제, 유화제, 방향제, 보존제, 용해보조제, 또는 기타 적당한 첨가제가 배합될 수 있다.In the case of aerosols, white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetyl alcohol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin, squalene, etc. as bases used in ointments, creams, gels, suspensions, emulsions, solutions, lotions, external powders, etc.; solvents and solubilizers such as oleic acid, isopropyl myristate, diisopropyl adipate, diisopropyl sebacate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, vegetable oils, etc.; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole; preservatives such as p-hydroxy benzoate; glycerin, propylene glycol, Humectants such as sodium hyaluronate, polyoxyethylene derivatives, glycerol esters of fatty acids, sucrose esters of fatty acids, sorbitan esters of fatty acids, propylene glycol of fatty acids, surfactants such as lecithin, thickeners such as carboxy vinyl polymer, xanthan gum, carboxymethyl cellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, propellants such as liquefied petroleum gas, liquefied carbon dioxide, dimethyl ether, nitrogen, kerosene, carbon dioxide, buffers, correctives, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, or other suitable additives can be blended.

한편, 본 발명의 피부 외용제 조성물은 화장품학 또는 피부과학적으로 허용 가능한 매질 또는 기제를 함유하여 피부 국소 도포용으로 제형화될 수 있다. 이는 국소적용에 적합한 모든 제형으로서, 예를 들면, 용액, 겔, 고체, 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀) 및 비이온형의 소낭 분산제의 형태로, 또는 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태로 제공될 수 있다. 또한, 폼(foam)의 형태로 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 사용될 수 있다. 이들 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다.Meanwhile, the external skin composition of the present invention may be formulated for topical application to the skin by containing a cosmetically or dermatologically acceptable medium or base. This may be any formulation suitable for topical application, for example, in the form of a solution, gel, solid, anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule, or an ionic (liposome) and nonionic vesicle dispersion, or in the form of a cream, a skin, a lotion, a powder, an ointment, a spray, or a concealer stick. It may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

또한, 본 발명의 피부 외용제 조성물은 지방 물질, 유기용매, 용해제, 농축제, 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품학 또는 피부과학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 상기 보조제는 화장품학 또는 피부과학 분야에서 일반적으로 사용되는 양으로 도입된다.In addition, the skin external composition of the present invention may contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics. The adjuvants are introduced in an amount commonly used in the fields of cosmetology or dermatology.

본 발명은 또한 비만세포의 조정 배지를 포함하는 피부 창상 예방 또는 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for preventing or improving skin wounds, which comprises a regulated medium of mast cells.

본 발명은 통상적으로 이용되는 식품으로써 일반적으로 사용될 수 있다. The present invention can be generally used as a commonly used food.

본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능식품"이라 함은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present invention can be used as a health functional food. The above "health functional food" means a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to the Health Functional Food Act, and "functionality" means that it is consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

본 발명의 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 "식품 첨가물"로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. The food composition of the present invention may contain conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

상기 "식품 첨가물 공전"에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Items listed in the above "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

본 발명의 식품 조성물은 조성물 총 중량에 대하여 비만세포의 조정배지를 0.01 내지 95 중량%, 바람직하게는 5 내지 90 중량%로 포함할 수 있다. The food composition of the present invention may contain a conditioned medium for mast cells in an amount of 0.01 to 95 wt%, preferably 5 to 90 wt%, based on the total weight of the composition.

또한, 본 발명의 식품 조성물은 피부 창상의 예방 및/또는 개선을 목적으로, 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc., for the purpose of preventing and/or improving skin wounds.

예를 들어, 캡슐 형태의 건강기능식품 중 경질캡슐제는 통상의 경질캡슐에 본 발명에 따른 비만세포의 조정배지, 및 부형제 등의 첨가제와의 혼합물을 충진하여 제조할 수 있으며, 연질캡슐제는 본 발명에 따른 식품 조성물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캡슐기제에 충진하여 제조할 수 있다. 상기 연질캡슐제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.For example, among health functional foods in capsule form, hard capsules can be manufactured by filling a mixture of the conditioned medium of mast cells according to the present invention and additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by filling a mixture of the food composition according to the present invention and additives such as excipients into a capsule base such as gelatin. The soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 (대한약전 해설편, 문성사, 한국약학대학협의회, 제5 개정판, p33-48, 1989).The definitions of terms for the above excipients, binders, disintegrants, lubricants, maturing agents, flavoring agents, etc. are described in literature known in the art and include those with the same or similar functions (Commentary on the Korean Pharmacopoeia, Munsungsa, Korean Pharmaceutical Colleges Council, 5th revised edition, p. 33-48, 1989).

상기 식품의 종류에는 특별한 제한이 없으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There are no special restrictions on the types of the above foods, and all health functional foods in the usual sense are included.

본 발명은 비만세포의 조정배지를 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 피부 창상의 예방 또는 치료방법을 제공한다.The present invention provides a method for preventing or treating skin wounds, comprising a step of administering a conditioned medium of mast cells to a subject in need thereof.

본 발명에 "비만세포의 조정배지", "피부 창상", "투여" 등의 용어는 상기에서 설명한 바와 동일하다.In the present invention, the terms “adjusted medium for mast cells,” “skin wound,” “administration,” etc. are the same as those described above.

상기 대상체는 동물을 말하며, 전형적으로 본 발명의 비만세포의 조정배지를 이용한 치료로 유익한 효과를 나타낼 수 있는 포유동물일 수 있다. 이러한 대상체의 바람직한 예로 인간과 같은 영장류가 포함될 수 있다. 또한 이와 같은 대상체들에는 피부 창상의 증상을 갖거나 이와 같은 증상을 가질 위험이 있는 대상체들이 모두 포함될 수 있다.The above subject refers to an animal, and may typically be a mammal that can show a beneficial effect by treatment using the mast cell-modified medium of the present invention. Preferred examples of such subjects may include primates such as humans. In addition, such subjects may include all subjects having symptoms of skin wounds or at risk of having such symptoms.

본 발명은 또한 피부 창상의 예방 또는 치료를 위한 약제의 제조에서 비만세포의 조정배지의 용도를 제공한다. The present invention also provides the use of a conditioned medium of mast cells in the manufacture of a medicament for the prevention or treatment of skin wounds.

본 발명은 또한 피부 창상의 예방 또는 치료에 사용하기 위한 비만세포의 조정배지를 포함하는 조성물을 제공한다. The present invention also provides a composition comprising a conditioned medium of mast cells for use in the prevention or treatment of skin wounds.

본 발명은 또한 피부 창상의 예방 또는 치료를 위한 비만세포의 조정배지의 용도를 제공한다. The present invention also provides the use of a mast cell-conditioned medium for the prevention or treatment of skin wounds.

본 발명의 피부 창상 예방, 개선 또는 치료용 조성물은 비만세포 조정배지를 포함하며, 사람 피부 각질세포의 세포이동을 촉진하고 창상 치유 과정 중 특히 염증의 개선 효과가 우수하여 창상 치료제로 활용 가치가 높다. The composition for preventing, improving or treating skin wounds of the present invention comprises a mast cell-regulated medium, promotes cell migration of human skin keratinocytes and has an excellent effect of improving inflammation, especially during the wound healing process, and thus has high value as a wound treatment agent.

도 1은 Scratch assay 이미지를 통해 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. Figure 1 shows the results of a scratch assay image confirming the effect of human-derived mast cell-conditioned medium on human skin keratinocyte migration.

도 2는 Scratch assay 정량화를 통해 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향 확인한 결과이다. (**p<0.01, ***p<0.001, ****p<0.0001)Figure 2 shows the results of confirming the effect of human-derived mast cell-conditioned medium on human skin keratinocyte migration through scratch assay quantification. (**p<0.01, ***p<0.001, ****p<0.0001)

도 3은 Transwell migration assay 이미지를 통해 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. Figure 3 shows the results of a Transwell migration assay image confirming the effect of conditioned medium containing human-derived mast cells on the migration of human skin keratinocytes.

도 4는 Transwell migration assay 정량화를 통해 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. (**p<0.01, ***p<0.001, ****p<0.0001)Figure 4 shows the results of confirming the effect of conditioned medium of human-derived mast cells on the migration of human skin keratinocytes through Transwell migration assay quantification. (**p<0.01, ***p<0.001, ****p<0.0001)

도 5는 Scratch assay 이미지를 통해 톨 유사 수용체 2와 6 작용제를 처리한 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. Figure 5 shows the results of a scratch assay image showing the effect of conditioned media of human mast cells treated with toll-like receptor 2 and 6 agonists on the migration of human skin keratinocytes.

도 6은 Scratch assay 정량화를 통해 톨 유사 수용체 2와 6 작용제를 처리한 사람 유래 비만세포의 조정배지가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. (**p<0.01, ***p<0.001, ****p<0.0001)Figure 6 shows the results of the Scratch assay quantification to determine the effect of conditioned media of human mast cells treated with toll-like receptor 2 and 6 agonists on human skin keratinocyte migration. (**p<0.01, ***p<0.001, ****p<0.0001)

도 7은 세 그룹의 마우스 창상 모델(Control, HMC-1 CM, TLR2/6 HMC-1 CM)의 치유 과정 이미지를 통해 창상 치유 효과를 비교한 결과이다. Figure 7 shows the results of comparing the wound healing effect through images of the healing process of three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM).

도 8은 세 그룹의 마우스 창상 모델(Control, HMC-1 CM, TLR2/6 HMC-1 CM)의 치유 정도 정량화를 통해 창상 치유 효과를 비교한 결과이다. (**p<0.01, ***p<0.001, ****p<0.0001)Figure 8 shows the results of comparing the wound healing effect through quantification of the degree of healing in three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM). (**p<0.01, ***p<0.001, ****p<0.0001)

도 9는 세 그룹의 마우스 창상 모델(Control, HMC-1 CM, TLR2/6 HMC-1 CM)의 피부조직에서 H&E 염색을 수행한 결과이다.Figure 9 shows the results of H&E staining on skin tissues of three groups of mouse wound models (Control, HMC-1 CM, TLR2/6 HMC-1 CM).

도 10은 비만세포 유래 조정배지(HMC-1 CM)과 TLR2/6 작용제로 자극된 비만세포의 조정배지 (T2/6 HMC-1 CM), 피부 각질 세포주 유래 조정배지(HaCaT CM), 단핵구 세포주 유래 조성배지(THP-1 CM), 완전한 음성 대조군으로서 IMDM 배지(IMDM)에서의 트립타제(tryptase) 함유량을 ELISA assay 통해 확인한 결과이다. (****p<0.0001)Figure 10 shows the results of ELISA assay for tryptase content in conditioned medium derived from mast cells (HMC-1 CM), conditioned medium of mast cells stimulated with TLR2/6 agonist (T2/6 HMC-1 CM), conditioned medium derived from skin keratinocyte cell line (HaCaT CM), conditioned medium derived from monocyte cell line (THP-1 CM), and IMDM medium (IMDM) as a complete negative control. (****p<0.0001)

도 11은 Scratch assay 이미지를 통해 트립타제(tryptase)가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. Figure 11 shows the results of a scratch assay image confirming the effect of tryptase on human skin keratinocyte migration.

도 12는 Scratch assay 정량화를 통해 트립타제(tryptase)가 사람 피부 각질세포 이동에 미치는 영향을 확인한 결과이다. (****p<0.0001)Figure 12 shows the results of the Scratch assay quantification to confirm the effect of tryptase on human skin keratinocyte migration. (****p<0.0001)

본 발명의 이해를 돕기 위하여 실시예, 제조예를 제시한다. 하기의 실시예, 제조예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예, 제조예에 의해 본 발명의 내용이 한정되는 것은 아니다.In order to help understand the present invention, examples and manufacturing examples are presented. The following examples and manufacturing examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the examples and manufacturing examples.

<실시예 1> 사람 유래 비만세포 조정배지의 제조<Example 1> Preparation of human-derived mast cell conditioned medium

사람 유래 비만세포로부터 조정배지를 얻기 위하여 10%의 혈청, 1% penicillin/streptomycin이 포함된 IMDM으로 사람 유래 비만세포를 배양하였다. 100mm cell culture plate에 confluency 80%가 되었을 때 세포를 걷어낸 후 다시 1x107개의 세포에 무혈청 IMDM 배지를 첨가하였다. 48시간 후, 배지를 걷어 5300 xg에서 1시간 30분동안 원심분리한 후 상층액을 여과하였다. 여과된 배지는 centrifugal filter (cut-off of 3K, Millipore)로 1/5로 농축하여 실험에 이용하거나 -80℃에서 보관하였다. 이를 통해 비만세포 조정배지를 제조하였다. To obtain conditioned media from human mast cells, human mast cells were cultured in IMDM containing 10% serum and 1% penicillin/streptomycin. When the confluency reached 80% in a 100 mm cell culture plate, the cells were harvested and serum-free IMDM medium was added to 1 x 10 7 cells again. After 48 hours, the medium was harvested, centrifuged at 5300 xg for 1 hour and 30 minutes, and the supernatant was filtered. The filtered medium was concentrated to 1/5 using a centrifugal filter (cut-off of 3K, Millipore) and used in the experiment or stored at -80℃. In this way, mast cell conditioned media was prepared.

TLR2/6 작용제를 처리하는 경우에는 1x107개의 세포에 TLR2/6 작용제를 50 ng/ml 농도로 처리 후 혈청 IMDM 배지에서 24시간 배양하였다. 그후 원심분리로 상층액을 걷어내고 1x107개의 세포를 무혈청 IMDM 배지에서 48시간 배양 후 배지를 걷어 5300 xg에서 1시간 30분동안 원심분리한 후 상층액을 여과하였다. 여과된 배지는 centrifugal filter (cut-off of 3K, Millipore)로 1/5로 농축하여 실험에 이용하거나 -80℃에서 보관하였다. 이를 통해 TLR2/6 작용제로 전처리된 비만세포의 조정 배지를 제조하였다. In the case of TLR2/6 agonist treatment, 1x107 cells were treated with TLR2/6 agonist at a concentration of 50 ng/ml and cultured in serum IMDM medium for 24 hours. The supernatant was then removed by centrifugation, and 1x107 cells were cultured in serum-free IMDM medium for 48 hours, the medium was removed, centrifuged at 5300 xg for 1 hour 30 minutes, and the supernatant was filtered. The filtered medium was concentrated to 1/5 using a centrifugal filter (cut-off of 3K, Millipore) and used in the experiment or stored at -80℃. Through this, the conditioned medium of mast cells pretreated with TLR2/6 agonist was prepared.

<실시예 2> 비만세포 조정배지에 의한 상처 이동 분석 (Scratch assay)<Example 2> Scratch assay for wound migration using mast cell-conditioned medium

사람 유래 비만세포 조정배지의 세포 이동 촉진효과를 확인하기 위해 Scratch assay를 실시하였다. Scratch assay was performed to confirm the cell migration promoting effect of human-derived mast cell-conditioned medium.

먼저, 사람 피부 각질세포를 12 웰 규격의 세포 배양 접시의 confluency 90%까지 37℃, 5% CO2 조건의 세포 배양기에서 배양하였다. 멸균된 200 ul 마이크로 파이펫 팁을 이용하여 배양 접시 바닥을 긁어 세포가 부착되지 않은 스크래치 상처를 만들었다. 떨어진 세포와 배지를 제거한 후 실시예 1에서 얻은 사람 유래 비만세포 조정 배지를 100 ul 첨가하였다. 대조군의 경우 비만세포 조정배지 대신 무혈청의 IMDM 배지를 100 ul 첨가하였다. 배지 첨가 이후 0시간, 24시간, 48시간이 지났을 때 상처의 이미지를 얻었으며, 이를 도 1에 나타내었다. 또한 각 이미지에서 상처의 열린 간격을 측정하였으며, 이를 도 2에 나타내었다. First, human skin keratinocytes were cultured in a 12-well cell culture dish at 37°C and 5% CO2 in a cell incubator until 90% confluency. A scratch wound to which cells did not attach was created by scratching the bottom of the culture dish using a sterilized 200 μl micropipette tip. After removing the detached cells and the medium, 100 μl of the human-derived mast cell-conditioned medium obtained in Example 1 was added. In the case of the control group, 100 μl of serum-free IMDM medium was added instead of the mast cell-conditioned medium. Wound images were obtained at 0, 24, and 48 hours after the addition of the medium, and the results are shown in Fig. 1. In addition, the wound opening gap was measured in each image, and the results are shown in Fig. 2.

도 1 및 2에서 사람 유래 비만세포 조정배지를 첨가한 경우 상처의 간격이 더 빨리 가까워지는 것을 확인할 수 있었으며, 이를 통해 사람 유래 비만세포 조정배지에 사람 각질 세포의 세포이동을 촉진하는 효과가 있음을 확인할 수 있었다.In Figures 1 and 2, it was confirmed that the gap between the wounds closed more quickly when the human-derived mast cell-conditioned medium was added, and through this, it was confirmed that the human-derived mast cell-conditioned medium has the effect of promoting cell migration of human keratinocytes.

<실시예 3> 비만세포 조정배지에 의한 생체 외 세포 이동 분석 (Transwell migration assay)<Example 3> In vitro cell migration analysis using mast cell conditioned medium (Transwell migration assay)

사람 유래 비만세포 조정배지의 세포 이동 촉진효과를 확인하기 위해 직경이 0.4 um인 다공성 막을 갖는 트렌스웰 플레이트를 이용해 Transwell migration assay를 실시하였다. To confirm the cell migration promoting effect of human-derived mast cell-conditioned medium, a Transwell migration assay was performed using a Transwell plate with a porous membrane with a diameter of 0.4 um.

트렌스웰의 상층 챔버에는 사람 피부 각질세포 2x105개를, 하층 챔버에는 사람 유래 비만세포 조정배지 50 ul와 2% 우태아혈청을 포함하는 IMDM 배지 550ul를 첨가한 후, 37℃, 5% CO2 조건의 세포 배양기에서 배양하였다. 배양 후 24시간 또는 48시간이 지났을 때 세포를 고정시키고 크리스탈 바이올렛으로 염색하여 현미경 이미지를 얻었으며, 이를 도 3에 나타내었다. 또한 각 이미지에서 염색된 세포의 면적을 측정하였으며, 이를 도 4에 나타내었다.In the upper chamber of the Transwell, 2x105 human skin keratinocytes were added, and in the lower chamber, 50 ul of human mast cell conditioned medium and 550 ul of IMDM medium containing 2% fetal bovine serum were added, and then cultured in a cell incubator under the conditions of 37℃ and 5% CO2 . After 24 or 48 hours of culture, the cells were fixed and stained with crystal violet to obtain microscopic images, which are shown in Fig. 3. In addition, the area of the stained cells in each image was measured, which is shown in Fig. 4.

도 3 및 4에서 사람 유래 비만세포 조정배지를 첨가한 경우 염색된 면적이 더 넓은 것을 확인할 수 있었으며, 이를 통해 사람 유래 비만세포 조정배지에 사람 각질 세포의 세포이동을 촉진하는 효과가 있음을 확인할 수 있었다.In Figures 3 and 4, it was confirmed that the stained area was wider when human-derived mast cell-conditioned medium was added, and through this, it was confirmed that human-derived mast cell-conditioned medium has the effect of promoting cell migration of human keratinocytes.

<실시예 4> 톨 유사 수용체 2와 6 작용제로 처리된 비만세포 조정 배지에 의한 상처 이동 분석 (Scratch assay)<Example 4> Scratch assay of wound migration by mast cell-conditioned medium treated with toll-like receptor 2 and 6 agonists

톨 유사 수용체 2와 6 작용제로 처리된 사람 유래 비만세포 조정배지의 세포 이동 촉진효과를 확인하기 위해 Scratch assay를 실시하였다.Scratch assay was performed to confirm the cell migration-promoting effect of human mast cell-conditioned medium treated with toll-like receptor 2 and 6 agonists.

실험 방법은 실시예 2와 동일하나, 스크래치 상처에 톨 유사 수용체 2와 6 작용제로 처리된 사람 유래 비만세포 조정배지를 100 ul 첨가하였다. 대조군의 경우 무혈청의 IMDM 배지를 100 ul 첨가하였다. 배지 첨가 이후 0시간, 48시간이 지났을 때 상처의 이미지를 얻었으며, 이를 도 5에 나타내었다. 또한 각 이미지에서 상처의 열린 간격을 측정하였으며, 이를 도 6에 나타내었다. The experimental method was the same as in Example 2, but 100 μl of human mast cell-conditioned medium treated with toll-like receptor 2 and 6 agonists was added to the scratch wound. For the control group, 100 μl of serum-free IMDM medium was added. Wound images were obtained 0 and 48 hours after the medium addition, and the results are shown in Fig. 5. In addition, the wound opening gap was measured in each image, and the results are shown in Fig. 6.

도 5 및 6에서 톨 유사 수용체 2와 6 작용제를 처리한 사람 유래 비만세포 조정배지를 첨가한 경우 48시간 안에 상처 간격이 100% 닫히는 것을 확인할 수 있었으며, 이를 통해 톨 유사 수용체 2와 6 작용제를 처리한 사람 유래 비만세포 조정배지가 사람 각질 세포의 세포 이동 능력을 가장 크게 향상시키는 것을 확인할 수 있었다.In Figures 5 and 6, it was confirmed that the wound gap was closed 100% within 48 hours when the human mast cell conditioned medium treated with toll-like receptor 2 and 6 agonists was added, confirming that the human mast cell conditioned medium treated with toll-like receptor 2 and 6 agonists most significantly enhanced the cell migration ability of human keratinocytes.

<실시예 5> 창상 마우스 모델에서의 창상 치유 효과 비교<Example 5> Comparison of wound healing effects in a wound mouse model

창상 마우스 모델에서 상처 치유 효과를 비교하기 위해 C57BL/6J (암컷, 9주령) 마우스를 아래 표 1과 같이 세 그룹으로 나누어 실험을 진행하였다.To compare the wound healing effects in a wound mouse model, C57BL/6J (female, 9 weeks old) mice were divided into three groups as shown in Table 1 below and the experiment was conducted.

Control 군Control group 마우스 창상에 무혈청 IMDM 배지를 처리한 그룹Group treated with serum-free IMDM medium on mouse wounds HMC-1 CM 군HMC-1 CM group 마우스 창상에 비만세포 조정 배지를 처리한 그룹Group treated with mast cell-modified medium on mouse wounds TLR 2/6 HMC-1 CM 군TLR 2/6 HMC-1 CM Group 마우스 창상에 TLR 2/6 agonist를 처리한 비만세포 조정 배지를 처리한 그룹Group treated with mast cell-conditioned medium containing TLR 2/6 agonist on mouse wounds

모든 마우스에 직경 8mm의 창상을 유발한 후, 무혈청 IMDM 배지 60ul를 처리한 그룹을 Control 그룹(n=5)으로, 비만세포 조정 배지 60ul(1.3x106/마우스)를 처리한 그룹을 HMC-1 CM 그룹(n=5)으로, 또는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포 조정배지 60ul(1.3x106/마우스)를 처리한 그룹을 TLR 2/6 HMC-1 CM 그룹(n=5)으로 분류하였다. 실험 시작일로부터 3일 후에 각 군에 해당하는 물질을 추가 주입하였으며, 총 12일 동안 마우스의 몸무게와 창상 회복 과정 등을 관찰하였다. 실험 기간동안 그룹별 마우스의 창상 이미지를 도 7에 나타내었다. 또한 각 이미지에서 상처의 열린 간격을 측정하였으며, 이를 도 8에 나타내었다.After inducing a wound of 8 mm in diameter on all mice, the group treated with 60 μl of serum-free IMDM medium was classified as the Control group (n = 5), the group treated with 60 μl of mast cell-conditioned medium (1.3 × 10 6 /mouse) was classified as the HMC-1 CM group (n = 5), or the group treated with 60 μl of mast cell-conditioned medium stimulated with toll-like receptor 2 and 6 agonists (1.3 × 10 6 /mouse) was classified as the TLR 2/6 HMC-1 CM group (n = 5). Three days after the start of the experiment, the corresponding substances were additionally injected into each group, and the body weights and wound healing processes of the mice were observed for a total of 12 days. The wound images of the mice by group during the experimental period are shown in Fig. 7. In addition, the wound opening gap was measured in each image, and this is shown in Fig. 8.

도 7 및 8에서 HMC-1 CM군 및 TLR 2/6 HMC-1 CM군의 경우 12일차의 상처 회복 면적이 100%에 가까운 것을 확인할 수 있었으며, 이를 통해 HMC-1 CM 및 TLR 2/6 HMC-1 CM이 우수한 창상 치료 효과를 갖는 것을 확인할 수 있었다. In Figures 7 and 8, it was confirmed that the wound healing area on the 12th day was close to 100% for the HMC-1 CM group and the TLR 2/6 HMC-1 CM group, which confirmed that HMC-1 CM and TLR 2/6 HMC-1 CM had excellent wound healing effects.

<실시예 6> H&E Staining을 통한 창상 치유 효과 비교<Example 6> Comparison of wound healing effects through H&E staining

창상 마우스 모델에서 상처 치유 효과를 비교하기 위해 동물실험 종료 후 H&E staining을 수행하였다. To compare the wound healing effect in a wound mouse model, H&E staining was performed after the end of the animal experiment.

창상 마우스 모델의 피부 조직 채취하여 포르말린에 고정하였다. 이후 파라핀 포매 슬라이드를 제작하고 H&E 염색을 실시하였으며 그룹별 마우스의 염색된 피부 조직 이미지를 도 9에 나타내었다. Skin tissues from the wound mouse model were collected and fixed in formalin. Paraffin-embedded slides were then prepared and H&E staining was performed. Images of the stained skin tissues of the mice in each group are shown in Figure 9.

도 9에서 HMC-1 CM군 및 TLR 2/6 HMC-1 CM군의 경우 상피화(epithelialization)와 육아조직형성(granulation tissue formation)이 관찰되었으며, 이를 통해 HMC-1 CM 및 TLR 2/6 HMC-1 CM이 우수한 창상 치료 효과를 갖는 것을 확인할 수 있었다. In Fig. 9, epithelialization and granulation tissue formation were observed in the HMC-1 CM group and the TLR 2/6 HMC-1 CM group, confirming that HMC-1 CM and TLR 2/6 HMC-1 CM have excellent wound healing effects.

실시예 1 내지 6의 결과로부터, 비만세포 조정 배지는 각질 세포의 세포 이동을 촉진하고 염증을 개선함으로써 피부 창상 치료에 우수한 효과를 갖는 것을 확인하였다. 또한, 상피화(epithelialization)와 육아조직형성(granulation tissue formation) 측면에서도 치료제 처리에 의해 개선된 효능을 나타내었다. From the results of Examples 1 to 6, it was confirmed that the mast cell-conditioned medium had an excellent effect on the treatment of skin wounds by promoting cell migration of keratinocytes and improving inflammation. In addition, it showed improved efficacy by treatment with the therapeutic agent in terms of epithelialization and granulation tissue formation.

더욱이, 톨 유사 수용체 2와 6 작용제로 비만세포를 자극할 경우, 조정배지의 피부 창상 치료 효과가 향상되는 것을 확인하였다.Furthermore, it was confirmed that the skin wound healing effect of the conditioned medium was enhanced when mast cells were stimulated with toll-like receptor 2 and 6 agonists.

<실시예 7> 조정배지 종류에 따른 트립타제(tryptase) 함유량 확인 (ELISA assay)<Example 7> Confirmation of tryptase content according to the type of conditioned medium (ELISA assay)

세포의 종류에 따른 tryptase 함유량 차이를 확인하기 위해, 비만세포 유래 조정배지(HMC-1 CM)과 TLR2/6 작용제로 자극된 비만세포의 조정배지 (T2/6 HMC-1 CM), 피부 각질 세포주 유래 조정배지(HaCaT CM), 단핵구 세포주 유래 조성배지(THP-1 CM), 완전한 음성 대조군으로서 IMDM 배지(IMDM)를 이용하여 ELISA assay를 실시하였다.To confirm the difference in tryptase content according to cell type, ELISA assays were performed using conditioned medium derived from mast cells (HMC-1 CM), conditioned medium from mast cells stimulated with a TLR2/6 agonist (T2/6 HMC-1 CM), conditioned medium derived from a skin keratinocyte cell line (HaCaT CM), conditioned medium derived from a monocyte cell line (THP-1 CM), and IMDM medium (IMDM) as a complete negative control.

구체적으로, 각 세포의 조정배지를 1/50로 희석한후 Human Tryptase Elisa kit (Innovative Research, Inc.)를 이용하여 시료 내에 존재하는 tryptase 농도를 측정하였으며, 이를 도 10에 나타내었다.Specifically, the conditioned medium of each cell was diluted 1/50, and the tryptase concentration in the sample was measured using a Human Tryptase Elisa kit (Innovative Research, Inc.), and the results are shown in Figure 10.

도 10에서 HMC-1 CM, T2/6 HMC-1 CM, HaCaT CM, THP-1 CM, IMDM에서의 tryptase 함유량 차이를 확인할 수 있었으며, HMC-1 CM, T2/6 HMC-1 CM에서 tryptase 함유량이 다른 배지에서의 함유량 보다 높았으며, 이를 통해 HMC-1 세포는 자극이 없는 평상시에도 tryptase를 높게 분비하는 것으로 나타났다.In Fig. 10, the difference in tryptase content in HMC-1 CM, T2/6 HMC-1 CM, HaCaT CM, THP-1 CM, and IMDM was confirmed, and the tryptase content in HMC-1 CM and T2/6 HMC-1 CM was higher than that in other media, indicating that HMC-1 cells secrete high levels of tryptase even under normal conditions without stimulation.

<실시예 8> 비만세포 조정배지와 톨 유사 수용체 2와 6 작용제로 자극된 비만세포 조정배지의 tryptase에 의한 세포 이동 촉진 효과 분석 (Scratch assay)<Example 8> Analysis of the effect of mast cell conditioned medium and mast cell conditioned medium stimulated with toll-like receptor 2 and 6 agonists on cell migration by tryptase (Scratch assay)

비만세포 조정배지와 톨 유사 수용체 2와 6 작용제로 자극된 비만세포 조정배지에 의한 HaCaT 세포 이동 촉진에 있어서 tryptase의 영향을 확인하기 위해 Scratch assay를 실시하였다.Scratch assays were performed to determine the effect of tryptase on the promotion of HaCaT cell migration by mast cell-conditioned medium and mast cell-conditioned medium stimulated with toll-like receptor 2 and 6 agonists.

구체적으로, 실시예 2와 동일한 방법으로 Scratch assay를 수행하되, 실험군은 대조군(control), 비만세포 조정배지(HMC-1 CM) 그룹 및 톨 유사 수용체 2와 6 작용제로 자극된 비만세포의 조정배지(T2/6 HMC-1 CM) 그룹과 각 그룹에 tryptase inhibitor(APC 366, trifluoroacetate salt, Sigma-Aldrich, SML2490)를 20 ug/ml 처리한 그룹(control+Inhibitor; HMC-1 CM+Inhibitor; 및 T2/6 HMC-1 CM+Inhibitor)으로 구성하였다. 각 그룹의 배지 첨가 이후 0시간, 24시간, 48시간이 지났을 때 상처의 이미지를 얻었으며, 이를 도 11에 나타내었다(40 배율). 또한 각 이미지에서 간격 길이를 여러 번 측정한 후 창상 봉합율을 정량화하였으며, 이를 도 12에 나타내었다.Specifically, the Scratch assay was performed in the same manner as in Example 2, but the experimental groups were composed of a control group, a mast cell-conditioned medium (HMC-1 CM) group, a mast cell-conditioned medium stimulated with toll-like receptor 2 and 6 agonists (T2/6 HMC-1 CM) group, and groups treated with 20 ug/ml of tryptase inhibitor (APC 366, trifluoroacetate salt, Sigma-Aldrich, SML2490) in each group (control+Inhibitor; HMC-1 CM+Inhibitor; and T2/6 HMC-1 CM+Inhibitor). Wound images were obtained 0 h, 24 h, and 48 h after the addition of the medium to each group, and the results are shown in Fig. 11 (40x magnification). In addition, the gap length was measured multiple times in each image, and the wound closure rate was quantified, which is shown in Fig. 12.

도 11 및 12에서 HMC-1 그룹 및 T2/6 HMC-1 CM 그룹은 시간이 지남에 따라 상처의 간격이 빠르게 가까워지는 것으로 나타났으며, tryptase inhibitor를 처리한 군(HMC-1 CM+Inhibitor; 및 T2/6 HMC-1 CM+Inhibitor)에서는 상처의 간격이 가까워지는 속도가 상대적으로 느려지는 것으로 나타났다. 즉, tryptase inhibitor 처리하는 경우 조정 배지에 의한 HaCaT 세포의 이동 촉진 효과가 제한되는 것으로 나타났다.In Figures 11 and 12, the HMC-1 group and the T2/6 HMC-1 CM group showed that the gap between the wounds quickly closed over time, while the groups treated with tryptase inhibitor (HMC-1 CM+Inhibitor; and T2/6 HMC-1 CM+Inhibitor) showed a relatively slow rate of closing the gap between the wounds. In other words, it appeared that the effect of the conditioned medium on promoting HaCaT cell migration was limited when treated with tryptase inhibitor.

이러한 결과를 통해, 본 발명의 비만세포 조정배지는 tryptase 작용 의존적으로 피부 상피세포의 이동을 촉진하는 것으로 나타났으며, 세포 이동을 촉진시키는데 있어서 tryptase가 주요 인자로 작용하고 있음을 확인할 수 있었다.Through these results, it was shown that the mast cell-conditioned medium of the present invention promotes the migration of skin epithelial cells in a tryptase action-dependent manner, and it was confirmed that tryptase acts as a major factor in promoting cell migration.

Claims (23)

비만세포의 조정배지를 포함하는 피부 창상 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating skin wounds, comprising a conditioned medium of mast cells. 제1항에 있어서, 상기 비만세포는 사람 유래 비만세포인, 약학 조성물.A pharmaceutical composition in claim 1, wherein the mast cells are human-derived mast cells. 제1항에 있어서, 상기 비만세포의 조정배지는 피부 각질세포의 세포 이동을 촉진하는, 약학 조성물.A pharmaceutical composition in claim 1, wherein the conditioned medium of the mast cells promotes cell migration of skin keratinocytes. 제1항에 있어서, 상기 비만세포의 조정배지는 트립타제(tryptase)를 포함하는 것인, 약학 조성물.A pharmaceutical composition in claim 1, wherein the conditioned medium of the mast cells contains tryptase. 제1항에 있어서, 상기 비만세포는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포인, 약학 조성물.A pharmaceutical composition in claim 1, wherein the mast cells are mast cells stimulated with toll-like receptor 2 and 6 agonists. 제5항에 있어서, 상기 톨 유사 수용체 2와 6 작용제는 FSL-1, MALP-2, CBLB613, 디아실화된 지질단백질 및 디아실화된 리포펩티드로 이루어진 군으로부터 선택되는 어느 하나 이상인, 약학 조성물.A pharmaceutical composition in claim 5, wherein the toll-like receptor 2 and 6 agonist is at least one selected from the group consisting of FSL-1, MALP-2, CBLB613, deacylated lipoprotein and deacylated lipopeptide. 제5항에 있어서, 상기 톨 유사 수용체 2와 6 작용제로 자극된 비만세포는 비만 세포를 1 내지 100 시간 동안 톨 유사 수용체 2와 6 작용제로 전처리한 것인, 약학 조성물.A pharmaceutical composition according to claim 5, wherein the mast cells stimulated with the toll-like receptor 2 and 6 agonists are mast cells pretreated with the toll-like receptor 2 and 6 agonists for 1 to 100 hours. 제7항에 있어서, 상기 톨 유사 수용체 2와 6 작용제를 10 내지 200 ng/ml 농도로 전처리한 것인, 약학 조성물.A pharmaceutical composition in claim 7, wherein the toll-like receptor 2 and 6 agonists are pretreated at a concentration of 10 to 200 ng/ml. 제5항에 있어서, 상기 톨 유사 수용체 2와 6으로 자극된 비만세포의 조정배지는 트립타제를 포함하는 것인, 약학 조성물.A pharmaceutical composition in claim 5, wherein the conditioned medium of mast cells stimulated with toll-like receptors 2 and 6 contains tryptase. 제1항에 있어서, 상기 피부 창상은 온도 손상, 외상, 버거병, 혈관 및 림프관 손상, 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 및 화학적 손상으로 이루어진 군으로부터 선택되는 어느 하나인, 약학 조성물.A pharmaceutical composition according to claim 1, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wound, stomatitis, bedsore, pressure ulcer, diabetic ulcer/gangrene, post-herpetic ulcer, drug-induced ulcer, skin ulcer, damage due to dermatitis, radiation damage, and chemical damage. 비만세포의 조정배지를 포함하는 피부 외용제 조성물.A composition for external application to the skin containing a conditioned medium of mast cells. 제11항에 있어서, 상기 비만세포는 사람 유래 비만세포인, 피부 외용제 조성물.A composition for external application to the skin, wherein the mast cells in claim 11 are human-derived mast cells. 제11항에 있어서, 상기 비만세포는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포인, 피부 외용제 조성물.A composition for external application to the skin, wherein the mast cells in claim 11 are mast cells stimulated with toll-like receptor 2 and 6 agonists. 비만세포의 조정배지를 포함하는 피부 창상 예방 또는 개선용 식품 조성물.A food composition for preventing or improving skin wounds, comprising a regulating medium for mast cells. 제14항에 있어서, 상기 비만세포는 톨 유사 수용체 2와 6 작용제로 자극된 비만세포인 식품 조성물.A food composition in claim 14, wherein the mast cells are mast cells stimulated with toll-like receptor 2 and 6 agonists. 비만세포의 조정배지를 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는 피부 창상의 예방 또는 치료 방법.A method for preventing or treating a skin wound, comprising the step of administering a mast cell-modified medium to a subject in need thereof. 제16항에 있어서, 상기 피부 창상은 온도 손상, 외상, 버거병, 혈관 및 림프관 손상, 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 및 화학적 손상으로 이루어진 군으로부터 선택되는 어느 하나인, 피부 창상 예방 또는 치료 방법.A method for preventing or treating a skin wound, in claim 16, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wound, stomatitis, bedsore, pressure ulcer, diabetic ulcer/gangrene, post-herpetic ulcer, drug-induced ulcer, skin ulcer, damage due to dermatitis, radiation damage, and chemical damage. 피부 창상의 예방 또는 치료를 위한 약제의 제조에서 비만세포의 조정배지의 용도.Use of a conditioned medium of mast cells in the manufacture of a medicament for the prevention or treatment of skin wounds. 제18항에 있어서, 상기 피부 창상은 온도 손상, 외상, 버거병, 혈관 및 림프관 손상, 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 및 화학적 손상으로 이루어진 군으로부터 선택되는 어느 하나인, 용도.The use of claim 18, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wounds, stomas, bedsores, pressure ulcers, diabetic ulcers/gangrene, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, and chemical damage. 피부 창상의 예방 또는 치료에 사용하기 위한 비만세포의 조정배지를 포함하는 조성물.A composition comprising a conditioned medium of mast cells for use in the prevention or treatment of skin wounds. 제20항에 있어서, 상기 피부 창상은 온도 손상, 외상, 버거병, 혈관 및 림프관 손상, 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 및 화학적 손상으로 이루어진 군으로부터 선택되는 어느 하나인, 조성물.A composition according to claim 20, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wound, stomatitis, bedsores, pressure ulcers, diabetic ulcers/gangrene, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, and chemical damage. 피부 창상의 예방 또는 치료를 위한 비만세포의 조정배지의 용도.Use of a mast cell-conditioned medium for the prevention or treatment of skin wounds. 제22항에 있어서, 상기 피부 창상은 온도 손상, 외상, 버거병, 혈관 및 림프관 손상, 수술 후 창상, 장루, 욕창, 압박성 궤양, 당뇨병성 궤양ㆍ탈저(gangrene), 대상포진 후 궤양, 약물성 궤양, 피부 궤양, 피부염에 의한 손상, 방사선 손상, 및 화학적 손상으로 이루어진 군으로부터 선택되는 어느 하나인, 용도.The use of claim 22, wherein the skin wound is any one selected from the group consisting of temperature damage, trauma, Buerger's disease, vascular and lymphatic damage, post-surgical wounds, stomas, bedsores, pressure ulcers, diabetic ulcers/gangrene, post-herpetic ulcers, drug-induced ulcers, skin ulcers, damage due to dermatitis, radiation damage, and chemical damage.
PCT/KR2024/002453 2023-03-03 2024-02-26 Composition comprising mast cell-conditioned medium for wound healing Pending WO2024186031A1 (en)

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FRUNGIERI B., WEIDINGER STEPHAN, MEINEKE VIKTOR, KÖHN FRANK M, MAYERHOFER ARTUR: "Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPAR␥: Possible relevance to human fibrotic disorders", PNAS, vol. 99, no. 23, 17 July 2002 (2002-07-17), pages 15072 - 15077, XP093208620 *
NUMATA TAKAFUMI, HARADA KAZUTOSHI, NAKAE SUSUMU: "Roles of Mast Cells in Cutaneous Diseases", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 13, Lausanne, CH , pages 1 - 12, XP093208627, ISSN: 1664-3224, DOI: 10.3389/fimmu.2022.923495 *
SONIA VARADARADJALOU; FRÉDÉRIC FÉGER; NATHALIE THIEBLEMONT; NADINE BEN HAMOUDA; JEAN‐MARIE PLEAU; MICHEL DY; MICHEL AROCK: "Toll‐like receptor 2 (TLR2) and TLR4 differentially activate human mast cells", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY-VCH, HOBOKEN, USA, vol. 33, no. 4, 18 March 2003 (2003-03-18), Hoboken, USA, pages 899 - 906, XP071222133, ISSN: 0014-2980, DOI: 10.1002/eji.200323830 *

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