WO2019107440A1 - Cholesterol synthesis promoter - Google Patents

Cholesterol synthesis promoter Download PDF

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Publication number
WO2019107440A1
WO2019107440A1 PCT/JP2018/043851 JP2018043851W WO2019107440A1 WO 2019107440 A1 WO2019107440 A1 WO 2019107440A1 JP 2018043851 W JP2018043851 W JP 2018043851W WO 2019107440 A1 WO2019107440 A1 WO 2019107440A1
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cells
extract
differentiation
dhcr7
epidermal
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PCT/JP2018/043851
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French (fr)
Japanese (ja)
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弓子 石松
勤 相馬
重良 藤原
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株式会社資生堂
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Priority to JP2019557285A priority Critical patent/JP7471085B2/en
Publication of WO2019107440A1 publication Critical patent/WO2019107440A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/74—Rubiaceae (Madder family)
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/81—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions involving only carbon-to-carbon unsaturated bonds
    • 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
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the present invention relates to a novel cholesterol synthesis promoter, a composition for promoting differentiation of epidermal cells comprising the cholesterol synthesis promoter, and a method of screening an agent for promoting epidermal cell differentiation.
  • Aging is progressing in organs throughout the body, but among them, the skin that can be seen visually, especially for the face that is particularly easy to concentrate, wrinkles and small wrinkles that occur with aging are many of the elderly people in the world. , Especially women. Recently, not only women but also men have a strong desire to look young, and in particular, it is desired to reduce skin wrinkles that appear with aging.
  • PRP therapy is a technology whose research and clinical application started from around 1985, and was originally used mainly for bone regeneration induction therapy in dental implant treatment, but then it is likely to be applied to cosmetic medicine It is mainly applied for eliminating wrinkles and slackness.
  • PRP therapy has not been discouraged from the point of foreign body injection because it returns its own blood, and it has also been found that the effect lasts for a long time.
  • PRP contains various factors derived from platelets, which act in concert with serum fibrin to activate fibroblasts in the dermis and promote collagen production etc., resulting in a fresh and firm skin. It is believed to contribute to At present, it has been suggested that PRP can be applied not only to the dermatology field but also to regeneration treatment of the liver and treatment of myocardial infarction in patients who have removed much of the liver due to liver cancer (Non-patent Document 1, Non-patent Document 1) 2, Non-Patent Document 3).
  • Non-Patent Document 1 PRP containing a large amount of leukocytes
  • W-PRP PRP containing a large amount of leukocytes
  • An object of the present invention is to provide a novel cholesterol synthesis promoter, a composition for promoting differentiation of epidermal cells containing the cholesterol synthesis promoter, and a method for screening a drug for promoting epidermal cell differentiation. is there.
  • the present inventors found that W-PRP treatment significantly increases DHCR7 gene expression.
  • the inventors of the present invention conducted extensive investigations and, surprisingly, nowadays, the expression of the DHCR7 gene in skin cells is closely involved in the differentiation of epidermal cells, and further, the extract of Asenyaku , A copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and an extract of Lycaenaceae promote the expression of the DHCR7 gene, leading to the completion of the present invention.
  • a cholesterol synthesis promoter comprising at least one component selected from the group consisting of acacia extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer extract.
  • a composition for promoting epidermal cell differentiation comprising the cholesterol synthesis promoter according to [1].
  • a method of screening for an agent that promotes epidermal cell differentiation which, when a candidate agent is caused to act on cultured cells, promotes the differentiation of epidermal cells by an agent that enhances the expression of the DHCR7 gene of the cell. A method characterized by selecting as a medicine.
  • a composition for promoting differentiation of epidermal cells at least one selected from the group consisting of acacia extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer tree extract Use of ingredients.
  • a composition for promoting the differentiation of epidermal cells which comprises an agent for enhancing the expression of DHCR7 gene.
  • a cosmetic or therapeutic method for promoting epidermal cell differentiation which comprises applying to a subject an agent that enhances the expression of DHCR7 gene.
  • the present invention it is possible to promote the differentiation of epidermal cells, thereby preventing, ameliorating or treating a condition or disease caused by suppression of epidermal cell differentiation.
  • FIG. 1 is a graph showing changes in DHCR7 gene expression before, two weeks after, and three months after treatment with W-PRP.
  • FIG. 2 is a photograph showing morphological changes associated with differentiation of human epidermal keratinocytes by a DHCR7 inhibitor (AY-9944).
  • FIG. 3 is a graph showing the expression change of epidermal cell molecular marker (keratin 10) by DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes on the 4th day of culture.
  • FIG. 4A is a graph showing the change in the concentration of cholesterol by human DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes 1 and 4 days after the initiation of differentiation.
  • FIG. 4B is a graph showing the cellular respiration activity of the DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes 1 day after the initiation of differentiation.
  • FIG. 5 is a graph showing the gene expression of DHCR7 by Asenyaku extract, MPC copolymer, retinol acetate, and raccoon bean extract at various concentrations in human epidermal keratinocytes.
  • FIG. 6 is a graph showing gene expression of epidermal differentiation marker (involucrin) by Asenyaku extract in human epidermal keratinocytes.
  • the DHCR7 gene is a gene encoding 7-dehydrocholesterol reductase (EC 1.3.1.21), which is mainly present in the intracellular endoplasmic reticulum membrane and known as an enzyme that converts 7-dehydrocholesterol to cholesterol.
  • the DHCR7 gene is known as the causative gene of Smith-Lemley Optiz syndrome (SOLS).
  • SOLS Smith-Lemley Optiz syndrome
  • Patients suffering from SOLS have symptoms such as mental retardation, facial atrophy, toe acupuncture, total forencephalopathy (if severe), etc., and it is also reported that photosensitivity to UVA is enhanced. Is thought to be due to the increase of 7-dehydrocholesterol in the blood, the increase of UVA-absorbing metabolites, and the production of active oxygen (Non-patent Document 7).
  • a cholesterol synthesis promoter comprising at least one component selected from the group consisting of aniseck extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer extract Provided.
  • Asenyaku extract is an extract derived from Asenyaku (scientific name: Uncarina gambir), which is a plant belonging to the Rubiaceae family distributed in Southeast Asia etc., and as a part to be used as an extraction material, for example, flower (carp) part , Leaf, branch, bark, root and the like, preferably leaf.
  • the asenyaku extract can be obtained by a conventional method, for example, after the site used as an extraction raw material is dried if necessary, it is immersed in the extraction solvent for a fixed period or contacted with the heating reflux refluxing extraction solvent and then filtered. It can be obtained by concentration.
  • any solvent which is usually used for extraction can be used, and examples thereof include water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, alcohols such as glycerin, chloroform, dichloroethane, Organic solvents such as carbon chloride, acetone and ethyl acetate can be used alone or in combination as appropriate.
  • the extract obtained by extraction with the above solvent is used as it is or a concentrated extract, or these extracts are adsorbed, for example, those from which impurities have been removed using an ion exchange resin, or porous polymers (eg Amberlite XAD- After being adsorbed on the column of 2), one eluted with methanol or ethanol and concentrated can also be used.
  • a distribution method such as an extract extracted with water / ethyl acetate.
  • the extract thus obtained can be used in the present invention as it is or after further dilution with ethanol or the like, or after solidification, the dried product as it is or the dried product is redissolved in, for example, ethanol.
  • the copolymer having a 2-methacryloyloxyethyl phosphorylcholine group is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic monomer, and has a weight average molecular weight of 600,000 to 700,000.
  • a copolymer of 2-methacryloyloxyethyl phosphoryl choline and butyl methacrylate (MPC copolymer) is used for a hand cream, a contact lens preservation solution, a shampoo rinse, etc. because it has an excellent moisturizing property.
  • Retinol acetate is an acetate ester of vitamin A and is used as a cosmetic ingredient such as moisturizing cream.
  • the extract of red bean is native to Southeast Asia and is an extract derived from red bean (scientific name: Psophocarpus tetragonolobus), which is also cultivated in Okinawa prefecture and Ogasawara Islands in Japan, and as a part to be used as an extraction raw material, for example , Anther, leaves, flowers, roots, whole grass, etc., but it is preferably a seed or anther.
  • the red bean extract can also be obtained by the conventional method described above.
  • a composition for promoting the differentiation of epidermal cells comprising the above-mentioned cholesterol synthesis promoter.
  • a composition for promoting the differentiation of epidermal cells comprising the above-mentioned cholesterol synthesis promoter.
  • Such a composition can be used as a cosmetic composition or a pharmaceutical composition, and can be used as a condition or disease caused by abnormal differentiation of epidermal cells due to a decrease in local cholesterol synthesis, for example, in the epidermis such as wrinkles and sags. It is effective for preventing, ameliorating or treating age-related changes of the skin accompanying cholesterol lowering.
  • the composition of the present invention can be appropriately determined for dosage, method of administration, dosage form according to the purpose of use.
  • the administration form of the composition of the present invention is not particularly limited, and may be oral, parenteral, external use, etc., but is preferably a skin external composition.
  • the dosage form include external preparations such as ointments, creams, emulsions, lotions, packs and bath preparations, parenteral preparations such as injections, drops, and suppositories, or tablets, powders, capsules, granules, An orally administered agent such as an extract, a syrup and the like can be mentioned.
  • the compounding quantity of the active ingredient of the composition of this invention can be suitably determined according to a use, in a composition whole quantity, the asenyaku extract is typically 0.0001%-10%, Preferably 0.001 % To 1%, optimally 0.05% to 0.5%.
  • Copolymers having 2-methacryloyloxyethyl phosphorylcholine groups are typically formulated at 0.0001% to 10%, preferably 0.001% to 1%, optimally 0.05% to 0.5%.
  • Retinol acetate is typically formulated at 0.000001% to 1%, preferably 0.00001% to 0.01%, optimally 0.00005% to 0.005%.
  • the red bean extract is typically formulated at 0.0001% to 10%, preferably 0.001% to 1%, optimally 0.05% to 0.5%.
  • composition of the present invention may, for example, be a skin-whitening agent usually used in cosmetics or pharmaceuticals, a moisturizer, an oil component, an ultraviolet light absorber, a surfactant, a thickener, alcohols, powders.
  • a skin-whitening agent usually used in cosmetics or pharmaceuticals
  • a moisturizer an oil component
  • an ultraviolet light absorber a surfactant
  • a thickener an alcohol
  • alcohols a surfactant
  • a thickener alcohols
  • ingredients, coloring agents, aqueous ingredients, water, various skin nutrients and the like can be appropriately blended as needed.
  • auxiliary agents commonly used for external preparations for skin such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconate And other sequestering agents, caffeine, tannin, verapamil, tranexamic acid and derivatives thereof, glabridin, hot water extract of cullin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizinic acid and its derivatives or salts thereof, vitamin C Ascorbic acid phosphate phosphate, ascorbic acid glucoside, albutin, kojic acid etc., whitening agents, glucose, fructose, mannose, sucrose, saccharides such as trehalose etc. can be suitably blended.
  • Enhancement of DHCR7 gene expression can be determined, for example, by directly measuring the amount of DHCR7 in cultured cells.
  • the cultured cells are typically skin cells, in particular epidermal cells, and include keratinocytes, granule cells, spinous cells and the like, and even if they are of human origin, other animals such as rats, mice, rabbits Or the like.
  • DHCR7 When measuring the amount of DHCR7 directly, preferably using an antibody specific for DHCR7, methods well known in the art, such as immunostaining methods using fluorescent substances, dyes, enzymes, etc., Western blotting, immunoassay methods For example, various methods such as ELISA and RIA can be used. Alternatively, it can be determined by extracting RNA from cultured cells and measuring the amount of mRNA encoding DHCR7. Extraction of mRNA and measurement of its amount are also well known in the art, for example, quantification of RNA is performed by quantitative polymerase chain reaction (PCR), such as real-time polymerase chain reaction (RT-PCR). Selection of appropriate primers for RT-PCR can be carried out by methods known to those skilled in the art.
  • PCR quantitative polymerase chain reaction
  • RT-PCR real-time polymerase chain reaction
  • RT-PCR analysis of DHCR7 gene for example, the following primers can be used.
  • Forward primer 5'-AGGGGAAGGTGGGCGCAGGAC-3 '(SEQ ID NO: 1)
  • Reverse primer 5'-TTGGGCCTCCCAGCCTCTTGC-3 '(SEQ ID NO: 2)
  • the expression of the DHCR7 gene in cultured cells is renewed with a significant difference compared to the control value, preferably 30% or more, more preferably 50% or more, still more preferably 70% or more, most preferably If it is increased by 100% or more, it may be judged as "a drug that promotes epidermal cell differentiation".
  • the control value may be, for example, but not limited to, an average value of expression levels of DHCR7 gene of cultured cells at the corresponding site of a statistically significant number of healthy persons.
  • the above screening method makes it possible to easily obtain novel drugs that promote epidermal cell differentiation.
  • W-PRP treatment is given to the anterior side of upper arm of two subjects, and 2 weeks after treatment, 3 months after treatment, skin tissue by 5 mm punch I took it. Each sample is disrupted by cryopress in liquid nitrogen, RNA in cells is extracted with Isogen (Nippon Gene), and a labeled probe is synthesized according to the manual (Agilent), and approximately 40,000 genes are spotted. The hybridization was performed on a DNA microarray (one-color, whole mouse 44K, Agilent).
  • the obtained signal was detected using a scanner Agilent dual-laser Microarray scanner G2565AA, and quantified using analysis software Feature Extraction Software 9.1 (Agilent) to obtain a gene expression index value. As shown in FIG. 1, it was found that W-PRP treatment enhances DHCR7 expression.
  • a DHCR7 inhibitor (AY-9944) (Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO) was added to a final concentration of 10 ⁇ M, and cultured for 4 days. Observation of cell state was performed under a phase contrast microscope. As a control, a sample to which DMSO was added in the same amount (final concentration 0.1%) was used. As shown in the photograph of FIG. 2, the inhibition of DHCR7 inhibited the morphological change associated with the differentiation of epidermal cells.
  • the synthesized cDNA is used as a template, primer pairs of SEQ ID NOS: 1 and 2 described below, reaction reagent QuantiFast SYBR Green RT-PCR kit (Qiagen) and reaction device Lightcycler (Roche), keratin 10 gene as a differentiation marker Quantitative real-time PCR was performed.
  • the composition conditions were according to the Qiagen protocol.
  • the PCR conditions were: initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, elongation at 72 ° C. for 12 seconds.
  • G3PDH was used as an internal standard, and the mRNA amount of the control group was correct
  • Keratin 10 Forward primer: 5'-CCATCGATGACCTTAAAAATCAG-3 '(SEQ ID NO: 1) Reverse primer: 5'-GCAGAGCATCCTC ATTCTCATACTT-3 '(SEQ ID NO: 2)
  • G3PDH Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 3) Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 4)
  • AY-9944 significantly reduced keratin 10, a marker of differentiation of epidermal cells.
  • DHCR7 Cholesterol-producing enzyme (DHCR7) production promotion
  • CnT-07 medium CELLnTEC
  • the cells of passage 3 are suspended in the same medium, collagen-coated 12-well multiplate (Asahi Glass) )
  • At a rate of 50,000 cells and cultured for 3-5 days until the cells reached confluence at 37 ° C. in the presence of 5% CO 2 .
  • the culture was further continued for 1 day.
  • RNA extraction reagent MagNA Pure LC mRNA HS kit (Roche) and automated nucleic acid extractor MagNA Pure LC 1.0 Instrument (Roche) from cells after culture
  • cDNA was synthesized from the RNA by reverse transcriptase Superscript II (Invitrogen) using random hexamer primers according to the Invitrogen protocol.
  • the primer pair of SEQ ID NOS: 1 and 2 described below the primer pair of SEQ ID NOS: 1 and 2 described below, the reaction reagent QuantiFast SYBR Green RT-PCR kit (Qiagen) and the reaction device Light cycler (Roche) quantitative real-time PCR of DHCR7 gene quantitative went.
  • Composition conditions followed the protocol of Qiagen.
  • the PCR conditions were: initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, elongation at 72 ° C. for 12 seconds.
  • G3PDH was used as an internal standard, and the mRNA amount of the control group was correct
  • DHCR7 Forward primer: 5'-AGGGGAAGGTGGGCGCAGGAC-3 '(SEQ ID NO: 1) Reverse primer: 5'-TTGGGCCTCCCAGCCTCTTGC-3 '(SEQ ID NO: 2)
  • G3PDH Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 3) Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 4)
  • four kinds of drugs such as Asen Yak extract (Maruzen Pharmaceutical Co., Ltd.), MPC copolymer (NIPPON Yushi Oil Co., Ltd.), retinol acetate (BASF) and deer bear extract (One circle Falcos Co., Ltd.) promote DHCR7 production It turned out to have an effect (FIG. 5).
  • RNA extraction reagent MagNA Pure LC mRNA HS kit (Roche) and automated nucleic acid extractor MagNA Pure LC 1.0 Instrument (Roche) from cells after culture
  • cDNA was synthesized from the RNA by reverse transcriptase Superscript II (Invitrogen) using random hexamer primers according to the Invitrogen protocol.

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Abstract

Provided is a new cholesterol synthesis promoter. This cholesterol synthesis promoter uses, as an effective component, at least one component selected from the group consisting of Uncaria gambir extracts, copolymers having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and Psophocarpus tetragonolobus extracts.

Description

コレステロール合成促進剤Cholesterol synthesis promoter
　本発明は、新規なコレステロール合成促進剤、該コレステロール合成促進剤を含む表皮細胞の分化を促進するための組成物、及び表皮細胞の分化を促進する薬剤をスクリーニングする方法に関する。 The present invention relates to a novel cholesterol synthesis promoter, a composition for promoting differentiation of epidermal cells comprising the cholesterol synthesis promoter, and a method of screening an agent for promoting epidermal cell differentiation.
　老化は全身の臓器で進行しているが、その中でも目で見ることができる皮膚、とりわけ特に意識が集中しやすい顔面については、老化とともに発生するシワ及び小ジワが、世の多くの中高年齢者、とりわけ女性を悩ませている。最近では女性だけでなく男性でも、若く見られたいという願望が強くなってきており、とりわけ老化に伴い現れる皮膚のシワを緩和することが望まれている。 Aging is progressing in organs throughout the body, but among them, the skin that can be seen visually, especially for the face that is particularly easy to concentrate, wrinkles and small wrinkles that occur with aging are many of the elderly people in the world. , Especially women. Recently, not only women but also men have a strong desire to look young, and in particular, it is desired to reduce skin wrinkles that appear with aging.
　このような要望に対し、従来、ボツリヌス毒素やヒアルロン酸などの注入が行われてきたが、このような施術においては、異物を注入する点から敬遠され、また、効果の持続性の点からも不満がもたれていた。これらの施術に代わり、近年では、自己血液中の血小板画分を濃縮し、それを自己の患部に注入する「多血小板血漿（Platelet Rich Plasma: PRP）療法」と称される施術が美容医療界に広まっている。ＰＲＰ療法は、1985年頃から研究や臨床応用が開始された技術であり、当初は主に歯科用のインプラント治療における骨再生誘導療法などに用いられていたが、その後、美容医療に応用されるようになり、主にシワやたるみの解消のために施されている。ＰＲＰ療法は、自己の血液を戻すことから異物注入という点から敬遠されることはなく、また、効果の点でも長期にわたって持続することが明らかになっている。ＰＲＰ中には血小板由来の各種因子が含有されており、これらが血清のフィブリンと協調的に作用し、真皮中の線維芽細胞を活性化し、コラーゲン産生等を促進して、みずみずしいハリのある肌に寄与するものと考えられている。現在では、ＰＲＰは、皮膚科領域にとどまらず、肝臓癌により肝臓の多くを摘出した患者における肝臓の再生治療や心筋梗塞治療にも応用できることが示唆されている（非特許文献１、非特許文献２、非特許文献３）。また、白血球を多く含むＰＲＰ（Ｗ－ＰＲＰ）は、従来のＰＲＰ技術と比較してより有効性が高いことが報告されており（非特許文献１、非特許文献２）、皮膚老化の改善に有用であることが示されている（非特許文献４、非特許文献５、非特許文献６）。 In response to such requests, botulinum toxin and hyaluronic acid have been conventionally injected, but in such procedures, it is avoided from the point of injecting a foreign substance, and also from the point of sustainability of the effect. I was dissatisfied. Instead of these treatments, in recent years, a treatment called "Platelet Rich Plasma (PRP) therapy", which concentrates the platelet fraction in autologous blood and injects it into a patient's own afflicted area, is a cosmetic medicine field It is widespread. PRP therapy is a technology whose research and clinical application started from around 1985, and was originally used mainly for bone regeneration induction therapy in dental implant treatment, but then it is likely to be applied to cosmetic medicine It is mainly applied for eliminating wrinkles and slackness. PRP therapy has not been discouraged from the point of foreign body injection because it returns its own blood, and it has also been found that the effect lasts for a long time. PRP contains various factors derived from platelets, which act in concert with serum fibrin to activate fibroblasts in the dermis and promote collagen production etc., resulting in a fresh and firm skin. It is believed to contribute to At present, it has been suggested that PRP can be applied not only to the dermatology field but also to regeneration treatment of the liver and treatment of myocardial infarction in patients who have removed much of the liver due to liver cancer (Non-patent Document 1, Non-patent Document 1) 2, Non-Patent Document 3). In addition, PRP containing a large amount of leukocytes (W-PRP) has been reported to be more effective than conventional PRP techniques (Non-Patent Document 1, Non-Patent Document 2), and for the improvement of skin aging. It is shown that it is useful (nonpatent literature 4, nonpatent literature 5, nonpatent literature 6).
　しかしながら、Ｗ－ＰＲＰの治療効果には個人差が大きく、また、Ｗ－ＰＲＰの治療メカニズムについては依然として十分に知られていない。 However, the therapeutic effects of W-PRP vary widely among individuals, and the treatment mechanism of W-PRP is still not sufficiently known.
　本発明の課題は、新規なコレステロール合成促進剤、該コレステロール合成促進剤を含む表皮細胞の分化を促進するための組成物、及び表皮細胞の分化を促進する薬剤をスクリーニングする方法を提供することにある。 An object of the present invention is to provide a novel cholesterol synthesis promoter, a composition for promoting differentiation of epidermal cells containing the cholesterol synthesis promoter, and a method for screening a drug for promoting epidermal cell differentiation. is there.
　本発明者らは、マイクロアレイを利用し、Ｗ－ＰＲＰの施術前と施術後における皮膚中の遺伝子発現プロファイルを調べた結果、Ｗ－ＰＲＰ施術によって、ＤＨＣＲ７遺伝子の発現が有意に増加されるという知見を見出した。かかる知見に基づき、本発明者らは、鋭意検討したところ、この度、驚くべきことに、皮膚細胞中のＤＨＣＲ７遺伝子の発現が表皮細胞の分化に密接に関与していること、さらには、アセンヤク抽出物、２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマー、酢酸レチノール、及びシカクマメ抽出物が、ＤＨＣＲ７遺伝子の発現を促進することを見出し、本発明を完成するに至った。 As a result of examining gene expression profiles in the skin before and after W-PRP treatment using the microarray, the present inventors found that W-PRP treatment significantly increases DHCR7 gene expression. Found out. Based on such findings, the inventors of the present invention conducted extensive investigations and, surprisingly, nowadays, the expression of the DHCR7 gene in skin cells is closely involved in the differentiation of epidermal cells, and further, the extract of Asenyaku , A copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and an extract of Lycaenaceae promote the expression of the DHCR7 gene, leading to the completion of the present invention.
　したがって、本願は以下の発明を包含する：
［１］アセンヤク抽出物、２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマー、酢酸レチノール、及びシカクマメ抽出物からなる群から選択される少なくとも１種類の成分を含むコレステロール合成促進剤。
［２］１に記載のコレステロール合成促進剤を含む、表皮細胞の分化を促進するための組成物。
［３］表皮細胞の分化を促進する薬剤をスクリーニングする方法であって、候補薬剤を培養細胞に作用させた場合に、該細胞のＤＨＣＲ７遺伝子の発現を亢進させる薬剤を表皮細胞の分化を促進する薬剤として選定することを特徴とする方法。
［４］前記培養細胞がヒト表皮角化細胞である、３に記載の方法。
［５］表皮細胞の分化を促進するための組成物の調製における、アセンヤク抽出物、２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマー、酢酸レチノール、及びシカクマメ抽出物からなる群から選択される少なくとも１種類の成分の使用。
［６］ＤＨＣＲ７遺伝子の発現を亢進させる薬剤を含む、表皮細胞の分化を促進するための組成物。
［７］表皮細胞の分化を促進するための美容的又は治療的な方法であって、ＤＨＣＲ７遺伝子の発現を亢進させる薬剤を対象に適用することを含む方法。
［８］表皮細胞の分化を促進するための組成物の調製における、ＤＨＣＲ７遺伝子の発現を亢進させる薬剤の使用。 Accordingly, the present application includes the following inventions:
[1] A cholesterol synthesis promoter comprising at least one component selected from the group consisting of acacia extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer extract.
[2] A composition for promoting epidermal cell differentiation, comprising the cholesterol synthesis promoter according to [1].
[3] A method of screening for an agent that promotes epidermal cell differentiation, which, when a candidate agent is caused to act on cultured cells, promotes the differentiation of epidermal cells by an agent that enhances the expression of the DHCR7 gene of the cell. A method characterized by selecting as a medicine.
[4] The method according to 3, wherein the cultured cells are human epidermal keratinocytes.
[5] In the preparation of a composition for promoting differentiation of epidermal cells, at least one selected from the group consisting of acacia extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer tree extract Use of ingredients.
[6] A composition for promoting the differentiation of epidermal cells, which comprises an agent for enhancing the expression of DHCR7 gene.
[7] A cosmetic or therapeutic method for promoting epidermal cell differentiation, which comprises applying to a subject an agent that enhances the expression of DHCR7 gene.
[8] Use of an agent that enhances DHCR7 gene expression in the preparation of a composition for promoting epidermal cell differentiation.
　本発明により、表皮細胞の分化を促進させ、これにより、表皮細胞の分化の抑制に起因する状態又は疾患を予防、改善又は治療することが可能となる。 According to the present invention, it is possible to promote the differentiation of epidermal cells, thereby preventing, ameliorating or treating a condition or disease caused by suppression of epidermal cell differentiation.
図１は、Ｗ－ＰＲＰの施術前、施術２週間後、及び施術３か月後におけるＤＨＣＲ７遺伝子の発現変化を示すグラフである。FIG. 1 is a graph showing changes in DHCR7 gene expression before, two weeks after, and three months after treatment with W-PRP. 図２は、ＤＨＣＲ７阻害剤（ＡＹ－９９４４）による、ヒト表皮角化細胞の分化に伴う形態変化を示す写真である。FIG. 2 is a photograph showing morphological changes associated with differentiation of human epidermal keratinocytes by a DHCR7 inhibitor (AY-9944). 図３は、培養４日目のヒト表皮角化細胞における、ＤＨＣＲ７阻害剤（ＡＹ－９９４４）による表皮細胞分子マーカー（ケラチン１０）の発現変化を示すグラフである。FIG. 3 is a graph showing the expression change of epidermal cell molecular marker (keratin 10) by DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes on the 4th day of culture. 図４Ａは、分化開始後１日目及び４日目のヒト表皮角化細胞における、ＤＨＣＲ７阻害剤（ＡＹ－９９４４）によるコレステロールの濃度変化を示すグラフである。図４Ｂは、分化開始後１日目のヒト表皮角化細胞における、ＤＨＣＲ７阻害剤（ＡＹ－９９４４）による細胞呼吸活性を示すグラフである。FIG. 4A is a graph showing the change in the concentration of cholesterol by human DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes 1 and 4 days after the initiation of differentiation. FIG. 4B is a graph showing the cellular respiration activity of the DHCR7 inhibitor (AY-9944) in human epidermal keratinocytes 1 day after the initiation of differentiation. 図５は、ヒト表皮角化細胞における、各濃度のアセンヤク抽出物、ＭＰＣコポリマー、酢酸レチノール、及びシカクマメ抽出物によるＤＨＣＲ７の遺伝子発現を示すグラフである。FIG. 5 is a graph showing the gene expression of DHCR7 by Asenyaku extract, MPC copolymer, retinol acetate, and raccoon bean extract at various concentrations in human epidermal keratinocytes. 図６は、ヒト表皮角化細胞における、アセンヤク抽出物による表皮分化マーカー（インボルクリン）の遺伝子発現を示すグラフである。FIG. 6 is a graph showing gene expression of epidermal differentiation marker (involucrin) by Asenyaku extract in human epidermal keratinocytes.
　ＤＨＣＲ７遺伝子は、主に細胞内の小胞体膜に存在し、７－デヒドロコレステロールをコレステロールに変換する酵素として知られる７－デヒドロコレステロールレダクターゼ（ＥＣ１．３．１．２１）をコードする遺伝子である。ＤＨＣＲ７遺伝子は、スミス－レムリーオプティズ症候群（ＳＯＬＳ）の原因遺伝子として知られている。ＳＯＬＳに罹患した患者は、精神遅延、顔面異型、つま先の合趾、全前脳症（重篤な場合）などの症状を有するほか、ＵＶＡに対する光感受性が亢進されることも報告されており、これは、血中の７－デヒドロコレステロールが増加からＵＶＡ吸収性の代謝産物が増加し、活性酸素が産生されることに起因するものと考えられている（非特許文献７）。 The DHCR7 gene is a gene encoding 7-dehydrocholesterol reductase (EC 1.3.1.21), which is mainly present in the intracellular endoplasmic reticulum membrane and known as an enzyme that converts 7-dehydrocholesterol to cholesterol. The DHCR7 gene is known as the causative gene of Smith-Lemley Optiz syndrome (SOLS). Patients suffering from SOLS have symptoms such as mental retardation, facial atrophy, toe acupuncture, total forencephalopathy (if severe), etc., and it is also reported that photosensitivity to UVA is enhanced. Is thought to be due to the increase of 7-dehydrocholesterol in the blood, the increase of UVA-absorbing metabolites, and the production of active oxygen (Non-patent Document 7).
　本発明の１つの態様においては、アセンヤク抽出物、２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマー、酢酸レチノール、及びシカクマメ抽出物からなる群から選択される少なくとも１種類の成分を含むコレステロール合成促進剤が提供される。 In one aspect of the present invention, there is provided a cholesterol synthesis promoter comprising at least one component selected from the group consisting of aniseck extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer extract Provided.
　アセンヤク抽出物は、東南アジア等に分布しているアカネ科に属する植物であるアセンヤク（学名：Uncarina gambir）に由来する抽出物であり、抽出原料として使用する部位としては、例えば、花（蕾）部、葉部、枝部、樹皮部、根部等が挙げられるが、好ましくは葉部である。アセンヤク抽出物は常法より得ることができ、例えば、抽出原料として使用する部位を必要により乾燥した後、抽出溶媒に一定期間浸漬するか、あるいは加熱還流している抽出溶媒と接触させ、次いで濾過し、濃縮して得ることができる。抽出溶媒としては、通常抽出に用いられる溶媒であれば任意に用いることができ、例えば、水、メタノール、エタノール、プロピレングリコール、１，３－ブチレングリコール、グリセリン等のアルコール類、クロロホルム、ジクロルエタン、四塩化炭素、アセトン、酢酸エチル等の有機溶媒を、それぞれ単独であるいは適宜組み合わせて用いることができる。上記溶媒で抽出して得た抽出液をそのまま、あるいは濃縮したエキスを用いるか、あるいはこれらエキスを吸着法、例えばイオン交換樹脂を用いて不純物を除去したものや、ポーラスポリマー（例えばアンバーライトＸＡＤ－２）のカラムにて吸着させた後、メタノールまたはエタノールで溶出し、濃縮したものも使用することができる。また分配法、例えば水／酢酸エチルで抽出した抽出物等も用いることができる。こうして得られる抽出物は、そのまま、あるいはエタノール等でさらに希釈し、または固化後、乾燥物をそのまま、もしくは乾燥物を例えばエタノールに再溶解して、本発明で用いることができる。 Asenyaku extract is an extract derived from Asenyaku (scientific name: Uncarina gambir), which is a plant belonging to the Rubiaceae family distributed in Southeast Asia etc., and as a part to be used as an extraction material, for example, flower (carp) part , Leaf, branch, bark, root and the like, preferably leaf. The asenyaku extract can be obtained by a conventional method, for example, after the site used as an extraction raw material is dried if necessary, it is immersed in the extraction solvent for a fixed period or contacted with the heating reflux refluxing extraction solvent and then filtered. It can be obtained by concentration. As the extraction solvent, any solvent which is usually used for extraction can be used, and examples thereof include water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, alcohols such as glycerin, chloroform, dichloroethane, Organic solvents such as carbon chloride, acetone and ethyl acetate can be used alone or in combination as appropriate. The extract obtained by extraction with the above solvent is used as it is or a concentrated extract, or these extracts are adsorbed, for example, those from which impurities have been removed using an ion exchange resin, or porous polymers (eg Amberlite XAD- After being adsorbed on the column of 2), one eluted with methanol or ethanol and concentrated can also be used. It is also possible to use a distribution method, such as an extract extracted with water / ethyl acetate. The extract thus obtained can be used in the present invention as it is or after further dilution with ethanol or the like, or after solidification, the dried product as it is or the dried product is redissolved in, for example, ethanol.
　２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマーは、２－メタクリロイルオキシエチルホスホリルコリンと疎水性モノマーとの共重合体であり、６０万～７０万の重量平均分子量を有する。例えば、２－メタクリロイルオキシエチルホスホリルコリンとメタクリル酸ブチルのコポリマー（ＭＰＣコポリマー）は、優れた保湿性を有することから、ハンドクリームやコンタクトレンズ保存液、シャンプー・リンスなどに使用されている。 The copolymer having a 2-methacryloyloxyethyl phosphorylcholine group is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic monomer, and has a weight average molecular weight of 600,000 to 700,000. For example, a copolymer of 2-methacryloyloxyethyl phosphoryl choline and butyl methacrylate (MPC copolymer) is used for a hand cream, a contact lens preservation solution, a shampoo rinse, etc. because it has an excellent moisturizing property.
　酢酸レチノールは、ビタミンＡの酢酸エステルであり、保湿クリームなどの化粧品成分として使用されている。 Retinol acetate is an acetate ester of vitamin A and is used as a cosmetic ingredient such as moisturizing cream.
　シカクマメ抽出物は、東南アジアを原産とし、日本の沖縄県や小笠原諸島などでも栽培されているシカクマメ（学名：Psophocarpus tetragonolobus）に由来する抽出物であり、抽出原料として使用する部位としては、例えば、種子、莢、葉、花、根、全草等が挙げられるが、好ましくは種子又は莢である。シカクマメ抽出物もまた、上述した常法により得ることができる。 The extract of red bean is native to Southeast Asia and is an extract derived from red bean (scientific name: Psophocarpus tetragonolobus), which is also cultivated in Okinawa prefecture and Ogasawara Islands in Japan, and as a part to be used as an extraction raw material, for example , Anther, leaves, flowers, roots, whole grass, etc., but it is preferably a seed or anther. The red bean extract can also be obtained by the conventional method described above.
　本発明のさらなる態様として、上記コレステロール合成促進剤を含む、表皮細胞の分化を促進するための組成物が提供される。このような組成物は、化粧料組成物又は医薬組成物として用いることができ、局所的なコレステロール合成の低下による表皮細胞の分化異常に起因する状態又は疾患、例えば、しわ、たるみなどの表皮内のコレステロール低下に伴う皮膚の加齢変化を予防、改善又は治療するために有効である。 As a further aspect of the present invention, there is provided a composition for promoting the differentiation of epidermal cells, comprising the above-mentioned cholesterol synthesis promoter. Such a composition can be used as a cosmetic composition or a pharmaceutical composition, and can be used as a condition or disease caused by abnormal differentiation of epidermal cells due to a decrease in local cholesterol synthesis, for example, in the epidermis such as wrinkles and sags. It is effective for preventing, ameliorating or treating age-related changes of the skin accompanying cholesterol lowering.
　本発明の組成物は、その使用目的に合わせて用量、用法、剤型を適宜決定することが可能である。例えば、本発明の組成物の投与形態は特に制限されるものではなく、経口、非経口、外用等であってよいが、好ましくは皮膚外用組成物である。剤型としては、例えば軟膏、クリーム、乳液、ローション、パック、浴用剤等の外用剤、注射剤、点滴剤、若しくは坐剤等の非経口投与剤、又は錠剤、粉剤、カプセル剤、顆粒剤、エキス剤、シロップ剤等の経口投与剤を挙げることができる。 The composition of the present invention can be appropriately determined for dosage, method of administration, dosage form according to the purpose of use. For example, the administration form of the composition of the present invention is not particularly limited, and may be oral, parenteral, external use, etc., but is preferably a skin external composition. Examples of the dosage form include external preparations such as ointments, creams, emulsions, lotions, packs and bath preparations, parenteral preparations such as injections, drops, and suppositories, or tablets, powders, capsules, granules, An orally administered agent such as an extract, a syrup and the like can be mentioned.
　本発明の組成物の活性成分の配合量は、用途に応じて適宜決定できるが、組成物全量中、アセンヤク抽出物は、典型的には０．０００１％～１０％、好適には０．００１％～１％、最適には０．０５％～０．５％で配合される。２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマーは、典型的には０．０００１％～１０％、好適には０．００１％～１％、最適には０．０５％～０．５％で配合される。酢酸レチノールは、典型的には０．０００００１％～１％、好適には０．００００１％～０．０１％、最適には０．００００５％～０．００５％で配合される。シカクマメ抽出物は、典型的には０．０００１％～１０％、好適には０．００１％～１％、最適には０．０５％～０．５％で配合される。 Although the compounding quantity of the active ingredient of the composition of this invention can be suitably determined according to a use, in a composition whole quantity, the asenyaku extract is typically 0.0001%-10%, Preferably 0.001 % To 1%, optimally 0.05% to 0.5%. Copolymers having 2-methacryloyloxyethyl phosphorylcholine groups are typically formulated at 0.0001% to 10%, preferably 0.001% to 1%, optimally 0.05% to 0.5%. Ru. Retinol acetate is typically formulated at 0.000001% to 1%, preferably 0.00001% to 0.01%, optimally 0.00005% to 0.005%. The red bean extract is typically formulated at 0.0001% to 10%, preferably 0.001% to 1%, optimally 0.05% to 0.5%.
　また、本発明の組成物には、上記活性成分以外に、例えば、化粧品や医薬品に通常用いられる美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色剤、水性成分、水、各種皮膚栄養剤等を必要に応じて適宜配合することができる。 In addition to the above active ingredients, the composition of the present invention may, for example, be a skin-whitening agent usually used in cosmetics or pharmaceuticals, a moisturizer, an oil component, an ultraviolet light absorber, a surfactant, a thickener, alcohols, powders. Ingredients, coloring agents, aqueous ingredients, water, various skin nutrients and the like can be appropriately blended as needed.
　さらに、本発明の組成物を皮膚外用剤として使用する場合、皮膚外用剤に慣用の助剤、例えばエデト酸二ナトリウム、エデト酸三ナトリウム、クエン酸ナトリウム、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸等の金属封鎖剤、カフェイン、タンニン、ベラパミル、トラネキサム酸及びその誘導体、グラブリジン、カリンの果実の熱水抽出物、各種生薬、酢酸トコフェロール、グリチルリチン酸及びその誘導体またはその塩等の薬剤、ビタミンＣ、アスコルビン酸リン酸マグネシウム、アスコルビン酸グルコシド、アルブチン、コウジ酸等の美白剤、グルコース、フルクトース、マンノース、ショ糖、トレハロース等の糖類なども適宜配合することができる。 Furthermore, when the composition of the present invention is used as an external preparation for skin, auxiliary agents commonly used for external preparations for skin, such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconate And other sequestering agents, caffeine, tannin, verapamil, tranexamic acid and derivatives thereof, glabridin, hot water extract of cullin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizinic acid and its derivatives or salts thereof, vitamin C Ascorbic acid phosphate phosphate, ascorbic acid glucoside, albutin, kojic acid etc., whitening agents, glucose, fructose, mannose, sucrose, saccharides such as trehalose etc. can be suitably blended.
　本発明の別の態様において、ＤＨＣＲ７遺伝子の発現の亢進を指標として用いた、表皮細胞の分化を促進する薬剤のスクリーニング方法が提供される。ＤＨＣＲ７遺伝子の発現の亢進は、例えば培養細胞中のＤＨＣＲ７の量を直接測定することにより決定することができる。培養細胞は、典型的には皮膚細胞、特には表皮細胞であり、角化細胞、顆粒細胞、有棘細胞等が含まれ、ヒト由来であっても、その他の動物、例えばラット、マウス、ウサギなどであってもよい。ＤＨＣＲ７の量を直接測定する場合、好ましくはＤＨＣＲ７に特異的な抗体を利用し、当業界において周知の方法、例えば蛍光物質、色素、酵素などを利用する免疫染色法、ウエスタンブロット法、免疫測定方法、例えばＥＬＩＳＡ法、ＲＩＡ法など、様々な方法により実施できる。あるいは、培養細胞からＲＮＡを抽出し、ＤＨＣＲ７をコードするｍＲＮＡの量を測定することにより決定することもできる。ｍＲＮＡの抽出、その量の測定も当業界において周知であり、例えばＲＮＡの定量は定量ポリメラーゼ連鎖反応法（ＰＣＲ）、例えばリアルタイムポリメラーゼ連鎖反応（ＲＴ－ＰＣＲ）により行われる。ＲＴ－ＰＣＲに適切なプライマーの選定は当業者に周知な方法により実施することができる。 In another aspect of the present invention, there is provided a method of screening for an agent that promotes differentiation of epidermal cells, using the enhancement of DHCR7 gene expression as an index. Enhancement of DHCR7 gene expression can be determined, for example, by directly measuring the amount of DHCR7 in cultured cells. The cultured cells are typically skin cells, in particular epidermal cells, and include keratinocytes, granule cells, spinous cells and the like, and even if they are of human origin, other animals such as rats, mice, rabbits Or the like. When measuring the amount of DHCR7 directly, preferably using an antibody specific for DHCR7, methods well known in the art, such as immunostaining methods using fluorescent substances, dyes, enzymes, etc., Western blotting, immunoassay methods For example, various methods such as ELISA and RIA can be used. Alternatively, it can be determined by extracting RNA from cultured cells and measuring the amount of mRNA encoding DHCR7. Extraction of mRNA and measurement of its amount are also well known in the art, for example, quantification of RNA is performed by quantitative polymerase chain reaction (PCR), such as real-time polymerase chain reaction (RT-PCR). Selection of appropriate primers for RT-PCR can be carried out by methods known to those skilled in the art.
　ＤＨＣＲ７遺伝子のＲＴ－ＰＣＲ分析には、例えば、以下のプライマーを使用することができる。
　　フォワードプライマー：5'-AGGGGAAGGTGGGCGCAGGAC-3'（配列番号１）
　　リバースプライマー：　5'-TTGGGCCCTCCAGCCTCTTGC-3'（配列番号２） For RT-PCR analysis of DHCR7 gene, for example, the following primers can be used.
Forward primer: 5'-AGGGGAAGGTGGGCGCAGGAC-3 '(SEQ ID NO: 1)
Reverse primer: 5'-TTGGGCCTCCCAGCCTCTTGC-3 '(SEQ ID NO: 2)
　上述の測定において、例えば培養細胞中のＤＨＣＲ７遺伝子の発現がコントロール値と比べ有意差を持って更新し、好ましくは３０％以上、より好ましくは５０％以上、さらに好ましくは７０％以上、最も好ましくは１００％以上亢進したら、「表皮細胞の分化を促進する薬剤」と判断する、としてよい。コントロール値としては、限定されるものではないが、例えば統計学的に有意な数の健常人の対応の部位における培養細胞のＤＨＣＲ７遺伝子の発現量の平均値であってよい。 In the above-mentioned measurement, for example, the expression of the DHCR7 gene in cultured cells is renewed with a significant difference compared to the control value, preferably 30% or more, more preferably 50% or more, still more preferably 70% or more, most preferably If it is increased by 100% or more, it may be judged as "a drug that promotes epidermal cell differentiation". The control value may be, for example, but not limited to, an average value of expression levels of DHCR7 gene of cultured cells at the corresponding site of a statistically significant number of healthy persons.
　上記スクリーニング方法によって、表皮細胞の分化を促進する新規な薬剤を容易に得ることが可能となる。 The above screening method makes it possible to easily obtain novel drugs that promote epidermal cell differentiation.
　以下の実施例により、本発明を更に具体的に説明する。なお、本発明はこれに限定されるものではない。 The present invention will be more specifically described by the following examples. The present invention is not limited to this.
１．Ｗ－ＰＲＰ施術により発現が亢進される遺伝子のマイクロアレイ解析
　二人の被験者の上腕前側部にＷ－ＰＲＰ施術を施し、施術前、施術２週間後、施術３か月後に５ｍｍパンチにより皮膚組織の採取を行った。それぞれの試料を液体窒素中でクライオプレスにより破砕し、Isogen(ニッポンジーン社)で細胞内のＲＮＡを抽出し、マニュアル(Agilent 社)に従い、ラベル化プローブを合成し、約４万個の遺伝子がスポットされた、ＤＮＡマイクロアレイ(one-color, whole mouse 44K、Agilent社)上で、ハイブリダイゼーションを行った。得られたシグナルはスキャナーAgilent dual-laser Microarray scanner G2565AA を用いて検出し、解析ソフトFeature Extraction Software 9.1 (Agilent 社)を用いて数値化し遺伝子発現指標値とした。
　図１に示されるとおり、Ｗ－ＰＲＰ施術により、ＤＨＣＲ７の発現が亢進することが判明した。 1. Microarray analysis of genes whose expression is enhanced by W-PRP treatment W-PRP treatment is given to the anterior side of upper arm of two subjects, and 2 weeks after treatment, 3 months after treatment, skin tissue by 5 mm punch I took it. Each sample is disrupted by cryopress in liquid nitrogen, RNA in cells is extracted with Isogen (Nippon Gene), and a labeled probe is synthesized according to the manual (Agilent), and approximately 40,000 genes are spotted. The hybridization was performed on a DNA microarray (one-color, whole mouse 44K, Agilent). The obtained signal was detected using a scanner Agilent dual-laser Microarray scanner G2565AA, and quantified using analysis software Feature Extraction Software 9.1 (Agilent) to obtain a gene expression index value.
As shown in FIG. 1, it was found that W-PRP treatment enhances DHCR7 expression.
２．ＤＨＣＲ７阻害剤による表皮角化細胞分化への効果
　ヒト表皮角化細胞をＣｎＴ－０７培地（CELLnTEC社）で継代培養し、継代４代目の細胞を同培地に懸濁して、コラーゲンコート６穴マルチプレート（旭硝子）に２×１０5 細胞／ウェルになるように播種し、５％ＣＯ2存在下、３７℃で細胞が集密に達するまで３～５日間の培養を行った。ＣｎＴ－０２培地（CELLnTEC社）に交換し、分化誘導を開始した。同時に、ジメチルスルホキシド（ＤＭＳＯ）中に溶解したＤＨＣＲ７阻害剤（ＡＹ－９９４４）（Sigma-Aldrich社）を最終濃度１０μＭとなるように添加し、４日間培養した。細胞の状態観察は位相差顕微鏡下で行った。コントロールとしては、ＤＭＳＯを同量（最終濃度０．１％）添加したサンプルを用いた。
　図２の写真に示されるとおり、ＤＨＣＲ７の阻害により、表皮細胞の分化にともなう形態の変化が阻害された。 2. Effect on epidermal keratinocyte differentiation by DHCR7 inhibitor Human epidermal keratinocytes are subcultured in CnT-07 medium (CELLnTEC Co., Ltd.), and the cells at passage 4 are suspended in the same medium, and collagen-coated 6 wells are used. The cells were seeded at 2 × 10 5 cells / well in Multiplate (Asahi Glass), and cultured for 3-5 days until the cells reached confluence at 37 ° C. in the presence of 5% CO 2 . It replaced | exchanged for CnT-02 culture medium (CELLnTEC), and differentiation induction was started. At the same time, a DHCR7 inhibitor (AY-9944) (Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO) was added to a final concentration of 10 μM, and cultured for 4 days. Observation of cell state was performed under a phase contrast microscope. As a control, a sample to which DMSO was added in the same amount (final concentration 0.1%) was used.
As shown in the photograph of FIG. 2, the inhibition of DHCR7 inhibited the morphological change associated with the differentiation of epidermal cells.
３．ＤＨＣＲ７阻害剤による表皮角化細胞分化マーカーの発現変化
　培養後の細胞からＲＮＡ抽出試薬MagNA Pure LC mRNA HSキット（Roche社）と自動核酸抽出装置 MagNA Pure LC 1.0 インスツルメント（Roche社）を用いて、提供されたプロトコールに従ってｍＲＮＡの抽出・精製を行った。上述のＤＨＣＲ７阻害剤（ＡＹ－９９４４）及びコントロールについて、逆転写酵素 Superscript II（Invitrogen社）によりＲＮＡからランダムヘキサマー・プライマーを用いて、Invitrogen社のプロトコールにしたがいｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、後述の配列番号１及び２のプライマーペア、反応試薬 QuantiFast SYBR Green RT-PCR キット（Qiagen社）及び反応装置 LightCycler（Roche社）を用いて、分化マーカーであるケラチン１０遺伝子の定量リアルタイムＰＣＲを行った。組成条件は Qiagen社のプロトコールに従った。また、ＰＣＲの条件は、初期変性９５℃で１０分、変性９５℃で１０秒、アニール６０℃で１０秒、伸長７２℃で１２秒とした。なお、Ｇ３ＰＤＨは内部標準として用い、これを用いてコントロール群のｍＲＮＡ量を補正した。
ケラチン１０：
　　フォワードプライマー：5'-CCATCGATGACCTTAAAAATCAG-3'（配列番号１）
　　リバースプライマー：5'-GCAGAGCTACCTCATTCTCATACTT-3'（配列番号２）
Ｇ３ＰＤＨ：
　　フォワードプライマー：5'-GCACCGTCAAGGCTGAGAAC-3'（配列番号３）
　　リバースプライマー：　5'-ATGGTGGTGAAGACGCCAGT-3'（配列番号４）
　図３のグラフに示されるとおり、ＡＹ－９９４４により、表皮細胞の分化マーカーであるケラチン１０が顕著に減少した。 3. Expression change of epidermal keratinocyte differentiation marker by DHCR7 inhibitor RNA extraction reagent from cultured cells MagNA Pure LC mRNA HS kit (Roche) and automatic nucleic acid extractor MagNA Pure LC 1.0 Instrument (Roche) Extraction and purification of mRNA were performed according to the provided protocol. For the above-mentioned DHCR7 inhibitor (AY-9944) and control, cDNA was synthesized from RNA with reverse transcriptase Superscript II (Invitrogen) using random hexamer primers according to the Invitrogen protocol. The synthesized cDNA is used as a template, primer pairs of SEQ ID NOS: 1 and 2 described below, reaction reagent QuantiFast SYBR Green RT-PCR kit (Qiagen) and reaction device Lightcycler (Roche), keratin 10 gene as a differentiation marker Quantitative real-time PCR was performed. The composition conditions were according to the Qiagen protocol. The PCR conditions were: initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, elongation at 72 ° C. for 12 seconds. In addition, G3PDH was used as an internal standard, and the mRNA amount of the control group was correct | amended using this.
Keratin 10:
Forward primer: 5'-CCATCGATGACCTTAAAAATCAG-3 '(SEQ ID NO: 1)
Reverse primer: 5'-GCAGAGCATCCTC ATTCTCATACTT-3 '(SEQ ID NO: 2)
G3PDH:
Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 3)
Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 4)
As shown in the graph of FIG. 3, AY-9944 significantly reduced keratin 10, a marker of differentiation of epidermal cells.
４．細胞コレステロール量の測定
　上記で調製した分化開始後１日目及び４日目の細胞を２００μｌのクロロフォルム：イソプロパノール：ＮＰ－４０（７：１１：０．１）で抽出した後、溶媒を蒸散させ、コレステロール／コレステロールエステル定量キット（BioVision社）に含まれるコレステロールアッセイバッファー中に懸濁して測定試料とした。キット付属の９６ウェルプレートに試料を添加し、各ウェルに５０μｌの反応ミックスを加え、３７℃で１時間インキュベートした後、プレートリーダーを用い波長５７０ｎｍの吸光度を測定した。キット付属の標準品を用いて検量線を作成し、サンプル中のコレステロール濃度を定量した。
　図４Ａに示されるとおり、ＡＹ－９９４４により表皮細胞に含有されるコレステロール量は減少した。 4. Measurement of Cellular Cholesterol Content After extracting the cells prepared on the first and fourth days after the initiation of differentiation with 200 μl of chloroform: isopropanol: NP-40 (7: 11: 0.1), the solvent is evaporated, The sample was suspended in cholesterol assay buffer contained in a cholesterol / cholesterol ester assay kit (BioVision) to prepare a measurement sample. The sample was added to the 96-well plate attached to the kit, 50 μl of the reaction mix was added to each well, and after incubating for 1 hour at 37 ° C., the absorbance at a wavelength of 570 nm was measured using a plate reader. A standard curve was prepared using a standard product supplied with the kit to quantify cholesterol concentration in the sample.
As shown in FIG. 4A, AY-9944 reduced the amount of cholesterol contained in epidermal cells.
５．細胞呼吸活性の測定
　上記で調製した分化開始後１日目の細胞培養液をalamarBlue(R)Cell Viability Reagent（invitrogen社）を１０％含有するＣｎＴ－０２培地（CELLnTEC社）に交換し、５％ＣＯ2存在下、３７℃で３時間インキュベーションした後、蛍光プレートリーダーを用い励起波長／蛍光波長：５４０／５９０ｎｍで計測した。
　図４Ｂに示されるとおり、ＡＹ－９９４４によって細胞増殖に変化は見られなかった。
　図４Ａ及び図４Ｂに示される結果から、実験２におけるケラチン１０の減少は細胞死によるものではないこと、及び、ＤＨＣＲ７の阻害により細胞のコレステロール含有量が確かに減少していることが確認された。 5. Measurement of Cell Respiratory Activity The cell culture solution prepared on the first day after the initiation of differentiation is replaced with CnT-02 medium (CELLnTEC) containing 10% of alamarBlue (R) Cell Viability Reagent (invitrogen), 5% After incubation at 37 ° C. for 3 hours in the presence of CO 2 , measurement was performed at an excitation wavelength / fluorescence wavelength: 540/590 nm using a fluorescence plate reader.
As shown in FIG. 4B, no change in cell proliferation was seen by AY-9944.
From the results shown in FIGS. 4A and 4B, it was confirmed that the reduction of keratin 10 in Experiment 2 was not due to cell death, and that the cholesterol content of the cell was indeed reduced by the inhibition of DHCR7. .
６．コレステロール産生酵素（ＤＨＣＲ７）産生促進
　ヒト表皮角化細胞をＣｎＴ－０７培地（CELLnTEC社）で継代培養し、継代３代目の細胞を同培地に懸濁して、コラーゲンコート１２穴マルチプレート（旭硝子）に５０，０００個の割合で播種し、５％ＣＯ2存在下、３７℃で細胞が集密に達するまで３～５日間の培養を行った。３０品目の候補薬剤を各評価対象サンプルとして、各サンプルを上記濃度となるように添加、又は各評価対象サンプルが溶解している溶媒であるＤＭＳＯを同量添加したＣｎＴ－０７培地に交換した後、更に１日間培養を行った。培養後の細胞からＲＮＡ抽出試薬MagNA Pure LC mRNA HSキット（Roche社）と自動核酸抽出装置 MagNA Pure LC 1.0 インスツルメント（Roche社）を用いて、提供されたプロトコールに従ってｍＲＮＡの抽出・精製を行った。各サンプルについて、逆転写酵素 Superscript II（Invitrogen社）によりＲＮＡからランダムヘキサマー・プライマーを用いて、Invitrogen社のプロトコールにしたがいｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、後述の配列番号１及び２のプライマーペア、反応試薬 QuantiFast SYBR Green RT-PCR キット（Qiagen社）及び反応装置 LightCycler（Roche社）を用いて、ＤＨＣＲ７遺伝子の定量リアルタイムＰＣＲを行った。組成条件はQiagen社のプロトコールに従った。また、ＰＣＲの条件は、初期変性９５℃で１０分、変性９５℃で１０秒、アニール６０℃で１０秒、伸長７２℃で１２秒とした。なお、Ｇ３ＰＤＨは内部標準として用い、これを用いてコントロール群のｍＲＮＡ量を補正した。
ＤＨＣＲ７：
　　フォワードプライマー：5'-AGGGGAAGGTGGGCGCAGGAC-3'（配列番号１）
　　リバースプライマー：　5'-TTGGGCCCTCCAGCCTCTTGC-3'（配列番号２）
Ｇ３ＰＤＨ：
　　フォワードプライマー：5'-GCACCGTCAAGGCTGAGAAC-3'（配列番号３）
　　リバースプライマー：　5'-ATGGTGGTGAAGACGCCAGT-3'（配列番号４）
　その結果、アセンヤク抽出物（丸善製薬（株））、ＭＰＣコポリマー（日本油脂（株））、酢酸レチノール（BASF社）及びシカクマメ抽出物（一丸ファルコス（株））の４種類の薬剤がＤＨＣＲ７産生促進効果を有することが判明した（図５）。 6. Cholesterol-producing enzyme (DHCR7) production promotion The human epidermal keratinocytes are subcultured in CnT-07 medium (CELLnTEC), the cells of passage 3 are suspended in the same medium, collagen-coated 12-well multiplate (Asahi Glass) ) At a rate of 50,000 cells, and cultured for 3-5 days until the cells reached confluence at 37 ° C. in the presence of 5% CO 2 . After replacing 30 candidate drug samples with each evaluation target sample, or adding CnT-07 medium containing the same amount of DMSO, which is the solvent in which each evaluation target sample is dissolved. The culture was further continued for 1 day. Perform extraction and purification of mRNA according to the provided protocol using RNA extraction reagent MagNA Pure LC mRNA HS kit (Roche) and automated nucleic acid extractor MagNA Pure LC 1.0 Instrument (Roche) from cells after culture The For each sample, cDNA was synthesized from the RNA by reverse transcriptase Superscript II (Invitrogen) using random hexamer primers according to the Invitrogen protocol. Using the synthesized cDNA as a template, the primer pair of SEQ ID NOS: 1 and 2 described below, the reaction reagent QuantiFast SYBR Green RT-PCR kit (Qiagen) and the reaction device Light cycler (Roche) quantitative real-time PCR of DHCR7 gene quantitative went. Composition conditions followed the protocol of Qiagen. The PCR conditions were: initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, elongation at 72 ° C. for 12 seconds. In addition, G3PDH was used as an internal standard, and the mRNA amount of the control group was correct | amended using this.
DHCR7:
Forward primer: 5'-AGGGGAAGGTGGGCGCAGGAC-3 '(SEQ ID NO: 1)
Reverse primer: 5'-TTGGGCCTCCCAGCCTCTTGC-3 '(SEQ ID NO: 2)
G3PDH:
Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 3)
Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 4)
As a result, four kinds of drugs such as Asen Yak extract (Maruzen Pharmaceutical Co., Ltd.), MPC copolymer (NIPPON Yushi Oil Co., Ltd.), retinol acetate (BASF) and deer bear extract (One circle Falcos Co., Ltd.) promote DHCR7 production It turned out to have an effect (FIG. 5).
７．アセンヤク抽出物による表皮分化マーカー産生促進
　ヒト表皮角化細胞をＣｎＴ－０７培地（CELLnTEC社）で継代培養し、継代３代目の細胞を同培地に懸濁して、コラーゲンコート１２穴マルチプレート（旭硝子）に５０，０００個の割合で播種し、５％ＣＯ2存在下、３７℃で細胞が集密に達するまで３～５日間の培養を行った。アセンヤク抽出物（丸善製薬）を０．２％となるように添加、又はコントロールとしてＤＭＳＯを同量添加したＣｎＴ－０７培地に交換した後、更に１日間培養を行った。培養後の細胞からＲＮＡ抽出試薬MagNA Pure LC mRNA HSキット（Roche社）と自動核酸抽出装置 MagNA Pure LC 1.0 インスツルメント（Roche社）を用いて、提供されたプロトコールに従ってｍＲＮＡの抽出・精製を行った。各サンプルについて、逆転写酵素 Superscript II（Invitrogen社）によりＲＮＡからランダムヘキサマー・プライマーを用いて、Invitrogen社のプロトコールにしたがいｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、後述の配列番号１及び２のプライマーペア、反応試薬 QuantiFast SYBR Green RT-PCR キット（Qiagen社）及び反応装置 LightCycler（Roche社）を用いて、分化マーカーであるインボルクリン（involucrin）遺伝Ｗ－ＰＲＰ施術により発現が亢進される遺伝子のマイクロアレイ解析子の定量リアルタイムＰＣＲを行った。組成条件は Qiagen社のプロトコールに従った。また、ＰＣＲの条件は、初期変性９５℃で１０分、変性９５℃で１０秒、アニール６０℃で１０秒、伸長７２℃で１２秒とした。なお、Ｇ３ＰＤＨは内部標準として用い、これを用いてコントロール群のｍＲＮＡ量を補正した。
インボルクリン：
　　フォワードプライマー：5'-CTGCCTCAGCCTTACTGTGA-3'（配列番号５）
　　リバースプライマー：　5'-TGGGTATTGACTGGAGGAGG-3'（配列番号６）
Ｇ３ＰＤＨ：
　　フォワードプライマー：5'-GCACCGTCAAGGCTGAGAAC-3'（配列番号３）
　　リバースプライマー：　5'-ATGGTGGTGAAGACGCCAGT-3'（配列番号４）
　図６に示されるとおり、アンヤク抽出物によって、インボルクリンの産生が促進されることが確認された。 7. Stimulation of epidermal differentiation marker production by Asen Yak extract Human epidermal keratinocytes are subcultured in CnT-07 medium (CELLnTEC), cells in passage 3 are suspended in the same medium, and collagen-coated 12-well multiplate ( The cells were seeded at a ratio of 50,000 cells in Asahi Glass, and cultured for 3-5 days until the cells reached confluence at 37 ° C. in the presence of 5% CO 2 . After replacing it with CnT-07 medium to which 0.2 g of Asen Yak extract (Maruzen Pharmaceutical) was added to 0.2%, or as a control, the same amount of DMSO was added, culture was further performed for 1 day. Perform extraction and purification of mRNA according to the provided protocol using RNA extraction reagent MagNA Pure LC mRNA HS kit (Roche) and automated nucleic acid extractor MagNA Pure LC 1.0 Instrument (Roche) from cells after culture The For each sample, cDNA was synthesized from the RNA by reverse transcriptase Superscript II (Invitrogen) using random hexamer primers according to the Invitrogen protocol. Using the synthesized cDNA as a template, the primer pair of SEQ ID NOS: 1 and 2 described below, the reaction reagent QuantiFast SYBR Green RT-PCR kit (Qiagen) and the reaction device Involucrin (involucrin) which is a differentiation marker using LightCycler (Roche). 2.) Quantitative real-time PCR of microarray analyzers of genes whose expression is enhanced by genetic W-PRP treatment was performed. The composition conditions were according to the Qiagen protocol. The PCR conditions were: initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, elongation at 72 ° C. for 12 seconds. In addition, G3PDH was used as an internal standard, and the mRNA amount of the control group was correct | amended using this.
Involucrin:
Forward primer: 5'-CTGCCTCCAGCTTTACTGTGA-3 '(SEQ ID NO: 5)
Reverse primer: 5'-TGGGTATTGACTGGAGGAGG-3 '(SEQ ID NO: 6)
G3PDH:
Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 3)
Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 4)
As shown in FIG. 6, it was confirmed that annyak extract promotes the production of involucrin.

Claims (4)

  1. 　アセンヤク抽出物、２－メタクリロイルオキシエチルホスホリルコリン基を有するコポリマー、酢酸レチノール、及びシカクマメ抽出物からなる群から選択される少なくとも１種類の成分を含むコレステロール合成促進剤。 A cholesterol synthesis promoter comprising at least one component selected from the group consisting of acenyaku extract, a copolymer having a 2-methacryloyloxyethyl phosphorylcholine group, retinol acetate, and a deer extract.
  2. 　請求項１に記載のコレステロール合成促進剤を含む、表皮細胞の分化を促進するための組成物。 A composition for promoting differentiation of epidermal cells, comprising the cholesterol synthesis promoter according to claim 1.
  3. 　表皮細胞の分化を促進する薬剤をスクリーニングする方法であって、候補薬剤を培養細胞に作用させた場合に、該細胞のＤＨＣＲ７遺伝子の発現を亢進させる薬剤を表皮細胞の分化を促進する薬剤として選定することを特徴とする方法。 A method of screening for an agent that promotes differentiation of epidermal cells, wherein, when a candidate agent is caused to act on cultured cells, an agent that enhances expression of DHCR7 gene of the cells is selected as an agent that promotes differentiation of epidermal cells How to do it.
  4. 　前記培養細胞がヒト表皮角化細胞である、請求項３に記載の方法。 The method according to claim 3, wherein the cultured cells are human epidermal keratinocytes.
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