JP2018203676A - Keratin 5 gene expression promoter, keratin 10 gene expression promoter, loricrin gene expression promoter, and transglutaminase gene expression promoter - Google Patents
Keratin 5 gene expression promoter, keratin 10 gene expression promoter, loricrin gene expression promoter, and transglutaminase gene expression promoter Download PDFInfo
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Abstract
Description
本発明は、フィチン酸を有効成分とするケラチン５遺伝子発現促進剤に関する。また、フィチン酸を有効成分とするケラチン１０遺伝子発現促進剤に関する。また、フィチン酸を有効成分とするロリクリン遺伝子発現促進剤に関する。また、フィチン酸を有効成分とするトランスグルタミナーゼ遺伝子発現促進剤に関する。 The present invention relates to a keratin 5 gene expression promoter containing phytic acid as an active ingredient. The present invention also relates to a keratin 10 gene expression promoter containing phytic acid as an active ingredient. The present invention also relates to a loricrin gene expression promoter containing phytic acid as an active ingredient. The present invention also relates to a transglutaminase gene expression promoter containing phytic acid as an active ingredient.
皮膚は体の最も外側に存在しており、温度、紫外線、物理的な衝撃といった外部刺激から生体を保護する機能や、生体内の水分蒸発を防ぐ保湿機能を有しており、生命活動に重要な役割を果たしている。皮膚は皮下組織、真皮、及び表皮の３層から構成され、表皮は内側から基底層、有棘層、顆粒層、角層の４層から構成される。中でも最外層を構成する角層は生体を強力に被覆し、侵入物に対する防御や水分の保持という役割を担っている。 Skin is located on the outermost side of the body, and has a function to protect the body from external stimuli such as temperature, ultraviolet rays, and physical impact, and a moisturizing function to prevent moisture evaporation in the body, which is important for life activities Plays an important role. The skin is composed of three layers of subcutaneous tissue, dermis, and epidermis, and the epidermis is composed of four layers from the inside: the basal layer, the spinous layer, the granular layer, and the horny layer. Among them, the stratum corneum constituting the outermost layer strongly covers the living body, and plays a role of protecting against intruders and retaining moisture.
表皮では、表皮角化細胞が基底層から、有棘層、顆粒層、及び角層へと分化・成熟しながら移行し、最終的には垢として剥がれ落ちる。表皮角化細胞が基底膜から角層まで移行することを角化という。また、表皮角化細胞の基底層での分裂から剥離するまでの表皮の生まれ変わりは、一般的にターンオーバーと呼ばれる。皮膚のターンオーバーを促進することで表皮角化細胞を新しく且つ正常な状態に保つことができ、その結果、皮膚バリア機能が向上する。 In the epidermis, epidermal keratinocytes migrate from the basal layer to the spiny layer, the granule layer, and the horny layer with differentiation and maturation, and eventually peel off as plaque. The transition of epidermal keratinocytes from the basement membrane to the stratum corneum is called keratinization. In addition, the reincarnation of the epidermis from the division in the basal layer of the epidermal keratinocytes to the detachment is generally referred to as turnover. Facilitating skin turnover can keep epidermal keratinocytes in a new and normal state, resulting in improved skin barrier function.
表皮角化細胞は角化の各過程において、種々のたんぱく質や酵素を産生しながら分化・成熟することが知られている。例えば、基底層ではケラチン５等、有棘層ではケラチン１０及びトランスグルタミナーゼ等、顆粒層ではロリクリン等が産生されており、これらをコードする遺伝子は、表皮角化細胞の分化に関連するマーカーとして知られている。 It is known that epidermal keratinocytes differentiate and mature while producing various proteins and enzymes in each process of keratinization. For example, keratin 5 etc. are produced in the basal layer, keratin 10 and transglutaminase etc. are produced in the spiny layer, lolicrin etc. are produced in the granule layer, and the genes encoding these are known as markers associated with differentiation of epidermal keratinocytes. It has been.
フィチン酸は玄米等の穀類に多く含まれる有機リン酸化合物である。フィチン酸はシグナル伝達物質としても知られ、その他にも血小板凝集を減少させることによる、血栓症やアテローム性動脈硬化症の予紡効果等様々な報告がなされている（非特許文献１及び２）。 Phytic acid is an organic phosphate compound that is abundant in grains such as brown rice. Phytic acid is also known as a signal transducing substance, and various reports have been made such as pre-spinning effect of thrombosis and atherosclerosis by reducing platelet aggregation (Non-patent Documents 1 and 2). .
上述の通り、フィチン酸の生理機能に関する研究は盛んに行われている。しかしながら、フィチン酸が表皮角化細胞の分化等に関連する遺伝子であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の発現を促進することについては知られていなかった。 As described above, research on the physiological function of phytic acid has been actively conducted. However, it has not been known that phytic acid promotes the expression of keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene, which are genes related to differentiation of epidermal keratinocytes.
よって、本発明の目的は、ケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の発現促進剤を提供することである。また、これらの遺伝子の発現を促進することにより、表皮角化細胞の角化促進、ターンオーバーの促進、皮膚バリア機能を強化させることが可能な促進剤を提供することである。 Therefore, an object of the present invention is to provide an expression promoter for keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene. Another object of the present invention is to provide an accelerator capable of promoting the keratinization of epidermal keratinocytes, promoting turnover, and enhancing the skin barrier function by promoting the expression of these genes.
上記の様な事情に鑑み、本発明者らは皮膚のターンオーバーのメカニズムに着目して鋭意研究を重ねた結果、フィチン酸が表皮角化細胞の分化に関連する特定の遺伝子の発現を促進することを見出して本発明を完成させた。 In view of the circumstances as described above, the present inventors have conducted extensive research focusing on the mechanism of skin turnover, and as a result, phytic acid promotes the expression of specific genes related to differentiation of epidermal keratinocytes. As a result, the present invention was completed.
すなわち、本発明は、フィチン酸を有効成分とするケラチン５遺伝子発現促進剤を提供する。また、フィチン酸を有効成分とするケラチン１０遺伝子発現促進剤を提供する。また、フィチン酸を有効成分とするロリクリン遺伝子発現促進剤を提供する。また、フィチン酸を有効成分とするトランスグルタミナーゼ遺伝子発現促進剤を提供する。 That is, the present invention provides a keratin 5 gene expression promoter containing phytic acid as an active ingredient. Moreover, the keratin 10 gene expression promoter which uses phytic acid as an active ingredient is provided. Moreover, the loricrin gene expression promoter which uses phytic acid as an active ingredient is provided. Moreover, the transglutaminase gene expression promoter which uses phytic acid as an active ingredient is provided.
本発明によれば、表皮角化細胞の分化に関連する遺伝子（以下、単に「分化関連遺伝子」と称することがある）であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の発現を促進する剤（発現促進剤）を提供することができる。また、前記の促進剤は表皮角化細胞の分化を促進することが可能であるため、ターンオーバーの促進、皮膚バリア機能の強化が可能である。 According to the present invention, expression of keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene, which are genes related to differentiation of epidermal keratinocytes (hereinafter sometimes simply referred to as “differentiation-related genes”). It is possible to provide an agent that promotes expression (expression promoter). Moreover, since the said promoter can accelerate | stimulate differentiation of an epidermal keratinocyte, promotion of turnover and reinforcement | strengthening of a skin barrier function are possible.
本発明は、フィチン酸を有効成分とするケラチン５遺伝子発現促進剤、フィチン酸を有効成分とするケラチン１０遺伝子発現促進剤、フィチン酸を有効成分とするロリクリン遺伝子発現促進剤、及びフィチン酸を有効成分とするトランスグルタミナーゼ遺伝子発現促進剤に関する。なお、これらの遺伝子発現促進剤を「本発明の促進剤」と称する場合がある。また、ケラチン５遺伝子を単に「Ｋ５」と、ケラチン１０遺伝子を単に「Ｋ１０」と、ロリクリン遺伝子を単に「ＬＯＲ」と、トランスグルタミナーゼ遺伝子を単に「ＴＧａｓｅ」と称することがある。 The present invention provides a keratin 5 gene expression promoter containing phytic acid as an active ingredient, a keratin 10 gene expression promoter containing phytic acid as an active ingredient, a loricrin gene expression promoter containing phytic acid as an active ingredient, and phytic acid as an effective ingredient The present invention relates to a transglutaminase gene expression promoter as a component. In addition, these gene expression promoters may be referred to as “the promoter of the present invention”. The keratin 5 gene may be simply referred to as “K5”, the keratin 10 gene as simply “K10”, the loricrin gene as simply “LOR”, and the transglutaminase gene as simply “TGase”.
本発明の促進剤は、フィチン酸を有効成分とすることを特徴とする。フィチン酸は、ＩＮＣＩ名（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｃｏｓｍｅｔｉｃ Ｉｎｇｒｅｄｉｅｎｔ Ｄｉｃｔｉｏｎａｒｙ ａｎｄ Ｈａｎｄｂｏｏｋ，第１６版，第２巻，２０１６年，ｐ．２７２４）で「Ｐｈｙｔｉｃ Ａｃｉｄ」と表記される化合物である。フィチン酸は塩を形成していても良く、フィチン酸塩としては、例えば、フィチン酸ナトリウム、フィチン酸カリウム等のフィチン酸アルカリ金属塩等が挙げられる。フィチン酸は市販品を用いることもでき、例えば、商品名「フィチン酸」（築野ライスファインケミカルズ（株）製）、商品名「５０％フィチン酸溶液」（和光純薬工業（株）製）が挙げられる。 The promoter of the present invention is characterized by containing phytic acid as an active ingredient. Phytic acid is a compound represented by “Physic Acid” under the INCI name (International Cosmetic Ingredient Dictionary and Handbook, 16th edition, Volume 2, 2016, p. 2724). Phytic acid may form a salt, and examples of phytate include phytic acid alkali metal salts such as sodium phytate and potassium phytate. As phytic acid, a commercially available product can be used. For example, the trade name “Phytic acid” (manufactured by Tsukino Rice Fine Chemicals Co., Ltd.), the trade name “50% phytic acid solution” (manufactured by Wako Pure Chemical Industries, Ltd.) Can be mentioned.
本発明の促進剤は表皮角化細胞の分化関連遺伝子であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の少なくとも１つの遺伝子の発現を促進する。本発明の促進剤は、経口、注射、外用のいずれでも効能を発揮するが、外用剤に配合して用いられることが好ましく、特に皮膚外用剤に配合して用いられることがより好ましい。皮膚外用剤には、皮膚用化粧料、皮膚洗浄剤、外用医薬部外品、医療用皮膚外用剤が含まれる。 The promoter of the present invention promotes the expression of at least one gene of keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene, which are differentiation-related genes of epidermal keratinocytes. The promoter of the present invention exhibits efficacy for oral, injection, and external use, but is preferably used by blending with an external preparation, and more preferably used by blending with a skin external preparation. Skin external preparations include skin cosmetics, skin cleansing agents, external quasi-drugs, and medical skin external preparations.
本発明の促進剤を含有する外用剤の剤形は特に限定されないが、例えば、液状、ミスト状、霧状、乳液状、クリーム状、ジェル状、ワックス状、フォーム状等の各種剤形に調製して使用できる。また、前記外用剤は、所望の効果の発現が阻害されない範囲であれば、例えば、水、低級アルコール、多価アルコール、糖アルコール、紫外線吸収剤、香料、防腐剤、キレート剤、抗菌剤、酸化防止剤、保湿剤、清涼剤、ビタミン類、界面活性剤、油性原料、カチオン性ポリマー、ノニオン性ポリマー、両性ポリマー、アニオン性ポリマー、植物抽出液、噴射剤、ｐＨ調整剤、アミノ酸、抗炎症剤、収斂剤、色素、増粘剤等のその他の成分と混合し、攪拌すること等により調製することができる。具体的には、化粧水、柔軟化粧品、収れん化粧水、ふき取り用化粧水、アフターシェーブローション、スキンローション、スキンクリーム、マッサージクリーム、マッサージクリーム、クレンジングクリーム、ミルキーローション、リップクリーム、洗顔料、シェービングフォーム、シェービングクリーム、ボディソープ、乳液類、制汗剤、デオドラント剤などの種々の形態の外用剤とすることができ、製剤化については、一般に知られている方法により製造すればよい。 The dosage form of the external preparation containing the accelerator of the present invention is not particularly limited. For example, it is prepared in various dosage forms such as liquid, mist, mist, emulsion, cream, gel, wax, and foam. Can be used. Further, the external preparation is within a range in which expression of a desired effect is not inhibited, for example, water, lower alcohol, polyhydric alcohol, sugar alcohol, ultraviolet absorber, fragrance, preservative, chelating agent, antibacterial agent, oxidation Inhibitor, moisturizer, refresher, vitamins, surfactant, oily raw material, cationic polymer, nonionic polymer, amphoteric polymer, anionic polymer, plant extract, propellant, pH adjuster, amino acid, anti-inflammatory agent It can be prepared by mixing with other components such as astringents, pigments, thickeners and stirring. Specifically, lotion, softening cosmetics, astringent lotion, wiping lotion, after shave lotion, skin lotion, skin cream, massage cream, massage cream, cleansing cream, milky lotion, lip balm, face wash, shaving foam, Various forms of external preparations such as shaving creams, body soaps, emulsions, antiperspirants, deodorants and the like can be used, and preparations may be produced by generally known methods.
本発明の促進剤を外用剤（特に皮膚外用剤）に使用する場合、外用剤におけるフィチン酸の濃度はその使用態様に応じて適宜調整されるが、遺伝子の発現促進の観点からは０．０１ｍＭ以上であることが好ましく、０．０５〜３．０ｍＭであることが好ましく、０．１〜２．０ｍＭであることが更に好ましい。 When the promoter of the present invention is used for external preparations (especially skin external preparations), the concentration of phytic acid in the external preparation is appropriately adjusted according to the use mode, but from the viewpoint of promoting gene expression, 0.01 mM. It is preferable that it is above, it is preferable that it is 0.05-3.0 mM, and it is still more preferable that it is 0.1-2.0 mM.
本発明の促進剤を外用剤（特に皮膚外用剤）に使用する場合、フィチン酸の配合量はその使用態様に応じて適宜調整されるが、遺伝子の発現促進の観点からは、外用剤全体に対して０．０００６質量％以上配合することが好ましく、０．００３〜０．２質量％の範囲で配合することがより好ましく、０．００６〜０．１５質量％の範囲で配合することが更に好ましい。 When the promoter of the present invention is used for an external preparation (especially a skin external preparation), the amount of phytic acid is appropriately adjusted according to the use mode. From the viewpoint of promoting gene expression, the entire external preparation is used. It is preferable to mix | blend 0.0006 mass% or more with respect to it, it is more preferable to mix | blend in the range of 0.003-0.2 mass%, and it is further mixing | blending in the range of 0.006-0.15 mass%. preferable.
本発明の促進剤は、分化関連遺伝子であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の少なくとも１つ、好ましくは２つ、より好ましくは３つ、特に好ましくは全ての遺伝子の発現を促進する剤である。 The promoter of the present invention contains at least one, preferably 2, more preferably 3, particularly preferably all genes of keratin 5 gene, keratin 10 gene, loricrin gene and transglutaminase gene which are differentiation-related genes. It is an agent that promotes expression.
以下に、実施例に基づいて本発明をより詳細に説明するが、本発明はこれらの実施例により限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.
［使用試薬］
本実施例にて用いた、エタノール、クロロホルム、２−プロパノール、Ｄｉｅｔｈｙｌｐｙｒｏｃａｒｂｏｎａｔｅ ｔｒｅａｔｅｄ ｗａｔｅｒ（ＤＥＰＣ ｗａｔｅｒ）、及び５０％フィチン酸溶液は和光純薬工業（株）より購入した。また、Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｄ ｓａｌｔｓ ｗｉｔｈｏｕｔ Ｃａ ａｎｄ Ｍｇ（ＰＢＳ（−））、ＲＮＡｉｓｏ Ｐｌｕｓ、ＳＹＢＲ Ｐｒｅｍｉｘ ＥＸ Ｔａｑ（Ｔｌｉ ＲＮａｓｅ Ｈ Ｐｌｕｓ）、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔはタカラバイオ（株）より購入した。また、Ｎｏｒｍａｌ ｈｕｍａｎ ｅｐｉｄｅｒｍａｌ ｋｅｒａｔｉｎｏｃｙｔｅ（ＮＨＥＫ；正常ヒト表皮角化細胞）、ＮＨＥＫ培養培地（ＨｕＭｅｄｉａ−ＫＧ２特注ＧＣ別添）は倉敷紡績（株）より購入した。
[Reagent used]
Ethanol, chloroform, 2-propanol, diethylpyrocarbonate treated water (DEPC water), and 50% phytic acid solution used in this example were purchased from Wako Pure Chemical Industries, Ltd. Phosphate buffer salts without Ca and Mg (PBS (-)), RNAiso Plus, SYBR Premix EX Taq (Tli RNase H Plus), PrimeScript RT Reagent Kit purchased from Takara Bio Inc. In addition, Normal human epidermal keratinocyte (NHEK; normal human epidermal keratinocytes) and NHEK culture medium (HuMedia-KG2 special order GC attachment) were purchased from Kurashiki Spinning Co., Ltd.
［遺伝子発現量測定］
ＮＨＥＫを、３７℃、５％ＣＯ2の条件下でＮＨＥＫ培養培地を用いて培養した。得られたＮＨＥＫを、ＮＨＥＫ培養培地が２ｍＬ注入された６ウェルプレート（Ｇｒｅｉｎｅｒ Ｂｉｏ−ｏｎｅ ＧｍｂＨ）に４．０×１０5細胞／ウェルとなるように播種し、８０％コンフルエントになるまで培養した。これに培地中のフィチン酸濃度が０．０５、０．１、１．０、及び５．０ｍＭとなるようにフィチン酸（５０％フィチン酸溶液をＮＨＥＫ培養培地で１０ｍＭに希釈したもの）をそれぞれ添加し、４８時間追培養後に総ＲＮＡを抽出した。総ＲＮＡの逆転写後、リアルタイムＰＣＲによりＫ５、Ｋ１０、ＬＯＲ、及びＴＧａｓｅの遺伝子発現量を測定・算出し、その結果を図１〜４に記載した。なお、フィチン酸を添加することなく同様の操作を行ったものをコントロールとした。ＲＮＡ抽出、ＲＮＡ逆転写、リアルタイムＰＣＲ等の操作については、以下の通りに行った。
[Measurement of gene expression level]
NHEK was cultured using NHEK culture medium under conditions of 37 ° C. and 5% CO 2 . The obtained NHEK was seeded at a concentration of 4.0 × 10 5 cells / well in a 6-well plate (Greiner Bio-one GmbH) into which 2 mL of NHEK culture medium had been injected, and cultured until 80% confluent. To this, phytic acid (50% phytic acid solution diluted to 10 mM with NHEK culture medium) was added so that the phytic acid concentration in the medium was 0.05, 0.1, 1.0, and 5.0 mM, respectively. Total RNA was extracted after additional culture for 48 hours. After reverse transcription of total RNA, gene expression levels of K5, K10, LOR, and TGase were measured and calculated by real-time PCR, and the results are shown in FIGS. In addition, what performed the same operation, without adding a phytic acid was made into control. Operations such as RNA extraction, RNA reverse transcription, and real-time PCR were performed as follows.
（ＲＮＡ抽出）
ＮＨＥＫをＰＢＳ（−）により洗浄した後、２５０μＬのＲＮＡｉｓｏ Ｐｌｕｓを添加し、その細胞表面全体に均等になじませ細胞を剥離させた。これをサンプリングチューブ（５０９−ＧＲＤ−Ｑ、ビーエム（株）製）に回収し、室温で５分間静置することにより、核酸から核タンパク質を遊離させた。その後、５０μＬのクロロホルムを加え、乳白状になるまでよく振り交ぜ、再び室温で５分間静置し、１２０００×ｇ、４℃、１５分間の条件でテーブルトップマイクロ冷却遠心機３５００（久保田商事（株）製）にて遠心した。得られた上清６０〜８０μＬを新しいサンプリングチューブに回収し、２５０μＬの２−プロパノールを加えて混合した後、室温で１０分間静置し、１２０００×ｇ、４℃、１０分間の条件で遠心した。遠心により生じたペレットに、オートクレーブ処理水で調整した７５％冷エタノールを加えて洗浄し、７５００×ｇ、４℃、５分間の条件で遠心した。その後、上清を捨てて乾燥し、２０μＬのＤＥＰＣ ｗａｔｅｒで溶解することにより、総ＲＮＡを抽出した。
(RNA extraction)
After NHEK was washed with PBS (−), 250 μL of RNAiso Plus was added, and the cells were exfoliated evenly over the entire cell surface. This was recovered in a sampling tube (509-GRD-Q, manufactured by BM Co., Ltd.) and allowed to stand at room temperature for 5 minutes to release nucleoprotein from the nucleic acid. Then, add 50 μL of chloroform, shake well until milky white, leave again at room temperature for 5 minutes, and table top micro-cooled centrifuge 3500 (Kubota Corp.) under the conditions of 12000 × g, 4 ° C., 15 minutes. )). 60-80 μL of the obtained supernatant was collected in a new sampling tube, mixed with 250 μL of 2-propanol, allowed to stand at room temperature for 10 minutes, and centrifuged under conditions of 12000 × g, 4 ° C., 10 minutes. . The pellet produced by centrifugation was washed with 75% cold ethanol adjusted with autoclaved water, and centrifuged at 7500 × g, 4 ° C. for 5 minutes. Thereafter, the supernatant was discarded and dried, and the total RNA was extracted by dissolving in 20 μL of DEPC water.
（ＲＮＡ逆転写）
逆転写反応はＰｒｉｍｅＳｃｒｉｐｔ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔを用いて行った。まず抽出したＲＮＡの濃度をＮａｎｏＤｒｏｐ １０００ ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ（サーモフィッシャーサイエンス（株）製）により測定し、ＤＥＰＣ ｗａｔｅｒにて５００ｎｇ／６．５μＬに調整した。得られた調整液６．５μＬに、２μＬの５×ＰｒｉｍｅＳｃｒｉｐｔ Ｂｕｆｆｅｒ（ｆｏｒ Ｒｅａｌ Ｔｉｍｅ）、０．５μＬのＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ＥｎｚｙｍｅＭｉｘ Ｉ、０．５μＬのＯｌｉｇｏ ｄＴ Ｐｒｉｍｅｒ（５０μＭ）、０．５μＬのＲａｎｄｏｍ ６ｍｅｒ（１００μＭ）を加え、１０μＬの逆転写反応溶液を調製した。なお、これらの調製は氷上で行った。調製した反応液はＶｅｒｉｔｉ ９６−ｗｅｌｌ サーマルサイクラー（サーモフィッシャーサイエンス（株）製）により３７℃・１５分、８５℃・５秒の条件で逆転写反応を行い、ｃＤＮＡ溶液を調製した。
(RNA reverse transcription)
Reverse transcription reaction was performed using PrimeScript RT Reagent Kit. First, the concentration of the extracted RNA was measured with NanoDrop 1000 spectrophotometer (manufactured by Thermo Fisher Science Co., Ltd.) and adjusted to 500 ng / 6.5 μL with DEPC water. To 6.5 μL of the obtained adjustment solution, 2 μL of 5 × PrimeScript Buffer (for Real Time), 0.5 μL of PrimeScript RT EnzymeMix I, 0.5 μL of Oligo dT Primer (50 μM) and 0.5 μL of 6 μM ) Was added to prepare 10 μL of a reverse transcription reaction solution. These preparations were performed on ice. The prepared reaction solution was subjected to a reverse transcription reaction at 37 ° C. for 15 minutes and 85 ° C. for 5 seconds using a Veriti 96-well thermal cycler (manufactured by Thermo Fisher Science Co., Ltd.) to prepare a cDNA solution.
（リアルタイムＰＣＲ）
リアルタイムＰＣＲはＳＹＢＲ Ｐｒｅｍｉｘ ＥＸ Ｔａｑを用いて行い、インターカレーター法により増幅するｃＤＮＡをリアルタイムに検出した。各分化関連遺伝子に対して使用したＦｏｒｗａｒｄプライマー、及びＲｅｖｅｒｓｅプライマーを表１に示す。
(Real-time PCR)
Real-time PCR was performed using SYBR Premix EX Taq, and cDNA amplified by the intercalator method was detected in real time. Table 1 shows Forward primers and Reverse primers used for each differentiation-related gene.
１０μＬのＳＹＢＲ Ｐｒｅｍｉｘ Ｅｘ Ｔａｑ（×２）、０．４μＬのＦｏｒｗａｒｄプライマー（１０μＭ）、０．４μＬのＲｅｖｅｒｓｅプライマー（１０μＭ）、０．４μＬのＲＯＸ Ｒｅｆｅｒｅｎｃｅ（５０×）、１μＬのｃＤＮＡ溶液、及び６．８μＬのｄＨ2０を混合することにより反応液を調製した。この反応液をＳｔｅｐ Ｏｎｅ Ｐｌｕｓ Ｒｅａｌ−ＴｉｍｅＣｙｃｌｅｒ（サーモフィッシャーサイエンス（株）製）によりリアルタイムＰＣＲを行った。この時の反応条件はＨｏｌｄｉｎｇ ｓｔａｇｅ：９５℃・３０秒、Ｃｙｃｌｉｎｇ ｓｔａｇｅ：９５℃・５秒、６０℃・３０秒を４０サイクル、Ｍｅｌｔ Ｃｕｒｖｅ ｓｔａｇｅ：９５℃・１５秒、６０℃・６０秒、９５℃・１５秒で行い、その解析はΔΔＣｔ法を用いた相対定量で行った。 5. 10 μL SYBR Premix Ex Taq (× 2), 0.4 μL Forward primer (10 μM), 0.4 μL Reverse primer (10 μM), 0.4 μL ROX Reference (50 ×), 1 μL cDNA solution, and A reaction solution was prepared by mixing 8 μL of dH 2 O. This reaction solution was subjected to real-time PCR using Step One Plus Real-TimeCycler (manufactured by Thermo Fisher Science Co., Ltd.). The reaction conditions at this time were: Holding stage: 95 ° C. for 30 seconds, Cycling stage: 95 ° C. for 5 seconds, 60 ° C. for 30 seconds for 40 cycles, Melt Curve stage: 95 ° C. for 15 seconds, 60 ° C. for 60 seconds, 95 The analysis was performed at 15 ° C. for 15 seconds, and the analysis was performed by relative quantification using the ΔΔCt method.
すべての結果は平均と標準偏差値で示し、統計ソフトウェア（ＳＡＳ Ｕｎｉｖｅｒｓｉｔｙ Ｅｄｉｔｉｏｎ（ＳＡＳ Ｉｎｓｔｉｔｕｔｅ））を用いたＤｕｎｎｅｔｔの検定で統計処理を行った。 All results are shown as mean and standard deviation values, and statistical processing was performed by Dunnett's test using statistical software (SAS University Edition (SAS Institute)).
［結果］
図１〜４に示される通り、フィチン酸が分化関連遺伝子であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子の発現を促進することが明らかになった。つまり、本発明の促進剤は上記の遺伝子群の少なくとも１つの発現を促進することが可能であることが明らかとなった。このため、フィチン酸は、１種だけではなく２種以上の分化関連遺伝子の発現を促進することが可能であることから、表皮角化細胞の分化を強力に促進することが示唆される。
[result]
1-4, it became clear that phytic acid promotes the expression of keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene, which are differentiation-related genes. That is, it became clear that the promoter of the present invention can promote the expression of at least one of the above gene groups. For this reason, phytic acid can promote the expression of not only one type but also two or more types of differentiation-related genes, suggesting that it strongly promotes the differentiation of epidermal keratinocytes.
さらに、全ての分化関連遺伝子（ケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、トランスグルタミナーゼ遺伝子）においてフィチン酸の濃度が０．１〜１．０ｍＭの間である場合に発現量の更なる増加が認められた（ｐ＜０．００１）。一方、前記の範囲に対してフィチン酸の濃度が５．０ｍＭである場合は発現量の増加は見られなかった。これはフィチン酸のキレート剤としての効力により細胞中のカルシウムイオン濃度が低下し、その結果、遺伝子発現量が減少したと考えられる。したがって、上記のフィチン酸の濃度範囲が分化関連遺伝子の発現促進に特に好ましいことが理解できる。 Further, in all differentiation-related genes (keratin 5 gene, keratin 10 gene, loricrin gene, transglutaminase gene), further increase in the expression level is observed when the concentration of phytic acid is between 0.1 and 1.0 mM. (P <0.001). On the other hand, when the concentration of phytic acid was 5.0 mM with respect to the above range, no increase in the expression level was observed. This is thought to be due to the decrease in the calcium ion concentration in the cells due to the efficacy of phytic acid as a chelating agent, resulting in a decrease in gene expression. Therefore, it can be understood that the above concentration range of phytic acid is particularly preferable for promoting the expression of differentiation-related genes.
以上より、本発明の促進剤が、分化関連遺伝子であるケラチン５遺伝子、ケラチン１０遺伝子、ロリクリン遺伝子、及びトランスグルタミナーゼ遺伝子からなる群より選択される少なくとも１つの遺伝子の発現を促進することが明らかとなった。また、本発明の促進剤が表皮角化細胞の分化を促進すること、これによりターンオーバーが促進されること、皮膚バリア機能が強化されることが示唆された。 From the above, it is clear that the promoter of the present invention promotes the expression of at least one gene selected from the group consisting of keratin 5 gene, keratin 10 gene, loricrin gene, and transglutaminase gene, which are differentiation-related genes. became. It was also suggested that the promoter of the present invention promotes differentiation of epidermal keratinocytes, thereby promoting turnover and enhancing the skin barrier function.
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