JP5275613B2 - Skin test method for predicting skin spot formation - Google Patents
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Description
本発明は、皮膚のシミ形成を予測するための皮膚検査方法に関する。 The present invention relates to a skin test method for predicting skin spot formation.
シミの形成は、紫外線やホルモンバランスの乱れなどによりメラノサイト（色素細胞）が活性化され、メラニン色素が局所的に過剰生成されることが主な原因とされている。活性化されたメラノサイト内では、メラニン色素の生成に関わる酵素であるチロシナーゼが活性化することによって、メラニン色素の過剰生成が起こると考えられる。そこで、メラニン色素の過剰生成を抑制するためにはチロシナーゼの活性化を抑制することが必要であるとの観点から、さまざまな手入れ方法が提案されている。また、既にできてしまったシミに対しては、肌の新陳代謝を促進して不要なメラニンを早く追い出すことや、余分なメラニンを作らないようにすることなどが必要となるため、できるだけ早めに手入れをする必要がある。従って、シミが形成される前に肌の手入れを行うことがより好ましい。しかしながら、シミのでき易さには個人差があり、またその原因となる条件もさまざまであるため、シミのでき易さを判断することは一般に困難である。よって、シミが形成される前にシミができ易い状態であるか否かを判断する手段があればシミの形成に対する判断において極めて有効である。 The formation of stains is mainly caused by the fact that melanocytes (pigment cells) are activated by ultraviolet rays or hormonal balance disturbance, and melanin pigments are locally overproduced. In activated melanocytes, tyrosinase, which is an enzyme involved in the production of melanin pigment, is activated, and melanin pigment overproduction is considered to occur. Therefore, various maintenance methods have been proposed from the viewpoint that it is necessary to suppress the activation of tyrosinase in order to suppress the excessive production of melanin pigment. In addition, it is necessary to promote the metabolism of the skin to expel unnecessary melanin as soon as possible and to prevent the creation of excess melanin for the already-developed spots, so take care as soon as possible. It is necessary to do. Therefore, it is more preferable to clean the skin before the stain is formed. However, it is generally difficult to judge the ease of stains because there are individual differences in the ease of stains and the conditions that cause them vary. Therefore, if there is a means for determining whether or not it is in a state where it is easy to make a stain before the stain is formed, it is very effective in determining the formation of the stain.
シミにはさまざまな種類があり、老人性色素班、肝班などが挙げられる。老人性色素班は、主に紫外線が原因となって誘発される色素班である。肝班は、中年以降の女性の顔面、特に頬部、前額部などに左右対称に生ずる比較的大型の色素班で、その形成には内分泌ホルモン（黄体ホルモン、卵胞ホルモン）が深く関与していると考えられている。 There are various types of stains, such as senile pigments and livers. Senile pigment spots are pigment spots that are mainly induced by ultraviolet rays. Liver patches are relatively large pigmented patches that appear symmetrically on the face of women after middle age, especially on the cheeks and forehead. Endocrine hormones (lutein hormones, follicular hormones) are deeply involved in their formation. It is thought that
しかしながら、シミの形成の原因は複雑で多岐にわたると考えられており、シミの生成機構についてはすべて解明されているわけではない。最近では、シミの生成機構に関する研究が盛んに行われており、従来の生化学的なアプローチに加えて、遺伝子工学的な手法を取り入れた解析が加わり、急速な進歩を遂げている。例えば、ヒトメラノサイト刺激ホルモン１受容体（ＭＣ１Ｒ）遺伝子の一塩基置換（ＳＮＰ）が、シミの形成に影響していることが知られている（特許文献１）。確かに、シミ部位の遺伝学的検査によって得られる情報（ＤＮＡ配列情報）は、シミの形成を予測することにとって重要なものである。しかしながら、遺伝子によっては、個人の生活習慣などの内的要因や気候などの外的要因のために生涯発現しない可能性も考えられること等を考慮すれば、シミの形成関連遺伝子のＤＮＡ配列情報だけでは、さまざまな要因が絡み合った結果として形成されるシミの形成し易さを的確に捉えることはできず、特に外的な要因によるシミの形成し易さの変化を理解することは不可能である。
本発明者は、皮膚にシミが形成される前に肌がシミのでき易い状態であるか否かを予測できる手法について鋭意検討した。そして、本発明者は、シミ部位におけるＭＩＦ（ｍａｃｒｏｐｈａｇｅ ｍｉｇｒａｔｉｏｎ ｉｎｈｉｂｉｔｏｒｙ ｆａｃｔｏｒ）の量が非シミ部位と比べて著しく多いことを見出した。また、本発明者は、ＭＩＦがメラノサイトを活性化することでメラニン色素の生成を亢進することを見出した。従って、皮膚におけるＭＩＦの量を調べることで、シミの形成が起こり易い皮膚であると判断することが可能であることが明らかとなった。 This inventor earnestly examined about the technique which can predict whether the skin is in the state which is easy to be made before a spot is formed in skin. The present inventor has found that the amount of MIF (macrophage migration inhibitory factor) at the spot site is significantly higher than that at the non-spot site. The present inventors have also found that MIF enhances the production of melanin pigment by activating melanocytes. Therefore, by examining the amount of MIF in the skin, it was revealed that it was possible to determine that the skin was susceptible to spot formation.
ＭＩＦは、マクロファージの遊走を阻止してマクロファージを集積させる分子量約１万２千のサイトカインであり、免疫応答や細胞の分化増殖能に関わる因子である。ＭＩＦは免疫系組織だけでなく、脳や腎臓、および皮膚等のさまざまな組織に発現していることが知られている。これまでに、皮膚におけるＭＩＦの増加により皮膚のコラゲナーゼ活性が増加することが示唆されているが（非特許文献１）、シミの形成に関する報告は存在しない。
本発明は、以下に列挙する皮膚検査方法を提供する。
［１］皮膚のシミ形成を予測する皮膚検査方法であって、皮膚中のＭＩＦの発現量が正常な皮膚中の発現量と比べて亢進している場合、シミの形成が起こり易い皮膚であると判断することを特徴とする皮膚検査方法。
［２］前記ＭＩＦの発現量の亢進が、角質細胞中のＭＩＦ量を測定することにより決定される、［１］に記載の皮膚検査方法。
［３］前記ＭＩＦの発現量の測定が、前記皮膚試料から抽出したＲＮＡを試料とし、ポリメラーゼ連鎖反応法（ＰＣＲ法）によって行われる、［２］に記載の皮膚検査方法。
［４］前記ＭＩＦの発現量の測定が、前記皮膚試料から抽出したタンパク質を試料とし、免疫染色法、ウエスタンブロット法又は免疫測定法によって行われる、［２］に記載の皮膚検査方法。
［５］シミの形成抑制因子及び／又はシミ除去因子をスクリーニングする方法であって、候補化合物をＭＩＦの発現及び／又は活性を阻害する能力について評価し、当該阻害能力を有するＭＩＦ抑制因子をシミの形成抑制因子及び／又はシミ除去因子として選定することを特徴とする皮膚検査方法。
The present invention provides the skin examination methods listed below.
[1] A skin test method for predicting skin spot formation, wherein skin formation is likely to occur when the expression level of MIF in the skin is increased compared to the expression level in normal skin. It is judged that the skin inspection method.
[2] The skin examination method according to [1], wherein the increase in the expression level of MIF is determined by measuring the amount of MIF in corneocytes.
[3] The skin test method according to [2], wherein the measurement of the expression level of the MIF is performed by a polymerase chain reaction method (PCR method) using RNA extracted from the skin sample as a sample.
[4] The skin test method according to [2], wherein the measurement of the expression level of the MIF is performed by immunostaining, Western blotting, or immunoassay using the protein extracted from the skin sample as a sample.
[5] A method for screening a spot formation inhibitor and / or spot remover, wherein a candidate compound is evaluated for its ability to inhibit the expression and / or activity of MIF, and a MIF inhibitor having the inhibitory ability is spotted. A skin test method, wherein the method is selected as a factor for inhibiting the formation of water and / or a factor for removing spots.
本発明により、被験対象部位の皮膚中のＭＩＦの量を測定することによって、シミの形成が起こり易い皮膚であると判断することを特徴とする皮膚のシミ形成を予測するための皮膚検査方法を提供することが可能となる。 According to the present invention, there is provided a skin inspection method for predicting skin spot formation, characterized in that it is determined that the skin is susceptible to spot formation by measuring the amount of MIF in the skin of the site to be tested. It becomes possible to provide.
本発明の第一の局面は、皮膚のシミ形成を予測するための検査方法を提供する。この方法は、皮膚中のＭＩＦの量が正常な皮膚と比べて多い場合、シミの形成が起こり易い皮膚であると判断することを特徴とする。 The first aspect of the present invention provides an inspection method for predicting skin spot formation. This method is characterized in that when the amount of MIF in the skin is larger than that of normal skin, it is determined that the skin is likely to be stained.
本発明のシミ形成を予測するための皮膚検査方法は、好ましくは以下の一連のステップ（１）〜（３）によって実施される。
（１）被験対象部位の皮膚試料を用意するステップ
（２）前記皮膚試料中のＭＩＦの量を測定するステップ
（３）前記発現量からシミの形成し易さを評価するステップ
The skin examination method for predicting spot formation according to the present invention is preferably performed by the following series of steps (1) to (3).
(1) A step of preparing a skin sample of a site to be tested (2) A step of measuring the amount of MIF in the skin sample (3) A step of evaluating the ease of forming a stain from the expression level
検査すべき皮膚は、例えば顔、首、腕、肢など、シミができ易く、またシミの形成が気になるあらゆる部位の皮膚であってよい。シミの形成が起こっていない正常皮膚、即ちコントロールの皮膚としては、同一個体の例えば紫外線に曝されにくく、比較的シミのできにくい部位の皮膚、例えば腹部、臀部の皮膚であってよい。また、かような試料（組織もしくは細胞）の培養物であってもよい。 The skin to be examined may be the skin of any part that is easily spotted, such as the face, neck, arms, and limbs, and is worried about spot formation. The normal skin in which no spot formation has occurred, that is, the control skin, may be the skin of a part of the same individual that is difficult to be exposed to, for example, ultraviolet rays and is relatively difficult to be stained, such as the skin of the abdomen and buttocks. Further, it may be a culture of such a sample (tissue or cell).
皮膚からの試料の採取方法は特に限定されない。例えば、外科的手段等の侵襲的な方法により採取されたものでよい。好ましくは非侵襲的な採取方法を採用することが望ましい。非侵襲的な方法としては、当該技術分野で常用されているテープストリッピングや擦過法等を挙げることができる。 The method for collecting the sample from the skin is not particularly limited. For example, it may be collected by an invasive method such as a surgical means. It is preferable to adopt a non-invasive sampling method. Non-invasive methods include tape stripping and rubbing methods that are commonly used in the art.
皮膚試料中のＭＩＦの測定方法は特に限定されない。例えば、ＭＩＦをコードするｍＲＮＡ発現量やＭＩＦのタンパク量を測定する方法が挙げられる。 The method for measuring MIF in the skin sample is not particularly limited. For example, a method for measuring the expression level of MIF-encoding mRNA or the amount of MIF protein can be mentioned.
皮膚試料中のＭＩＦｍＲＮＡ発現量は、皮膚試料からＲＮＡを抽出し、逆転写反応を行った後にＰＣＲ法を行うことにより決定することができる。ＲＮＡの抽出方法、ＰＣＲ法は当業者にとって周知であり、常法のプロトコール、又は常法のプロトコールを適宜修正・変更したプロトコールによって適切な測定系を構築し、そして実施することができる。 The expression level of MIF mRNA in the skin sample can be determined by extracting RNA from the skin sample and performing a reverse transcription reaction followed by a PCR method. RNA extraction methods and PCR methods are well known to those skilled in the art, and an appropriate measurement system can be constructed and implemented by a conventional protocol or a protocol that is appropriately modified or changed from a conventional protocol.
皮膚試料中のＭＩＦタンパク量の測定には、例えばＭＩＦに特異的な抗体を使用し、蛍光物質、色素、酵素等を利用する免疫染色法、ウエスタンブロット法、免疫測定法（例えばＥＬＩＳＡ法、ＥＩＡ法）等を用いることができる。これらの方法についても常法に従って実施すればよい。各方法についてさまざまなプロトコールが報告されており、当業者であれば常法のプロトコールに従い、又は常法のプロトコールを適宜修正・変更したプロトコールによって適切な測定系を構築し、そして実施することができる。 For the measurement of the amount of MIF protein in a skin sample, for example, an antibody specific for MIF is used, and an immunostaining method using a fluorescent substance, a dye, an enzyme, etc., a Western blot method, an immunoassay method (for example, ELISA method, EIA) Method) or the like. These methods may be carried out according to a conventional method. Various protocols have been reported for each method, and those skilled in the art can construct and implement an appropriate measurement system according to a conventional protocol, or a protocol that is appropriately modified or changed according to a conventional protocol. .
シミの形成の起こり易さの評価は、被験対象部位の皮膚中のＭＩＦ量が正常部位の皮膚中ＭＩＦ量と比べて１０％以上、又は２０％以上、又は３０％以上、又は５０％以上、又は７０％以上、又は１００％以上多い場合、「シミの形成が起こり易い皮膚である」と判断する、としてよい。 Evaluation of the likelihood of the formation of a stain is 10% or more, or 20% or more, or 30% or more, or 50% or more, compared to the amount of MIF in the skin of the normal site, the amount of MIF in the skin of the test target site, Alternatively, when it is 70% or more, or 100% or more, it may be determined that “the skin is likely to be stained”.
本発明の第二の局面は、シミの形成抑制因子及び／又はシミ除去因子をスクリーニングする方法を提供する。この方法は、候補化合物をＭＩＦの発現及び／又は活性を阻害する能力について評価し、当該阻害能力を有するＭＩＦ抑制因子をシミの形成抑制因子及び／又はシミ除去因子として選定することを特徴とする。 The second aspect of the present invention provides a method of screening for a spot formation inhibitor and / or spot remover. This method is characterized in that a candidate compound is evaluated for the ability to inhibit the expression and / or activity of MIF, and an MIF inhibitor having the inhibitory ability is selected as a stain formation inhibitor and / or a stain removal factor. .
例えば、被験対象部位の皮膚に候補化合物、又は候補化合物を含む製剤を適用し、適用前後のＭＩＦ量を測定することにより、当該阻害能力の評価をすることができる。被験対象部位への候補化合物の適用方法は、塗布や接種等、皮膚への直接的な適用に限定されることはなく、例えば、経口や経鼻等の間接的な適用であってもよい。また、被験対象部位の皮膚試料（組織もしくは細胞）の培養物に候補化合物を適用し、適用の有無によるＭＩＦ量を測定することにより、当該阻害能力の評価を行うこともできる。 For example, the inhibitory ability can be evaluated by applying a candidate compound or a preparation containing the candidate compound to the skin of the site to be tested and measuring the amount of MIF before and after application. The method for applying the candidate compound to the site to be tested is not limited to direct application to the skin, such as application or inoculation, and may be indirect application such as oral or nasal, for example. Alternatively, the inhibitory ability can be evaluated by applying a candidate compound to a culture of a skin sample (tissue or cell) at a site to be tested and measuring the amount of MIF depending on whether or not the sample is applied.
以下、本発明を効果的に説明するために、実験例を挙げる。なお、本発明はこれにより限定されるものではない。 In order to effectively explain the present invention, experimental examples are given below. In addition, this invention is not limited by this.
実験例１ 老人性色素班部位におけるＭＩＦｍＲＮＡ発現量の増加
被験者皮膚の老人性色素班部位と非老人性色素班部位にそれぞれ半径１ｃｍの円状のテープ（３Ｍ、Ｂｌｅｎｄｅｒｍ）を一枚貼り付け、十分に接着していることを確認した後、テープを剥がし取り、角質を含むテープを得た。得られた角質に含まれるＲＮＡをＲＮＡ抽出キット（Ａｍｂｉｏｎ社製、ＲＮＡｑｕｅｏｕｓ−４ＰＣＲ Ｋｉｔ）を用いて抽出した。すなわち、角質の付着したテープをテープごと３５０μＬのＬｙｓｉｓ／Ｂｉｎｄｉｎｇ Ｓｏｌｕｔｉｏｎに入れ、ｐｒｏｔｅｉｎａｓｅ Ｋ（Ｉｎｖｉｔｒｏｇｅｎ社製）を添加し、５６℃で１時間インキュベートした。その後、超音波破砕機を用いて分散させた後、３５０μＬの６４％エタノールを加えて攪拌し、テープを取り除き、角質抽出液を得た。付属のＦｉｌｔｅｒ ＣａｒｔｒｉｇｅにＲＮＡを吸着させた後に、１００μＬの抽出液でＲＮＡを溶出した。得られた溶出液にＤＮａｓｅＩを加え、残存しているＤＮＡを完全に除去した後、エタノールを用いてＲＮＡを沈殿させてＲＮＡを濃縮した。得られたＲＮＡのペレットを必要量の水に溶解して、角質に含まれる残存ＲＮＡを得た。各ＲＮＡをＲＴ−ＰＣＲキット（Ｉｎｖｉｔｒｏｇｅｎ社製、ＳｕｐｅｒＳｃｒｉｐｔ ＩＩＩ Ｐｌａｔｉｎｕｍ ＳＹＢＲ Ｇｒｅｅｎ Ｔｗｏ−ｓｔｅｐ ｑＲＴ−ＰＣＲ ｋｉｔ）を用いた定量ＰＣＲ反応に供し、ＭＩＦ遺伝子の発現量を定量した。ＭＩＦ遺伝子の発現量はβ−アクチン遺伝子の発現量で規格化（ノーマライズ）した。なお、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
Experimental Example 1 Increase in MIF mRNA expression level in senile pigment spot site Affix a sheet of circular tape (3M, Blenderm) with a radius of 1 cm to the senile pigment spot site and non-senile pigment spot site in the subject skin. After confirming that it was adhered to the tape, the tape was peeled off to obtain a tape containing keratin. RNA contained in the obtained keratin was extracted using an RNA extraction kit (manufactured by Ambion, RNAqueous-4PCR Kit). Specifically, the keratin-attached tape was placed in 350 μL of Lysis / Binding Solution together with proteinase K (manufactured by Invitrogen), and incubated at 56 ° C. for 1 hour. Then, after making it disperse | distribute using an ultrasonic crusher, 350 microliters 64% ethanol was added and stirred, the tape was removed, and the keratin extract was obtained. After adsorbing RNA to the attached Filter Cartridge, the RNA was eluted with 100 μL of the extract. DNase I was added to the obtained eluate to completely remove the remaining DNA, and then RNA was precipitated using ethanol to concentrate the RNA. The obtained RNA pellet was dissolved in a necessary amount of water to obtain residual RNA contained in the stratum corneum. Each RNA was subjected to a quantitative PCR reaction using an RT-PCR kit (Superscript III Platinum SYBR Green Two-step qRT-PCR kit, manufactured by Invitrogen), and the expression level of the MIF gene was quantified. The expression level of the MIF gene was normalized (normalized) with the expression level of the β-actin gene. The primers used for measuring the expression level of each gene are as follows.
ＭＩＦ用のプライマーセット
ＡＧＡＡＣＣＧＣＴＣＣＴＡＣＡＧＣＡＡＧ（配列番号１）
ＣＴＴＡＧＧＣＧＡＡＧＧＴＧＧＡＧＴＴＧ（配列番号２）
β−アクチン用のプライマーセット
ＣＡＣＴＣＴＴＣＣＡＧＣＣＴＴＣＣＴＴＣＣ（配列番号３）
ＧＴＧＴＴＧＧＣＧＴＡＣＡＧＧＴＣＴＴＴＧ（配列番号４）
Primer set AGAACCGCTCTCTACAGCAAG for MIF (SEQ ID NO: 1)
CTTAGGCGAAGGTGGAGTTG (SEQ ID NO: 2)
Primer set CACTCTCCCAGCCTCTCTCCC for β-actin (SEQ ID NO: 3)
GTGTTGCGCGTACAGGTCTTTG (SEQ ID NO: 4)
定量化はΔΔＣｔ法にて行い、非老人性色素班部位のＭＩＦｍＲＮＡ発現量に対するシミ部位のＭＩＦｍＲＮＡ発現量を算出した。その結果を表１に示す。図示の通り、非老人性色素班部位と比較して老人性色素班部位ではＭＩＦｍＲＮＡ発現量が大きいことが明らかとなった。 Quantification was performed by the ΔΔCt method, and the MIF mRNA expression level at the spot site was calculated relative to the MIF mRNA expression level at the non-senile pigment spot site. The results are shown in Table 1. As shown in the figure, it was revealed that the expression level of MIF mRNA was larger in the senile pigment spot site than in the non-senile pigment spot site.
実験例２ 肝班部位におけるＭＩＦｍＲＮＡ発現量の増加
女性被験者の顔部皮膚の肝班部位と非肝班部位にそれぞれ半径１ｃｍの円状のテープ（３Ｍ、Ｂｌｅｎｄｅｒｍ）を一枚貼り付け、十分に接着していることを確認した後、テープを剥がし取り、角質を含むテープを得た。以下、実験例１の方法によりＭＩＦｍＲＮＡの発現量を測定した。
Experimental example 2 Increase in the expression level of MIF mRNA in the liver area A single piece of circular tape (3M, Blenderm) with a radius of 1 cm is applied to the liver area and the non-liver area of the female subject's face skin, and fully adhered After confirming that this was done, the tape was peeled off to obtain a tape containing keratin. Hereinafter, the expression level of MIF mRNA was measured by the method of Experimental Example 1.
定量化はΔΔＣｔ法にて行い、非肝班部位のＭＩＦｍＲＮＡ発現量に対する肝班部位のＭＩＦｍＲＮＡ発現量を算出した。その結果を表２に示す。図示の通り、非肝班部位と比較して肝班部位ではＭＩＦｍＲＮＡ発現量が大きいことが明らかとなった。 Quantification was performed by the ΔΔCt method, and the expression level of MIF mRNA at the liver segment relative to the expression level of MIF mRNA at the non-liver segment was calculated. The results are shown in Table 2. As shown in the figure, it was revealed that the expression level of MIF mRNA was larger in the liver area than in the non-liver area.
実験例３ ＭＩＦによるチロシナーゼタンパク量の増加
直径６０ｍｍ ｄｉｓｈ（ＦＡＬＣＯＮ社製）でセミコンフルエントになるまで培養した正常ヒトメラノサイト（クラボウ社製）にｒｅｃｏｍｂｉｎａｎｔ ｈｕｍａｎ ＭＩＦ（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社製）を添加し、４８時間後にタンパク抽出試薬（ニッポンジーン社製、ＩＳＯＧＥＮ）を用いてタンパクを抽出した。チロシナーゼタンパク量の測定はウエスタンブロット法により行った。すなわち、正常ヒトメラノサイトから抽出したタンパクをタンパク濃度０．１ｍｇ／ｍＬに統一し、同量のサンプルバッファーを加えて１００℃で５分間処理した。そのうちの１０μＬをＳＤＳ電気泳動に供し、電気泳動後のゲルをトランスファー用メンブレン（Ｍｉｌｌｉｐｏｒｅ社製、ＩＰＶＨ０９１２０）にブロッティングした。次に、１％スキムミルク溶液（ブロッキング溶液）でメンブレンを１時間ブロッキングした後、ブロッキング溶液で２００倍希釈したａｎｔｉ−ｔｙｒｏｓｉｎａｓｅ抗体（ＬＡＢ ＶＩＳＩＯＮ社製、ＭＳ−８００−Ｐ１）と室温で１時間反応させた。さらに、ブロッキング溶液で１０，０００倍希釈したＰｅｒｏｘｉｄａｓｅ−ｃｏｎｊｕｇａｔｅｄ ａｎｔｉ−ｍｏｕｓｅ ＩｇＧ（Ｊａｃｋｓｏｎ Ｉｍｍｕｎｏｒｅｓｅａｒｃｈ社製、１１５−０３６−００３）と室温で１時間反応させた後、ウエスタンブロッティング検出試薬（Ａｍａｒｓｈａｍ社製、ＲＰＮ２２０９）と室温で１分間反応させ、ライトキャプチャー（ＡＴＴＯ社製）にて発光パターンを撮影した。撮影後、７５ｋＤａ付近に現れたチロシナーゼタンパクによるバンドを解析ソフト（ＡＴＴＯ社製）にて定量化した。
Experimental Example 3 Increase in the amount of tyrosinase protein by MIF Recombinant human MIF (manufactured by R & D Systems) was added to normal human melanocytes (manufactured by Kurabo Corp.) cultured to a semi-confluent state with a 60 mm diameter dish (manufactured by FALCON), for 48 hours. Later, the protein was extracted using a protein extraction reagent (Nippon Gene, ISOGEN). The amount of tyrosinase protein was measured by Western blotting. That is, proteins extracted from normal human melanocytes were standardized to a protein concentration of 0.1 mg / mL, and the same amount of sample buffer was added, followed by treatment at 100 ° C. for 5 minutes. 10 μL of the sample was subjected to SDS electrophoresis, and the gel after electrophoresis was blotted onto a transfer membrane (Millipore, IPVH09120). Next, the membrane was blocked with a 1% skim milk solution (blocking solution) for 1 hour, and then reacted with an anti-tyrosinase antibody (manufactured by LAB VISION, MS-800-P1) diluted 200 times with the blocking solution at room temperature for 1 hour. It was. Further, after reacting with Peroxidase-conjugated anti-mouse IgG (Jackson Immunoresearch, 115-036-003) diluted 10,000 times with a blocking solution at room temperature for 1 hour, Western blotting detection reagent (Amarsham, RPN2209) ) And room temperature for 1 minute, and a light emission pattern was photographed with light capture (manufactured by ATTO). After imaging, the band of tyrosinase protein that appeared in the vicinity of 75 kDa was quantified with analysis software (manufactured by ATTO).
ＭＩＦ無添加（コントロール）に対するチロシナーゼタンパク量（％）を算出した。結果を表３に示す。図示の通り、ＭＩＦによるチロシナーゼタンパク量の増加が認められた。 The amount of tyrosinase protein (%) relative to the absence of MIF (control) was calculated. The results are shown in Table 3. As shown in the figure, an increase in the amount of tyrosinase protein due to MIF was observed.
本発明のシミ形成を予測する皮膚検査方法によれば、シミの形成が起こり易いか否かを判断することができる。また、シミが形成する前からシミに対する手入れを行うことで、今まで以上の効果が期待できる。さらに、シミの形成抑制因子及び／又はシミ除去因子を適切に選定することができる。 According to the skin inspection method for predicting spot formation of the present invention, it is possible to determine whether or not spot formation is likely to occur. In addition, by taking care of the stain before the stain is formed, an effect more than ever can be expected. Furthermore, a stain formation inhibiting factor and / or a stain removing factor can be appropriately selected.
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