JP5292341B2 - Protein measurement method for skin stratum corneum - Google Patents

Protein measurement method for skin stratum corneum Download PDF

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JP5292341B2
JP5292341B2 JP2010052724A JP2010052724A JP5292341B2 JP 5292341 B2 JP5292341 B2 JP 5292341B2 JP 2010052724 A JP2010052724 A JP 2010052724A JP 2010052724 A JP2010052724 A JP 2010052724A JP 5292341 B2 JP5292341 B2 JP 5292341B2
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keratin
protein
expression level
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stratum corneum
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知永 安田
央 栢沼
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Fancl Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a housekeeping protein having a fixed manifestation amount in skin corneum. <P>SOLUTION: The invention relates to a method for measuring a total protein amount in the skin corneum by measuring keratin 2e or keratin 3, whereas, when searching for a housekeeping protein having the same behavior as the total protein amount in the skin corneum, it is found that keratin 2e or keratin 3 is pertinent thereto, or relates to a method for performing relative evaluation of a specific protein. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

本発明は、皮膚角質層中のタンパク質測定方法に関する。より詳しくはｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３を用いた皮膚角質層中のタンパク質測定方法に関する。   The present invention relates to a method for measuring protein in the stratum corneum. More specifically, the present invention relates to a method for measuring protein in the horny layer of skin using keratin 2e and keratin 3.

ハウスキーピングタンパク質とは、実験系で発現量が変動しないタンパク質のことをいい、一般に、細胞に刺激やストレスを与えても比較的変動しにくいタンパク質とされている。したがって、所望の実験系で細胞刺激などによって変動するその他のタンパク質との対照として、補正目的（ノーマライゼーション）に使われることが多い。
ここで、ハウスキーピングタンパク質として、細胞内の繊維タンパク質であるβ−ａｃｔｉｎや糖からエネルギーを作る解糖系に関わるタンパク質であるＧ３ＰＤＨが代表的である（非特許文献１，２）。
しかし、β−ａｃｔｉｎやＧ３ＰＤＨは細胞にある刺激が加わるとタンパク質の発現変化がおこることが知られており、実験系によっては、正確なタンパク質の測定ができないおそれがある。たとえば、皮膚角質層を用いた実験系においても、一般的にＧ３ＰＤＨ、β−ａｃｔｉｎがハウスキーピングタンパク質として使用されることが多い。しかし、後述する比較例に示すようにこの２種類のタンパク質の発現量は角質層中の全タンパク量と低い相関関係しか示さないことがわかった。
したがって、角質層中のタンパク質を測定する場合には、適切なハウスキーピングタンパク質の選択が必要である。 A housekeeping protein refers to a protein whose expression level does not vary in an experimental system, and is generally considered to be a protein that is relatively unlikely to fluctuate even when cells are stimulated or stressed. Therefore, it is often used for correction purposes (normalization) as a control for other proteins that vary due to cell stimulation or the like in a desired experimental system.
Here, typical housekeeping proteins include β-actin, which is an intracellular fiber protein, and G3PDH, which is a protein related to a glycolysis system that generates energy from sugar (Non-patent Documents 1 and 2).
However, β-actin and G3PDH are known to cause changes in protein expression when a certain stimulus is applied to cells, and there is a possibility that accurate protein measurement may not be possible depending on the experimental system. For example, even in an experimental system using a skin stratum corneum, G3PDH and β-actin are generally often used as housekeeping proteins. However, as shown in Comparative Examples described later, it was found that the expression levels of these two types of proteins showed only a low correlation with the total protein level in the stratum corneum.
Therefore, when measuring proteins in the stratum corneum, it is necessary to select an appropriate housekeeping protein.

Yamazaki et al,Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein,J.Biol.Chem., 279, 32191-32195, 2004Yamazaki et al, Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein, J. Biol. Chem., 279, 32191-32195, 2004 Hanley et al, Farnesol stimulates differentiation in epidermal keratinocytes via PPARalpha ,J.Biol.Chem, 275, 11484-11491, 2000Hanley et al, Farnesol stimulates differentiation in epidermal keratinocytes via PPARalpha, J. Biol. Chem, 275, 11484-11491, 2000

これまでに皮膚角質層において発現量が一定であるハウスキーピングタンパク質の報告例はない。
本発明者らは、皮膚角質層中のタンパク量と挙動が同じハウスキーピングタンパク質を探索したところ、ｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３がこれに該当することをつきとめ、本発明を完成するに至った。 There has been no report on a housekeeping protein whose expression level is constant in the skin stratum corneum.
The present inventors searched for a housekeeping protein having the same amount of protein and behavior in the skin stratum corneum, and found that keratin 2e or keratin 3 corresponds to this and completed the present invention.

本発明は以下の構成を有する。
〔１〕以下の工程を有する、採取した皮膚角質層の特定のタンパク質量の評価方法。
（１）採取した角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した角質層の特定のタンパク質の発現量を測定する工程
（３）（２）で求めた特定のタンパク質の発現量を（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程
〔２〕特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程が特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で除する工程である〔１〕に記載の方法。
〔３〕特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程が、
特定のタンパク質の発現量を、以下の工程により求めた全タンパク質量で除する工程である〔１〕に記載の方法。
（１）採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した皮膚角質層の全タンパク質量と（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量の一次関数を求める工程
（３）（２）で求めた１次関数を用いて、採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量から採取した皮膚角質層の全タンパク質量を求める工程。
〔４〕以下の工程を有する、採取した皮膚角質層の全タンパク質量を測定する方法。
（１）採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した皮膚角質層の全タンパク質量と（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量の一次関数を求める工程
（３）（２）で求めた１次関数を用いて、採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量から採取した皮膚角質層の全タンパク質量を求める工程。
〔５〕タンパク質の発現量が、抗体を用いて測定した測定強度である〔１〕から〔４〕のいずれかに記載の方法。
〔６〕タンパク質の発現量が、ＥＬＩＳＡ発光強度である〔１〕から〔４〕のいずれかに記載の方法。 The present invention has the following configuration.
[1] A method for evaluating a specific amount of protein in a collected skin stratum corneum having the following steps.
(1) A step of measuring the expression level of keratin 2e or keratin 3 in the collected stratum corneum (2) A step of measuring the expression level of a specific protein in the collected stratum corneum (3) The specific protein obtained in (2) The step of correcting the expression level of keratin 2e or keratin 3 determined in (1) [2] the step of correcting the expression level of a specific protein with the expression level of keratin 2e or keratin 3 The method according to [1], which is a step of dividing the amount by the expression amount of keratin 2e or keratin 3.
[3] The step of correcting the expression level of a specific protein with the expression level of keratin 2e or keratin 3,
The method according to [1], which is a step of dividing the expression level of a specific protein by the total protein amount determined by the following steps.
(1) The step of measuring the expression level of keratin 2e or keratin 3 in the collected skin stratum corneum (2) The total amount of protein in the collected skin stratum corneum and the primary expression level of keratin 2e or keratin 3 determined in (1) Step of obtaining function (3) Step of obtaining the total amount of protein in the horny layer collected from the expression level of keratin 2e or keratin 3 in the collected horny layer using the linear function obtained in (2).
[4] A method for measuring the total protein amount of the collected skin stratum corneum, comprising the following steps.
(1) The step of measuring the expression level of keratin 2e or keratin 3 in the collected skin stratum corneum (2) The total amount of protein in the collected skin stratum corneum and the primary expression level of keratin 2e or keratin 3 determined in (1) Step of obtaining function (3) Step of obtaining the total amount of protein in the horny layer collected from the expression level of keratin 2e or keratin 3 in the collected horny layer using the linear function obtained in (2).
[5] The method according to any one of [1] to [4], wherein the protein expression level is a measured intensity measured using an antibody.
[6] The method according to any one of [1] to [4], wherein the protein expression level is ELISA luminescence intensity.

本発明によれば、皮膚角質層中の全タンパク質量の測定を、ｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３の測定により正確かつ簡易に行うことができた。したがって、皮膚角質層中の全タンパク質量を、Ｌｏｗｒｙ法などにより測定する必要がなく、ＥＬＩＳＡ法など、抗体を用いて簡易にｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３を測定することによって、正確に測定することが可能となった。
また、評価したい特定のタンパク質について、当該タンパク質の発現量相対値をｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３の発現量相対値で除して、全タンパク質量の補正をすることで、皮膚角質層の特定のタンパク質の量を容易に比較評価できるようになった。
また、ｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３の発現量相対値とＬｏｗｒｙ法などの既知の方法で求めた全タンパク質量の関係を一次関数により最適化し、その一次関数を用いて、ｋｅｒａｔｉｎ ２ｅ又はｋｅｒａｔｉｎ ３の発現量相対値から全タンパク質量を算出し、その値で特定のタンパク質の発現量相対値を除して、全タンパク質量の補正をすることで、皮膚角質層の特定のタンパク質の量をより正確に、容易に比較評価することもできる。 According to the present invention, the total protein amount in the skin stratum corneum can be accurately and easily measured by measuring keratin 2e or keratin 3. Therefore, it is not necessary to measure the total amount of protein in the stratum corneum by the Lowry method, etc., and it is possible to accurately measure keratin 2e or keratin 3 using an antibody such as ELISA. It became.
In addition, for a specific protein to be evaluated, the relative expression level of the protein is divided by the relative expression level of keratin 2e or keratin 3, and the total protein amount is corrected, whereby the specific protein of the skin stratum corneum is corrected. The amount can be easily compared and evaluated.
Further, the relationship between the relative value of the expression level of keratin 2e or keratin 3 and the total protein amount obtained by a known method such as the Lowry method is optimized by a linear function, and the expression level of keratin 2e or keratin 3 is obtained using the linear function. By calculating the total protein amount from the relative value, dividing the relative expression level of the specific protein by that value, and correcting the total protein amount, the specific protein amount in the skin stratum corneum is more accurately determined. A comparative evaluation can be easily performed.

角質層から抽出した全タンパク質量とＧ３ＰＤＨの発現量との相関を調べた結果を示す図である。It is a figure which shows the result of having investigated the correlation with the total protein amount extracted from the stratum corneum, and the expression level of G3PDH. 角質層から抽出した全タンパク質量とβ−ａｃｔｉｎの発現量との相関を調べた結果を示す図である。It is a figure which shows the result of having investigated the correlation with the total protein amount extracted from the stratum corneum, and the expression level of (beta) -actin. 角質層から抽出した全タンパク質量とｋｅｒａｔｉｎ ２ｅの発現量との相関を調べた結果を示す図である。It is a figure which shows the result of having investigated the correlation with the total protein amount extracted from the stratum corneum, and the expression level of keratin 2e. 角質層から抽出した全タンパク質量とｋｅｒａｔｉｎ ３の発現量との相関を調べた結果を示す図である。It is a figure which shows the result of having investigated the correlation with the total protein amount extracted from the stratum corneum, and the expression level of keratin 3. ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）ｋａｌｌｉｋｌｅｉｎ ７の発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したｋａｌｌｉｋｌｅｉｎ ７相対量（２）ｋａｌｌｉｋｌｅｉｎ ７の発現量をｋｅｒａｔｉｎ ２ｅ発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ２ｅ発現量から求めた全タンパク質量で除して補正したｋａｌｌｉｋｌｅｉｎ ７相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) The relative amount of kalliklein 7 corrected by dividing the expression level of kalliklein 7 by the total protein amount determined by the Lowry method. (2) The expression level of kalliklein 7 is expressed using the keratin 2e expression amount and the linear function of the total protein amount. Kalliklein 7 relative amount corrected by dividing by the total protein amount obtained from the expression amount of keratin 2e ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）ｋａｌｌｉｋｌｅｉｎ ７の発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したｋａｌｌｉｋｌｅｉｎ ７相対量（２）ｋａｌｌｉｋｌｅｉｎ ７の発現量を、ｋｅｒａｔｉｎ ３発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ３発現量から求めた全タンパク質量で除して補正したｋａｌｌｉｋｌｅｉｎ ７相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) Kalliklein 7 relative amount corrected by dividing the expression level of kalliklein 7 by the total protein amount obtained by the Lowry method (2) The expression level of kalliklein 7 is expressed using a linear function of keratin 3 expression amount and total protein amount. Kalliklein 7 relative amount corrected by dividing by the total protein amount determined from the expression amount of keratin 3 ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）Ｇ３ＰＤＨの発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したＧ３ＰＤＨ相対量（２）Ｇ３ＰＤＨの発現量を、ｋｅｒａｔｉｎ ２ｅ発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ２ｅ発現量から求めた全タンパク質量で除して補正したＧ３ＰＤＨ相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) G3PDH relative amount corrected by dividing the expression amount of G3PDH by the total protein amount obtained by the Lowry method (2) The expression amount of G3PDH is calculated using the keratin 2e expression amount and a linear function of the total protein amount. Relative amount of G3PDH corrected by dividing by the total protein amount obtained from the expression level ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）Ｇ３ＰＤＨの発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したＧ３ＰＤＨ相対量（２）Ｇ３ＰＤＨの発現量を、ｋｅｒａｔｉｎ ３発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ３発現量から求めた全タンパク質量で除して補正したＧ３ＰＤＨ相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) G3PDH relative amount corrected by dividing the expression amount of G3PDH by the total protein amount obtained by the Lowry method (2) The expression amount of G3PDH is calculated using a linear function of keratin 3 expression amount and total protein amount, keratin 3 Relative amount of G3PDH corrected by dividing by the total protein amount obtained from the expression level ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）β−ａｃｔｉｎの発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したβ−ａｃｔｉｎ相対量（２）β−ａｃｔｉｎの発現量を、ｋｅｒａｔｉｎ ２ｅ発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ２ｅ発現量から求めた全タンパク質量で除して補正したβ−ａｃｔｉｎ相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) β-actin relative amount corrected by dividing the expression amount of β-actin by the total protein amount obtained by the Lowry method (2) The expression amount of β-actin is the primary amount of keratin 2e expression amount and total protein amount Β-actin relative amount corrected by dividing by the total protein amount obtained from the expression amount of keratin 2e using a function ２８名の被験者について、以下（１）と（２）との比較を示す図である。（１）β−ａｃｔｉｎの発現量をＬｏｗｒｙ法で求めた全タンパク質量で除して補正したβ−ａｃｔｉｎ相対量（２）β−ａｃｔｉｎの発現量を、ｋｅｒａｔｉｎ ３発現量と全タンパク質量の一次関数を用いてｋｅｒａｔｉｎ ３発現量から求めた全タンパク質量で除して補正したβ−ａｃｔｉｎ相対量It is a figure which shows the comparison with (1) and (2) below about 28 test subjects. (1) β-actin relative amount corrected by dividing the expression amount of β-actin by the total protein amount determined by the Lowry method (2) The expression amount of β-actin is the primary amount of keratin 3 expression amount and total protein amount Β-actin relative amount corrected by dividing by the total protein amount obtained from the expression amount of keratin 3 using the function

本発明に用いられる皮膚角質層中のタンパク質のハウスキーピングタンパク質としては、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３を用いることができる。皮膚角質層は皮膚の最外層に存在する１０層前後の層で表皮全体の厚みの１０分の１に相当する。皮膚角質層には表皮の最下層から分化してきた扁平な表皮角化細胞が脱核した状態で存在している。ｋｅｒａｔｉｎ ２ｅは分子量６５ＫＤａのタンパク質でｋｅｒａｔｉｎ １０とヘテロテトラマーを形成し、皮膚の顆粒層、有棘層に存在する。皮膚角質層の終分化に関与するといわれており、表皮角化細胞の活性や増殖、ケラチン化にも影響している。ｋｅｒａｔｉｎ ３は分子量６４ＫＤａのタンパク質でｋｅｒａｔｉｎ １２とヘテロテトラマーを形成しており、角質層特異的に発現している。これらのタンパク質の発現量を調べるには市販の抗体を使用して測定することができ、例えば、ＥＬＩＳＡ法、ウェスタンブロッティング法、ドットブロット法、抗体チップ等により測定することができる。   As a housekeeping protein of the protein in the skin stratum corneum used in the present invention, keratin 2e or keratin 3 can be used. The skin stratum corneum is a layer of about 10 layers existing in the outermost layer of the skin and corresponds to 1/10 of the thickness of the entire epidermis. In the skin stratum corneum, flat epidermal keratinocytes that have differentiated from the lowermost layer of the epidermis are present in an enucleated state. keratin 2e is a protein with a molecular weight of 65 KDa and forms a heterotetramer with keratin 10 and is present in the granular and spinous layers of the skin. It is said to be involved in the terminal differentiation of the skin stratum corneum, and also affects the activity and proliferation of keratinocytes and keratinization. Keratin 3 is a protein having a molecular weight of 64 KDa and forms a heterotetramer with keratin 12 and is expressed specifically in the stratum corneum. In order to investigate the expression level of these proteins, it can measure using a commercially available antibody, for example, can measure by ELISA method, Western blotting method, dot blot method, an antibody chip, etc.

本発明のタンパク質の測定の意味は、広義の意味で解釈するものとし、タンパク質濃度、タンパク質量の定量測定のほか、タンパク質の有無、量の多寡を検出するための定性的な測定も含まれる。本明細書では、タンパク質量の相対的な測定のことを、タンパク質量の評価ということがある。   The meaning of the protein measurement of the present invention is to be interpreted in a broad sense, and includes qualitative measurement for detecting the presence or absence of protein and the amount of protein in addition to quantitative measurement of protein concentration and protein amount. In the present specification, the relative measurement of protein amount is sometimes referred to as evaluation of protein amount.

本発明のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３をハウスキーピングタンパク質として用いる皮膚角質層中のタンパク質の測定例としては以下に示すものがある。
（１）皮膚角質層を含む試料中の全タンパク質量の測定において、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定することで試料中の全タンパク質の濃度に置き換えることができる。すなわち、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３に対する抗体を用いて、ＥＬＩＳＡ法等により、試料中のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量（例えばＥＬＩＳＡ発光強度）を求め、あらかじめ求めておいた全タンパク質の濃度とｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量との関係式に基づいて全タンパク質の濃度に補正換算することにより試料中の全タンパク質の濃度を求めることができる。なお、全タンパク質の相対量比較をする場合、全タンパク質量とｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量は比例関係にあるので、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を全タンパク質の相対量とすることもできる。
（２）皮膚角質層を含む試料中の特定のタンパク質の測定において、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を用いて特定のタンパク質の発現量を補正し、定量的な評価をすることができる。すなわち、測定対象・検査対象となる特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で除することにより、皮膚角質層の採取量を補正し、一定の皮膚角質層のタンパク質量に対する、特定のタンパク質の相対量を評価することができる。すなわち、Ｌｏｗｒｙ法等により全タンパク質の量を測定しなくても、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定すれば、異なる部位あるいは異なる被験者の皮膚角質層に存在する特定のタンパク質量の多寡を、比較評価できる。
さらに、（１）に記載した関係式により、ｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を全タンパク質量に換算し、その値を用いて特定のタンパク質の発現量を補正することにより、より正確に特定のタンパク質の相対量を評価することができる。
測定対象・検査対象となる特定のタンパク質としては、皮膚角質層中に存在するタンパク質であればいずれでも本発明を用いた測定をすることができ、例えばＨＳＰ２７、ＭＭＰ２、ｆｉｌａｇｇｒｉｎ等が挙げられる。 Examples of measurement of proteins in the skin stratum corneum using keratin 2e or keratin 3 of the present invention as housekeeping proteins include the following.
(1) In the measurement of the total protein amount in the sample including the skin stratum corneum, the expression level of keratin 2e or keratin 3 can be measured to replace the total protein concentration in the sample. That is, by using an antibody against keratin 2e or keratin 3, the expression level of keratin 2e or keratin 3 (for example, ELISA luminescence intensity) in a sample is determined by ELISA, etc., and the concentration of keratin 2e and keratin 2e determined in advance are obtained. Alternatively, the concentration of the total protein in the sample can be determined by performing correction conversion to the concentration of the total protein based on the relational expression with the expression level of keratin 3. When comparing the relative amount of all proteins, the total protein amount and the expression amount of keratin 2e or keratin 3 are proportional to each other. Therefore, the expression amount of keratin 2e or keratin 3 can also be used as the relative amount of all proteins. .
(2) In the measurement of a specific protein in a sample containing a skin stratum corneum, the expression level of the specific protein can be corrected using the expression level of keratin 2e or keratin 3, and quantitative evaluation can be performed. That is, by dividing the expression level of a specific protein to be measured / inspected by the expression level of keratin 2e or keratin 3, the amount of skin stratum corneum collected is corrected, The relative amount of a particular protein can be assessed. That is, even if the amount of keratin 2e or keratin 3 is measured without measuring the total protein amount by the Lowry method or the like, the amount of a specific protein amount present in the skin stratum corneum of a different site or a different subject, Comparative evaluation is possible.
Furthermore, according to the relational expression described in (1), the expression level of keratin 2e or keratin 3 is converted into the total protein level, and the expression level of a specific protein is corrected using that value, thereby allowing the specific protein to be identified more accurately. The relative amount of protein can be assessed.
As the specific protein to be measured / inspected, any protein present in the skin stratum corneum can be measured using the present invention, and examples thereof include HSP27, MMP2, and filaggrin.

〔比較例１〕
Ｇ３ＰＤＨ、β−ａｃｔｉｎ発現量と角質層タンパク質量との相関性
一般的にハウスキーピングタンパク質として用いられるＧ３ＰＤＨ、β−ａｃｔｉｎの２種類のタンパク質について、これらのタンパク質の発現量と角質層中の全タンパク質量との相関性をＥＬＩＳＡ法を用いて調べた。
（１）試験方法
（１−１）サンプルの調整
被験者２８名の頬から５枚テープストリッピングを行い、２００μｌの０．１％ＳＤＳ／ＰＢＳで角質層タンパク質を抽出し、サンプルとして用いた。
（１−２）Ｇ３ＰＤＨ、β−ａｃｔｉｎの発現量の測定（ＥＬＩＳＡ法）
（ｉ）（１−１）で調整した被験者２８名の各サンプル（１０μｌ）と０．１％ＳＤＳ／ＰＢＳ（４０μｌ）を混合した溶液をＥＬＩＳＡプレートの各ウェルに添加し、４℃で一晩静置し、固相化した。
（ｉｉ）１％ＢＳＡ／ＰＢＳを各ウェルに１５０μｌずつ加え、３７℃、１時間ブロッキングを行った。
（ｉｉｉ）各ウェルに２００μｌずつ０．０５％ Ｔｗｅｅｎ２０／ＰＢＳを入れて３回洗浄した。
（ｉｖ）１次抗体として抗Ｇ３ＰＤＨ抗体（Ａｂｆｒｏｎｔｉｅｒ社）、β−ａｃｔｉｎ（Ａｂｃａｍ 社）を用い、これらの抗体を０．０５％ Ｔｗｅｅｎ２０／ＰＢＳで１０００倍希釈した抗体希釈液を前記各ウェルに添加して、３７℃，で１時間反応を行った。
（ｖ）各ウェルに２００μｌずつ０．０５ ％ Ｔｗｅｅｎ２０／ＰＢＳを入れて３回洗浄した。
（ｖｉ）２次抗体としてＨＲＰ標識抗マウスＩｇＧ抗体（Ｚｙｍｅｄ社）を用い、本抗体を０．０５％ Ｔｗｅｅｎ２０／ＰＢＳで１０００倍希釈した抗体希釈液を前記各ウェルに添加して、３７℃で１時間反応を行った。
（ｖｉｉ）各ウェルに２００μｌずつ０．０５ ％ Ｔｗｅｅｎ２０／ＰＢＳを入れて３回洗浄した。
（ｖｉｉｉ）ＥＣＬ＋（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）を１００μｌ／ｗｅｌｌずつ添加し、５分間反応させて発光で検出した。検出は発光検出装置ＬＭａｘＩＩ３８４（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ社製）を用いた。
（１−３）角質層中のタンパク質の定量
サンプル中の全タンパク質量の定量はＬｏｗｒｙ法で行った。サンプル５μｌと試薬（Ｂｉｏ−Ｒａｄ社製Ｄｃ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ）を混ぜ合わせて１５分間反応させた後、７５０ｎｍで吸光度を測定した。
（２）試験結果
角質層から抽出した全タンパク質量とＧ３ＰＤＨ、β−ａｃｔｉｎの発現量（ＥＬＩＳＡ発光強度）との相関を調べた結果を図１、図２に示す。Ｇ３ＰＤＨ発現量と全タンパク質量の関係は、Ｒ２＝０．３３、β−ａｃｔｉｎ発現量と全タンパク質量の関係は、Ｒ２＝０．０８であった。これらの結果より、Ｇ３ＰＤＨ、β−ａｃｔｉｎの発現量と角質層全タンパク質量との相関性が高くないことがわかった。従って、Ｇ３ＰＤＨ、β−ａｃｔｉｎは、角質層タンパク質測定におけるインターナルコントロールとしての有用性は低いと考える。 [Comparative Example 1]
Correlation between G3PDH, β-actin expression level and stratum corneum protein level For two types of G3PDH, β-actin proteins generally used as housekeeping proteins, the expression level of these proteins and the total protein in the stratum corneum The correlation with the amount was examined by using the ELISA method.
(1) Test method (1-1) Sample adjustment Five tape strips were performed from the cheeks of 28 subjects, and the stratum corneum protein was extracted with 200 μl of 0.1% SDS / PBS and used as a sample.
(1-2) Measurement of expression level of G3PDH and β-actin (ELISA method)
(I) A solution prepared by mixing each sample (10 μl) of 28 subjects prepared in (1-1) and 0.1% SDS / PBS (40 μl) was added to each well of an ELISA plate, and overnight at 4 ° C. Allowed to stand and immobilize.
(Ii) 150 μl of 1% BSA / PBS was added to each well, and blocking was performed at 37 ° C. for 1 hour.
(Iii) Each well was washed 3 times with 200 μl of 0.05% Tween 20 / PBS.
(Iv) Anti-G3PDH antibodies (Abfrontier) and β-actin (Abcam) were used as primary antibodies, and antibody dilutions obtained by diluting these antibodies 1000 times with 0.05% Tween20 / PBS were added to each well. The reaction was conducted at 37 ° C. for 1 hour.
(V) Each well was washed 3 times with 200 μl of 0.05% Tween20 / PBS.
(Vi) An HRP-labeled anti-mouse IgG antibody (Zymed) was used as a secondary antibody, and an antibody dilution obtained by diluting the antibody 1000 times with 0.05% Tween20 / PBS was added to each of the wells. The reaction was carried out for 1 hour.
(Vii) Each well was washed 3 times with 200 μl of 0.05% Tween20 / PBS.
(Viii) ECL + (manufactured by GE Healthcare) was added in an amount of 100 μl / well, reacted for 5 minutes, and detected by luminescence. For detection, a luminescence detection device LMaxII384 (manufactured by Molecular Devices) was used.
(1-3) Quantification of protein in stratum corneum Quantification of the total protein amount in the sample was performed by the Lowry method. 5 μl of the sample and a reagent (Dc Protein Assay Kit manufactured by Bio-Rad) were mixed and reacted for 15 minutes, and then the absorbance was measured at 750 nm.
(2) Test results The results of examining the correlation between the total protein amount extracted from the stratum corneum and the expression levels of G3PDH and β-actin (ELISA luminescence intensity) are shown in FIG. 1 and FIG. The relationship between the G3PDH expression level and the total protein amount was R 2 = 0.33, and the relationship between the β-actin expression level and the total protein amount was R 2 = 0.08. From these results, it was found that the correlation between the expression level of G3PDH and β-actin and the total amount of stratum corneum protein was not high. Therefore, G3PDH and β-actin are considered to be less useful as an internal control in stratum corneum protein measurement.

〔試験例１〕
角質層タンパク中での標準タンパク質の同定
被験者の頬の試料より抽出した角質層タンパク質を利用して、全タンパク質量を一定にして測定した場合常に発現量が一定となるタンパク質のスクリーニングを行った。
（１）試験方法
（１−１）サンプルの調整および角質層中のタンパク質の定量
参考例１に同じ。
（１−２）標準蛋白質マーカーの検索・同定
（１−１）で調整した被験者２８名の各サンプルを各レーン１μｇ分のタンパク質量になるように調整し、ＳＤＳ−ＰＡＧＥを行った。電気泳動後、ゲルを銀染色して発現パターンがどのサンプルでも同じバンドを検索し、発現パターン（発現変動が見られない）が同じ４本のバンドについてＭＡＬＤＩ−ＴＯＦ−ＭＳを用いて同定を行った。
（２）試験結果
上記試験方法により、角質層タンパク質量を同量にして電気泳動を行い、発現量が一定のバンドのタンパク質（４種類）を同定した。４種類のタンパク質は以下のとおりであった。なお、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ６２ ｈｏｍｏｌｏｇ、ｐｈｏｓｐｈｏｐｒｏｔｅｉｎについては市販の抗体がなかったため以下の試験対象からは外した。
・Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ６２ ｈｏｍｏｌｏｇ
・ｋｅｒａｔｉｎ ２ｅ
・ｋｅｒａｔｉｎ ３
・ｐｈｏｓｐｈｏｐｒｏｔｅｉｎ [Test Example 1]
Identification of standard protein in stratum corneum protein Using a stratum corneum protein extracted from a sample of a subject's cheek, a protein having a constant expression amount when screened with a constant total protein amount was screened.
(1) Test method (1-1) Preparation of sample and quantification of protein in stratum corneum Same as Reference Example 1.
(1-2) Search and Identification of Standard Protein Markers Each sample of 28 subjects adjusted in (1-1) was adjusted to have a protein amount of 1 μg in each lane, and SDS-PAGE was performed. After electrophoresis, the gel is stained with silver to search for the same band in any sample, and the four bands with the same expression pattern (expression fluctuation is not observed) are identified using MALDI-TOF-MS. It was.
(2) Test results According to the test method described above, electrophoresis was carried out with the same amount of stratum corneum protein, and proteins (4 types) having a constant expression level were identified. The four types of proteins were as follows. Zinc finger protein 62 homolog and phosphoprotein were excluded from the following test subjects because there was no commercially available antibody.
・ Zinc finger protein 62 homolog
・ Keratin 2e
・ Keratin 3
・ Phosphoprotein

〔実施例１〕
ｋｅｒａｔｉｎ ２ｅ，ｋｅｒａｔｉｎ ３の発現量と角質層タンパク質量との相関性
一定量のタンパク質中における発現量の変化が見られなかったｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３の２種類のタンパク質について、ＥＬＩＳＡ法を用いてこれらのタンパク質の発現量（ＥＬＩＳＡ発光強度）と角質層中の全タンパク質量との相関性を調べた。
（１）試験方法
（１−１）サンプルの調整
比較例１と同じ。
（１−２）ｋｅｒａｔｉｎ ２ｅ，ｋｅｒａｔｉｎ ３の発現量の測定（ＥＬＩＳＡ法）
１次抗体として抗ｋｅｒａｔｉｎ ２ｅ抗体（Ｐｒｏｇｅｎ社）、抗Ｋｅｒａｔｉｎ３抗体（Ｍｉｌｌｉｐｏｒｅ 社）を用いた以外は比較例１と同じ。
（１−３）角質層中のタンパク質の定量
比較例１と同じ。
（２）試験結果
角質層から抽出した全タンパク質量とｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３の発現量（ＥＬＩＳＡ発光強度）との相関を調べた結果を図３、図４に示す。ｋｅｒａｔｉｎ ２ｅ発現量と全タンパク質量の関係は、Ｒ２＝０．７０９３、ｋｅｒａｔｉｎ ３発現量と全タンパク質量の関係は、Ｒ２＝０．５２６６であった。これらの結果より、ｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３の発現量と角質層全タンパク質量との相関性が高いことがわかった。従って、ｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３をハウスキーピングタンパク質として用いることで角質層中のタンパク質を測定することができることがわかった。
図３の測定結果より、ｋｅｒａｔｉｎ ２ｅの発現量を用いて、全タンパク質量は以下の式により求めることができる。
全タンパク質量（ｍｇ／ｍｌ）＝０．００２４４×ｋｅｒａｔｉｎ ２ｅの発現量＋０．１９・・・（１）
図４の測定結果より、ｋｅｒａｔｉｎ ３の発現量を用いて、全タンパク質量は以下の式により求めることができる。
全タンパク質量（ｍｇ／ｍｌ）＝０．００９２０×ｋｅｒａｔｉｎ ３の発現量−０．３９・・・（２）
尚、上記の２つの計算式は、頬の角質層の測定結果から求めたが、腕の角質層についても適用できることを確認している。 [Example 1]
Correlation between the expression level of keratin 2e and keratin 3 and the amount of stratum corneum protein Two types of proteins, keratin 2e and keratin 3, in which no change in the expression level in a certain amount of protein was observed, were detected using the ELISA method. The correlation between the protein expression level (ELISA luminescence intensity) and the total protein level in the stratum corneum was examined.
(1) Test method (1-1) Sample adjustment Same as Comparative Example 1.
(1-2) Measurement of expression levels of keratin 2e and keratin 3 (ELISA method)
The same as Comparative Example 1 except that anti-keratin 2e antibody (Progen) and anti-Keratin 3 antibody (Millipore) were used as primary antibodies.
(1-3) Quantification of protein in stratum corneum Same as Comparative Example 1.
(2) Test results The results of examining the correlation between the total protein amount extracted from the stratum corneum and the expression levels of keratin 2e and keratin 3 (ELISA luminescence intensity) are shown in FIGS. The relationship between the keratin 2e expression level and the total protein level was R 2 = 0.7093, and the relationship between the keratin 3 expression level and the total protein level was R 2 = 0.5266. From these results, it was found that the expression level of keratin 2e and keratin 3 and the stratum corneum total protein amount are highly correlated. Therefore, it was found that the protein in the stratum corneum can be measured by using keratin 2e and keratin 3 as housekeeping proteins.
From the measurement results of FIG. 3, the amount of total protein can be determined by the following equation using the expression level of keratin 2e.
Total protein amount (mg / ml) = 0.244 × Keratin 2e expression level + 0.19 (1)
From the measurement results of FIG. 4, the amount of total protein can be determined by the following equation using the expression level of keratin 3.
Total protein amount (mg / ml) = 0.920 × Keratin 3 expression level−0.39 (2)
The above two formulas were obtained from the measurement results of the cheek stratum corneum, but it has been confirmed that it can also be applied to the stratum corneum of the arm.

〔実施例２〕
Ｌｏｗｒｙ法による補正と本発明の補正との比較
比較例１および試験例１において２８名の被験者から採取した皮膚角質層について、Ｌｏｗｒｙ法による全タンパク質量、ｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３、Ｇ３ＰＤＨ，およびβ−ａｃｔｉｎの発現量（ＥＬＩＳＡ発光強度）を求めている。そこで、ｋａｌｌｉｋｌｅｉｎ ７の発現量も測定し、すでに測定したＧ３ＰＤＨ，β−ａｃｔｉｎの発現量と併せた３種のタンパク質について、Ｌｏｗｒｙ法による全タンパク質量で除して補正して相対量を求めた場合と、ｋｅｒａｔｉｎ ２ｅの発現量、ｋｅｒａｔｉｎ ３の発現量から、実施例１の式（１），（２）により、全タンパク質量を求めて、その値で除して補正して相対量を求めた場合とを比較した。
（１）ｋａｌｌｉｋｌｅｉｎ ７の発現量の測定方法（ＥＬＩＳＡ法）
１次抗体として抗ｋａｌｌｉｋｌｅｉｎ ７抗体（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社）を用いた以外は比較例１と同じ方法により同じ被験者から採取した皮膚角質層について測定を行った。
（２）試験結果
結果を図５から１０に示す。ｋａｌｌｉｋｌｅｉｎ ７、Ｇ３ＰＤＨ，β−ａｃｔｉｎの補正後の相対量について、Ｌｏｗｒｙ法による全タンパク質量で補正した相対量の小さい被験者から大きい被験者へと順番に並べた。（被験者によって、ｋａｌｌｉｋｌｅｉｎ ７、Ｇ３ＰＤＨ、β−ａｃｔｉｎそれぞれの多寡は異なるので、それぞれについて被験者の並び順は異なる。）
図からわかるように、Ｌｏｗｒｙ法による全タンパク質量補正とｋｅｒａｔｉｎ ２ｅの発現量、ｋｅｒａｔｉｎ ３の発現量から、実施例１の式により求めた全タンパク質量補正の結果は良く対応している。ｋｅｒａｔｉｎ ２ｅを用いた補正とｋｅｒａｔｉｎ ３を用いた補正を比較すると、ｋｅｒａｔｉｎ ２ｅを用いた補正の方がＬｏｗｒｙ法による補正と良く対応している。
尚、ｋａｌｌｉｋｌｅｉｎ ７、Ｇ３ＰＤＨ、β−ａｃｔｉｎの発現量をｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３の発現量で直接、除する補正も可能である。 [Example 2]
Comparison of Correction by Lowry Method and Correction of the Present Invention For the skin stratum corneum collected from 28 subjects in Comparative Example 1 and Test Example 1, total protein amount, keratin 2e, keratin 3, G3PDH, and β- The expression level of actin (ELISA emission intensity) is determined. Therefore, when the expression level of kalliklein 7 was also measured, and the three proteins combined with the already measured expression levels of G3PDH and β-actin were divided by the total protein amount by the Lowry method and corrected to obtain the relative amount And the expression amount of keratin 2e, the expression amount of keratin 3, and the expression (1) and (2) of Example 1, the total protein amount was obtained, divided by the value, corrected to obtain the relative amount. The case was compared.
(1) Method for measuring the expression level of kalliklein 7 (ELISA method)
The skin stratum corneum collected from the same subject was measured by the same method as Comparative Example 1 except that anti-kalliklein 7 antibody (R & D Systems) was used as the primary antibody.
(2) Test results The results are shown in FIGS. About the relative amount after correction | amendment of kalliklein 7, G3PDH, and beta-actin, it arranged in order from the test subject with the small relative amount corrected with the total protein amount by the Lowry method to the large subject. (Kalliklein 7, G3PDH, and β-actin have different levels depending on the subject, and the order of subjects is different for each.)
As can be seen from the figure, the results of the total protein amount correction obtained by the formula of Example 1 correspond well from the total protein amount correction by the Lowry method, the expression amount of keratin 2e, and the expression amount of keratin 3. Comparing the correction using keratin 2e and the correction using keratin 3, the correction using keratin 2e corresponds better to the correction by the Lowry method.
In addition, the correction | amendment which remove | divides directly the expression level of kalliklein 7, G3PDH, and (beta) -actin by the expression level of keratin 2e and keratin 3 is also possible.

ｋｅｒａｔｉｎ ２ｅ、ｋｅｒａｔｉｎ ３をハウスキーピングタンパク質として用いれば、皮膚角質層中のタンパク質を簡易、かつ正確に測定または評価することができる。   When keratin 2e and keratin 3 are used as housekeeping proteins, proteins in the skin stratum corneum can be measured or evaluated easily and accurately.

Claims (6)

以下の工程を有する、採取した皮膚角質層の特定のタンパク質量の評価方法。
（１）採取した角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した角質層の特定のタンパク質の発現量を測定する工程
（３）（２）で求めた特定のタンパク質の発現量を（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程 The evaluation method of the specific protein amount of the extract | collected skin stratum corneum which has the following processes.
(1) A step of measuring the expression level of keratin 2e or keratin 3 in the collected stratum corneum (2) A step of measuring the expression level of a specific protein in the collected stratum corneum (3) The specific protein obtained in (2) Correcting the expression level of keratin with the expression level of keratin 2e or keratin 3 determined in (1) 特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程が特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で除する工程である請求項１に記載の方法。 The method according to claim 1, wherein the step of correcting the expression level of the specific protein by the expression level of keratin 2e or keratin 3 is a step of dividing the expression level of the specific protein by the expression level of keratin 2e or keratin 3. 特定のタンパク質の発現量をｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量で補正する工程が、
特定のタンパク質の発現量を、以下の工程により求めた全タンパク質量で除する工程である請求項１に記載の方法。
（１）採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した皮膚角質層の全タンパク質量と（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量の一次関数を求める工程
（３）（２）で求めた１次関数を用いて、採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量から採取した皮膚角質層の全タンパク質量を求める工程。 The step of correcting the expression level of a specific protein with the expression level of keratin 2e or keratin 3,
The method according to claim 1, which is a step of dividing the expression level of a specific protein by the total protein amount determined by the following steps.
(1) The step of measuring the expression level of keratin 2e or keratin 3 in the collected skin stratum corneum (2) The total amount of protein in the collected skin stratum corneum and the primary expression level of keratin 2e or keratin 3 determined in (1) Step of obtaining function (3) Step of obtaining the total amount of protein in the horny layer collected from the expression level of keratin 2e or keratin 3 in the collected horny layer using the linear function obtained in (2). 以下の工程を有する、採取した皮膚角質層の全タンパク質量を測定する方法。
（１）採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量を測定する工程
（２）採取した皮膚角質層の全タンパク質量と（１）で求めたｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量の一次関数を求める工程
（３）（２）で求めた１次関数を用いて、採取した皮膚角質層のｋｅｒａｔｉｎ ２ｅまたはｋｅｒａｔｉｎ ３の発現量から採取した皮膚角質層の全タンパク質量を求める工程。 A method for measuring the total protein amount of a collected skin stratum corneum having the following steps.
(1) The step of measuring the expression level of keratin 2e or keratin 3 in the collected skin stratum corneum (2) The total amount of protein in the collected skin stratum corneum and the primary expression level of keratin 2e or keratin 3 determined in (1) Step of obtaining function (3) Step of obtaining the total amount of protein in the horny layer collected from the expression level of keratin 2e or keratin 3 in the collected horny layer using the linear function obtained in (2). タンパク質の発現量が、抗体を用いて測定した測定強度である請求項１から４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the protein expression level is a measured intensity measured using an antibody. タンパク質の発現量が、ＥＬＩＳＡ発光強度である請求項１から４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the protein expression level is ELISA luminescence intensity.
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