JP2015210199A - Evaluation method for degree of carbonylation - Google Patents
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Abstract
Description
本発明は、頭皮のカルボニル化度の評価方法に関するものである。 The present invention relates to a method for evaluating the degree of carbonylation of the scalp.
皮膚の最外層である角層においては、この角層を構成するタンパク質が酸化され易い状況にある。この酸化は、過酸化脂質の分解物であるアクロレインなどのアルデヒドのタンパク質への付加や、タンパク質への直接的な酸化ストレス作用によるカルボニル化によるものである。皮膚がカルボニル化したものであるかは、採取した角層の蛍光標識を行えば確認可能であると知られている。 In the stratum corneum, which is the outermost layer of the skin, the protein constituting the stratum corneum is easily oxidized. This oxidation is due to addition of an aldehyde such as acrolein, which is a degradation product of lipid peroxide, to a protein, or carbonylation by direct oxidative stress action on the protein. It is known that whether the skin is carbonylated can be confirmed by fluorescently labeling the collected stratum corneum.
ところで、特許文献１〜２には、皮膚のカルボニル化の確認に関する開示があるが、両文献共に、頭皮に着目した内容を開示していない。頭皮においても角層は存在し、頭皮のカルボニル化の状況を知ることができれば、今後の研究開発活動の一助になると考えられる。 By the way, Patent Documents 1 and 2 have disclosures regarding confirmation of skin carbonylation, but neither of these documents discloses content focused on the scalp. The stratum corneum also exists in the scalp, and it is thought that if we can know the state of carbonylation of the scalp, it will contribute to future research and development activities.
本発明は、上記事情に鑑み、頭皮における角層のカルボニル化の程度を評価する方法の提供を目的とする。 In view of the above circumstances, an object of the present invention is to provide a method for evaluating the degree of carbonylation of the stratum corneum in the scalp.
本発明者等が鋭意検討を行った結果、頭部及び頸部の２か所以上から採取した角層のカルボニルタンパク質に蛍光物質を接触させた後、各々の角層に標識した蛍光物質の蛍光を対比すれば、頭皮のカルボニル化度の評価に適することを見出し、本発明を完成するに至った。 As a result of intensive studies by the present inventors, after the fluorescent substance was brought into contact with the carbonyl protein of the stratum corneum collected from two or more locations of the head and neck, the fluorescence of the fluorescent substance labeled on each stratum corneum Was found to be suitable for evaluation of the degree of carbonylation of the scalp, and the present invention was completed.
すなわち、本発明に係るカルボニル化度の評価方法は、頭部又は頸部から採取した角層（Ｉ）に対して、カルボニルタンパク質のカルボニル基を蛍光標識可能な蛍光物質を接触させる標識工程（Ｉ）、前記角層（Ｉ）の採取部位とは異なる頭部の部位から採取した角層（II）に対して、カルボニルタンパク質のカルボニル基を蛍光標識可能な蛍光物質を接触させる標識工程（II）、及び、前記標識工程（Ｉ）後の角層（Ｉ）に標識した蛍光物質の蛍光と、前記標識工程（II）後の角層（II）に標識した蛍光物質の蛍光とを対比する対比工程、を備えることを特徴とする。 That is, in the method for evaluating the degree of carbonylation according to the present invention, the stratum corneum (I) collected from the head or neck is contacted with a fluorescent substance capable of fluorescently labeling the carbonyl group of the carbonyl protein (I ), A labeling step (II) in which a stratum corneum (II) collected from a site of the head different from the sampling site of the stratum corneum (I) is contacted with a fluorescent substance capable of fluorescently labeling the carbonyl group of the carbonyl protein And a comparison between the fluorescence of the fluorescent substance labeled on the stratum corneum (I) after the labeling step (I) and the fluorescence of the fluorescent substance labeled on the stratum corneum (II) after the labeling step (II) A process.
前記標識工程（Ｉ）及び前記標識工程（II）における蛍光物質は、ヒドラジノ基を備えるものであると良い。 The fluorescent substance in the labeling step (I) and the labeling step (II) is preferably provided with a hydrazino group.
本発明に係るカルボニル化度の評価方法において、前記角層（Ｉ）が頭部のネープ又は頸部から採取したものであり、前記角層（II）が頭部のトップ又はミドルから採取したものであると良い。好ましくは、前記角層（Ｉ）が頭部のトップ以外から採取したものであり、前記角層（II）が頭部のトップから採取したものである。頭部のトップに近づく程に紫外線により皮脂が過酸化脂質になり易くなると考えられるから、その過酸化脂質による角層（II）のタンパク質のカルボニル化が進行している場合には、対比工程で対比する傾向強度の差を確認し易くなる。 In the method for evaluating the degree of carbonylation according to the present invention, the stratum corneum (I) is collected from the nape or neck of the head, and the stratum corneum (II) is collected from the top or middle of the head. Good to be. Preferably, the stratum corneum (I) is collected from other than the top of the head, and the stratum corneum (II) is collected from the top of the head. Since the sebum is likely to become lipid peroxide due to ultraviolet rays as it gets closer to the top of the head, when the carbonylation of the protein of the stratum corneum (II) by the lipid peroxide is proceeding, It becomes easy to confirm the difference in tendency strength to be compared.
本発明に係るカルボニル化度の評価方法を、頭皮の老化指標として用いると良い。ここでは、本発明に係る評価方法における対比工程での結果が、老化指標の基礎となる。角層（Ｉ）と角層（II）のカルボニル化度の差は、年齢を重ねることに伴って生じやすくなる一般的傾向があることを見出しており、本発明の評価方法は、頭皮の老化指標の一つとして役立つ。 The method for evaluating the degree of carbonylation according to the present invention is preferably used as an index of aging of the scalp. Here, the result in the contrast process in the evaluation method according to the present invention is the basis of the aging index. The difference in the degree of carbonylation between the stratum corneum (I) and the stratum corneum (II) has been found to have a general tendency that it tends to occur with increasing age, and the evaluation method of the present invention is based on the aging of the scalp. Useful as an indicator.
本発明に係るカルボニル化度の評価方法によれば、採取した角層（Ｉ）に標識した蛍光物質の蛍光と、角層（Ｉ）とは異なる部位から採取した角層（II）に標識した蛍光物質の蛍光との対比を行うから、個人における頭皮のカルボニル化度の評価を行うことができる。 According to the method for evaluating the degree of carbonylation according to the present invention, the fluorescence of the fluorescent substance labeled on the collected stratum corneum (I) and the stratum corneum (II) collected from a site different from the stratum corneum (I) are labeled. Since the contrast with the fluorescence of the fluorescent substance is performed, the degree of carbonylation of the scalp in an individual can be evaluated.
標識工程（Ｉ）、標識工程（II）、及び対比工程を備える本実施形態のカルボニル化度の評価方法に基づき、本発明を以下に説明する。 The present invention will be described below based on the method for evaluating the degree of carbonylation of this embodiment comprising a labeling step (I), a labeling step (II), and a comparison step.
（標識工程（Ｉ））
標識工程（Ｉ）では、頭部又は頭部と胴体との間である頸部から採取した角層（Ｉ）に対して、蛍光物質を接触させる。
(Labeling step (I))
In the labeling step (I), the fluorescent material is brought into contact with the stratum corneum (I) collected from the head or the neck between the head and the trunk.
ここで、頭部のトップ、ミドル、及びネープの各部位について、図３に従って説明を加える。先ず、トップとは、オーバーとも呼ばれる部分であり、頭頂からテンプルポイントを通過する水平線の間の領域である。テンプルポイントとは、前髪の生え際の１番飛び出している部分のことである。次に、ミドルとは、テンプルポイントを通過する水平線と、ぼんの窪みを通過する水平線との間の領域である。ぼんの窪みとは、後頭部のもっともくぼんだ部分のことである。最後に、ネープとは、ぼんの窪みを通過する水平線と、みつえりを通過する水平線との間の領域である。みつえりとは、えり足の両端の１番下がった部分のことである。 Here, the top, middle, and nape parts of the head will be described with reference to FIG. First, the top is a portion called over and is a region between the top of the head and the horizontal line passing through the temple point. The temple point is the most protruding part of the hairline. Next, the middle is an area between a horizontal line passing through the temple point and a horizontal line passing through the depression. The dent of the bon is the most indented part of the back of the head. Finally, the nape is the area between the horizontal line that passes through the dent and the horizontal line that passes through the sight. Mitsuri is the part of the bottom end of the collar that has been lowered.
角層（Ｉ）の採取元となる頭部等の部位は、後記角層（II）とは異なるものとする。このように角層（II）に応じて角層（Ｉ）の採取元を決定すれば良く、角層（Ｉ）は、例えば頭部のネープ又は頸部（毛髪の生え際又はその周辺であると良い）から採取したものであると良く、頭部のトップ以外から採取したものであっても良い。 The part such as the head from which the stratum corneum (I) is collected is different from the stratum corneum (II) described later. In this way, the source of the stratum corneum (I) may be determined according to the stratum corneum (II), and the stratum corneum (I) is, for example, a head nape or neck (at the hairline or its periphery) Good) and may be collected from other than the top of the head.
角層（Ｉ）を採取する方法は、角層を採取する方法として公知のものを採用すると良い。その公知の方法としては、セロハンテープなどの粘着テープを使用するテープストリッピングが挙げられる。テープストリッピングでは、角層（Ｉ）の採取元の皮膚に粘着テープを貼り付けてから剥がし、粘着テープに角層を付着させる。 As a method for collecting the stratum corneum (I), a known method may be adopted as a method for collecting the stratum corneum. The known method includes tape stripping using an adhesive tape such as a cellophane tape. In tape stripping, the adhesive tape is applied to the skin from which the stratum corneum (I) is collected, and then peeled off to adhere the stratum corneum to the adhesive tape.
本標識工程（Ｉ）で使用する蛍光物質は、カルボニルタンパク質のカルボニル基を蛍光標識可能な公知のものとする。例えば、カルボニル基と結合するヒドラジノ基（―ＮＨＮＨ２）を備えるフルオレセイン−５−チオセミカルバジド、テキサスレッドヒドラジド、ルシファーイエローヒドラジドなどが、標識工程（Ｉ）で使用する蛍光物質として挙げられる。 The fluorescent substance used in this labeling step (I) is a known substance that can fluorescently label the carbonyl group of the carbonyl protein. For example, fluorescein-5-thiosemicarbazide having a hydrazino group (—NHNH 2 ) bonded to a carbonyl group, Texas red hydrazide, lucifer yellow hydrazide and the like can be mentioned as the fluorescent substance used in the labeling step (I).
標識工程（Ｉ）では、例えば上記蛍光物質を溶解させた緩衝液に角層（Ｉ）を適宜な時間浸漬することで、角層（Ｉ）への蛍光物質の接触を行う。テープストリッピングで角層（Ｉ）を採取した場合には、粘着テープに付着したままの角層（Ｉ）に蛍光物質を接触させると良い。この接触後、カルボニルタンパク質を蛍光標識していない蛍光物質を洗浄除去すると良い。 In the labeling step (I), for example, the stratum corneum (I) is immersed in a buffer solution in which the fluorescent substance is dissolved, for an appropriate time, so that the fluorescent substance is brought into contact with the stratum corneum (I). When the stratum corneum (I) is collected by tape stripping, the fluorescent substance may be brought into contact with the stratum corneum (I) that remains attached to the adhesive tape. After this contact, the fluorescent substance that is not fluorescently labeled with the carbonyl protein is preferably washed away.
（標識工程（II））
標識工程（II）では、角層（Ｉ）の採取方法と同様にテープストリッピングなどの公知の方法により頭部から採取した角層（II）に対して、標識工程（Ｉ）で使用する蛍光物質と同じ蛍光物質を、標識工程（Ｉ）と同様の方法により接触させると良い。この接触後、角層（II）のカルボニルタンパク質を蛍光標識していない蛍光物質を洗浄除去すると良い。
(Labeling process (II))
In the labeling step (II), the fluorescent substance used in the labeling step (I) for the stratum corneum (II) collected from the head by a known method such as tape stripping as in the method of collecting the stratum corneum (I) The same fluorescent substance may be contacted by the same method as in the labeling step (I). After this contact, the fluorescent substance not fluorescently labeled with the carbonyl protein in the stratum corneum (II) may be washed away.
角層（II）は、角層（Ｉ）の採取元とは異なる部位から採取したものであれば、頭部のトップ、ミドル、及びネープのいずれから採取したものでも良い。例えば、角層（II）は、角層（Ｉ）が頭部のネープ又は頸部から採取したものである場合には、頭部のトップ又はミドルから採取したものであると良く、角層（Ｉ）が頭部のトップ以外から採取したものである場合には、トップから採取したものであると良い。角層（II）と角層（Ｉ）の採取部位を異にすることで、個人の頭皮におけるカルボニルタンパク質の分布を確認できる。また、年齢を重ねると、個人差はあるが、頭部のトップの角層は頭部のトップ以外の角層よりもカルボニルタンパク質が多くなる一般的傾向がある知見を得ており、頭部のトップから採取したものを角層（II）、頭部のトップ以外から採取したものを角層（Ｉ）とすれば、個人における頭皮の老化指標を定めやすい。 The stratum corneum (II) may be collected from any of the top, middle, and nape of the head as long as the stratum corneum (II) is collected from a site different from the source of the stratum corneum (I). For example, when the stratum corneum (II) is collected from the nape or neck of the head, the stratum corneum (I) may be collected from the top or middle of the head. When I) is collected from other than the top of the head, it may be collected from the top. By differently collecting the stratum corneum (II) and the stratum corneum (I), the distribution of the carbonyl protein in the individual's scalp can be confirmed. In addition, with age, there are individual differences, but the stratum corneum at the top of the head has a general tendency to have more carbonyl protein than the stratum corneum other than the top of the head. If the sample collected from the top is the stratum corneum (II) and the sample collected from other than the top of the head is the stratum corneum (I), it is easy to determine the aging index of the scalp in an individual.
（対比工程）
対比工程では、上記標識工程（Ｉ）後の角層（Ｉ）に標識した蛍光物質の蛍光と、上記標識工程（II）後の角層（II）に標識した蛍光物質の蛍光との対比を行う。
(Contrast process)
In the comparison step, the fluorescence of the fluorescent substance labeled on the stratum corneum (I) after the labeling step (I) is compared with the fluorescence of the fluorescent substance labeled on the stratum corneum (II) after the labeling step (II). Do.
上記の蛍光の対比は、蛍光顕微鏡による撮影像の蛍光輝度を目視対比することで行える。また、蛍光輝度を画像解析により算出できることは公知であるから、その画像解析による算出結果の対比をもって対比工程を行っても良い。 The above-described fluorescence can be contrasted by visually comparing the fluorescence brightness of the image taken by the fluorescence microscope. Further, since it is known that the fluorescence luminance can be calculated by image analysis, the comparison process may be performed by comparing the calculation results by the image analysis.
本実施形態のカルボニル化度の評価方法は、以上の通りである。当該方法を、頭皮の老化の指標として用いても良い。この指標は、対比工程までの実行による結果を用いるものであり、例えば次の（１）〜（３）の手順で行うものであり、（１）様々な年齢の個人の頭皮について、対比工程までの実行による結果を得る、（２）その結果に基づき、年齢、年代毎又は年齢層毎とカルボニル化度の線形回帰式を求めることで、頭皮老化の基準を設ける、（３）その基準を指標として、個人の頭皮老化（頭皮年齢）を定めるものである。 The method for evaluating the degree of carbonylation of the present embodiment is as described above. The method may be used as an index of scalp aging. This index uses the result of execution up to the comparison process, and is performed, for example, by the following procedures (1) to (3). (1) For the scalp of individuals of various ages, until the comparison process (2) Establish a scalp aging standard by obtaining a linear regression equation of age, age or age group and carbonylation degree based on the result, and (3) index the criterion As an example, it determines the individual's scalp aging (scalp age).
また、本実施形態の評価方法を、頭皮のカルボニル化の抑制や、頭皮老化の抑制のために有効となる成分のスクリーニングに使用することが考えられる。このスクリーニングは、例えば次の（１）〜（３）の手順で行うものであり、（１）対比工程までの実行による対比結果を得る、（２）有効成分となるかの確認を行う被験物質を含むもの（単一成分の溶液、複数成分を含む組成物やエキスなど）を、上記（１）で使用した角層の被採取者の頭皮に一回又は複数回塗布し、一定期間経過後に再び対比工程までの実行による対比を行う、（３）上記（１）の対比結果と上記（２）の対比結果を比較し、上記（２）の対比結果の方が、角層（Ｉ）に標識した蛍光物質の蛍光と角層（II）に標識した蛍光物質の蛍光との輝度の差が小さいときには、被験物質を有効成分と判断するものである。なお、上記（２）の塗布部位は、カルボニル化が進行し易い頭部のトップが好ましい。 Moreover, it is possible to use the evaluation method of this embodiment for screening of the component effective for suppression of scalp carbonylation and scalp aging. This screening is performed, for example, according to the following procedures (1) to (3). (1) Obtain a comparison result by execution up to the comparison step. (2) Test substance for confirming whether it is an active ingredient. (Including single-component solutions, multiple-component compositions and extracts) are applied once or multiple times to the scalp of the stratum corneum used in (1) above, and after a certain period of time The comparison by the execution up to the comparison process is performed again. (3) The comparison result of (1) is compared with the comparison result of (2), and the comparison result of (2) is more in the stratum corneum (I). When the difference in luminance between the fluorescence of the labeled fluorescent substance and the fluorescence of the fluorescent substance labeled on the stratum corneum (II) is small, the test substance is judged as an active ingredient. In addition, the application | coating site | part of said (2) has a preferable top of the head where carbonylation advances easily.
以下、実施例に基づき本発明を詳述するが、この実施例の記載に基づいて本発明が限定的に解釈されるものではない。 EXAMPLES Hereinafter, although this invention is explained in full detail based on an Example, this invention is not interpreted limitedly based on description of this Example.
（カルボニル化度の評価）
以下の標識工程（Ｉ）、標識工程（II）、及び対比工程により、女性９０名の頭皮についてのカルボニル化度の評価を行った。
(Evaluation of degree of carbonylation)
The scalp degree of 90 female scalps was evaluated by the following labeling step (I), labeling step (II), and comparison step.
標識工程（Ｉ）
頭部のネープにおける頭皮にセロハンテープを貼り付けて、当該テープに角層（Ｉ）を付着させた。このテープをスライドガラスに貼り付けてから、テープの粘着剤を溶解させるためにキシレンに１時間浸漬し、キシレンを蒸発させた。その後、テープが付いた状態のスライドガラスを、室温のフルオレセイン−５−チオセミカルバジドが２０μＭである０．１Ｍモルホリノエタンスルホン酸ナトリウム塩緩衝液（ｐＨ５．５）に１時間浸漬してから、カルボニルタンパク質を蛍光標識していないフルオレセイン−５−チオセミカルバジドをＰＢＳ（リン酸緩衝生理食塩水）で洗浄した。
Labeling step (I)
A cellophane tape was affixed to the scalp of the head nape, and the stratum corneum (I) was adhered to the tape. The tape was affixed to a slide glass and then immersed in xylene for 1 hour in order to dissolve the adhesive on the tape to evaporate xylene. Thereafter, the glass slide with the tape attached was immersed in 0.1M morpholinoethanesulfonic acid sodium salt buffer (pH 5.5) containing 20 μM fluorescein-5-thiosemicarbazide at room temperature for 1 hour, and then carbonyl protein. Fluorescein-5-thiosemicarbazide that was not fluorescently labeled was washed with PBS (phosphate buffered saline).
標識工程（II）
頭部の頭頂部における頭皮にセロハンテープを貼り付けて、当該テープに角層（II）を付着させた。これを、標識工程（Ｉ）と同様の浸漬と洗浄を行った。
Labeling process (II)
Cellophane tape was affixed to the scalp at the top of the head, and the stratum corneum (II) was adhered to the tape. This was dipped and washed in the same manner as in the labeling step (I).
対比工程
標識工程（Ｉ）及び標識工程（II）後、角層（Ｉ）に標識した蛍光物質の蛍光及び角層（II）に標識した蛍光物質の蛍光について、蛍光顕微鏡を使用して蛍光画像を取得すると共に、画像解析により蛍光輝度の平均値を算出した。
Contrast process After the labeling process (I) and the labeling process (II), the fluorescence of the fluorescent substance labeled on the stratum corneum (I) and the fluorescence of the fluorescent substance labeled on the stratum corneum (II) using a fluorescence microscope And the average value of fluorescence luminance was calculated by image analysis.
図１は、上記取得した蛍光画像の例であり、上段、中段、下段の夫々は、同一人から採取した角層のカルボニル化度を評価したものである。図１においては、同一人であってもトップとネープの角層のカルボニル化度が異なることが、蛍光画像の輝度の相違から確認できる。 FIG. 1 is an example of the acquired fluorescence image, and each of the upper, middle, and lower stages is an evaluation of the degree of carbonylation of the stratum corneum collected from the same person. In FIG. 1, it can be confirmed from the difference in the brightness of the fluorescence image that the carbonylation degree of the stratum corneum of the top and the nape is different even for the same person.
下記表１は、上記の算出した蛍光輝度の平均値であり、図２は、その平均値と角層提供者である女性の年齢との関係を表すグラフである。図２に記載の回帰直線によれば、年齢を重ねるに伴い、トップの蛍光強度（カルボニル化度）がネープよりも高まっている一般的傾向が認められる。このことは、頭皮老化の指標として本実施例の方法を使用できることを示す。
Claims (5)
前記角層（Ｉ）の採取部位とは異なる頭部の部位から採取した角層（II）に対して、カルボニルタンパク質のカルボニル基を蛍光標識可能な蛍光物質を接触させる標識工程（II）、及び、
前記標識工程（Ｉ）後の角層（Ｉ）に標識した蛍光物質の蛍光と、前記標識工程（II）後の角層（II）に標識した蛍光物質の蛍光とを対比する対比工程、を備えることを特徴とする
カルボニル化度の評価方法。 A labeling step (I) in which a stratum corneum (I) collected from the head or neck is contacted with a fluorescent substance capable of fluorescently labeling a carbonyl group of a carbonyl protein;
A labeling step (II) in which a stratum corneum (II) collected from a site of the head different from the sampling site of the stratum corneum (I) is contacted with a fluorescent substance capable of fluorescently labeling the carbonyl group of the carbonyl protein; ,
A comparison step of comparing the fluorescence of the fluorescent substance labeled on the stratum corneum (I) after the labeling step (I) and the fluorescence of the fluorescent substance labeled on the stratum corneum (II) after the labeling step (II). A method for evaluating the degree of carbonylation, comprising:
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