JP2013257347A - Screening method and antiaging composition - Google Patents
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- JP2013257347A JP2013257347A JP2013201619A JP2013201619A JP2013257347A JP 2013257347 A JP2013257347 A JP 2013257347A JP 2013201619 A JP2013201619 A JP 2013201619A JP 2013201619 A JP2013201619 A JP 2013201619A JP 2013257347 A JP2013257347 A JP 2013257347A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
本発明は、化粧料原料等として好適な抗老化作用を有する成分のスクリーニング方法、並びに当該抗老化作用を有する成分を含有してなる抗老化用の組成物に関する。 The present invention relates to a method for screening an ingredient having an anti-aging action suitable as a cosmetic raw material and the like, and an anti-aging composition comprising the ingredient having the anti-aging action.
真核生物の核を細胞質から隔離する役割を果たす核膜は、内膜と外膜よりなる脂質二重膜構造を有すると共に、核ラミナと呼ばれる成分が核膜構造の形成・維持に重要な役割を果たしている。核ラミナは、ラミンやラミン結合蛋白質により形成され、内膜の内側（核質側）の中間径フィラメントによる格子状の裏打ち構造を構築し、核の状態を保持するほか、クロマチンの構造変化の動態や機能制御（例えば、非特許文献１を参照）にも関与することが知られている。このため、ラミン等の核膜上の蛋白質成分の異常は、核膜異常が関連する組織特異的な遺伝病や老化などの多細胞生物に特有な問題を引き起こす生体分子として注目されている。 The nuclear membrane, which plays a role in isolating the eukaryotic nucleus from the cytoplasm, has a lipid bilayer structure consisting of inner and outer membranes, and a component called nuclear lamina plays an important role in the formation and maintenance of the nuclear membrane structure. Plays. Nuclear lamina is formed by lamin and lamin-binding protein, and builds a lattice-like backing structure with intermediate filaments inside the inner membrane (nuclear side) to maintain the nuclear state and dynamics of chromatin structural changes It is also known to be involved in function control (for example, see Non-Patent Document 1). For this reason, abnormalities in protein components on the nuclear membrane such as lamin are attracting attention as biomolecules that cause problems peculiar to multicellular organisms such as tissue-specific genetic diseases and aging associated with nuclear membrane abnormalities.
前記の核膜異常が関連する疾患としては、先天的遺伝子異常を原因とするハッチソン・ギルフォード・プロジェリア症候群（ＨＧＰＳ）が有名である。ＨＧＰＳは、原因遺伝子の特定により、ヒト１番染色体上にあるラミンＡ（ＬＭＮＡ）遺伝子の異常プロセッシングにより、プロジェリンと呼ばれる異常タンパクが産生され、核膜に異常が生じることにより、老化促進が引き起こされることが明らかにされている。本疾患の主要な症状としては、身長、体重の発育が乏しいことのほか、症状の進行に伴い皮膚の老化、高コレステロール血症、動脈硬化の亢進、糖尿病、骨粗鬆症、老年性の白内障、白髪、脱毛などの早老変化が顕著になることが挙げられる。このことは、核膜異常が、様々な老化現象の発現に深く関与していることを示唆している。また近年、ＨＧＰＳに観察されるのと類似の核膜異常が正常老化皮膚細胞や、紫外線を照射した正常皮膚細胞において確認されたこと（例えば、非特許文献２、３を参照）により、核膜異常現象と正常皮膚老化との関連性に注目が集まっている。しかしながら、核膜異常現象が、いかなる生体反応を経由し老化現象を促進・顕在化させるかに関する研究は、はじまったばかりであり、その詳細な機構に付いては、得られた知見も限られているのが現状である（例えば、非特許文献４を参照）。 As the disease associated with the nuclear membrane abnormality, Hutchison-Gilford Progeria syndrome (HGPS) caused by a congenital genetic abnormality is famous. In HGPS, due to the identification of the causative gene, abnormal processing of the lamin A (LMNA) gene on human chromosome 1 produces an abnormal protein called progelin, which causes abnormalities in the nuclear membrane, leading to accelerated aging. It has been revealed that The main symptoms of this disease include poor growth in height and weight, as well as aging of the skin, hypercholesterolemia, increased arteriosclerosis, diabetes, osteoporosis, senile cataract, gray hair, It is mentioned that changes in premature aging such as hair loss become prominent. This suggests that nuclear membrane abnormalities are deeply involved in the development of various aging phenomena. In recent years, nuclear membrane abnormalities similar to those observed in HGPS have been confirmed in normal aging skin cells and normal skin cells irradiated with ultraviolet rays (see, for example, Non-Patent Documents 2 and 3). Attention has been focused on the relationship between abnormal phenomena and normal skin aging. However, research on what kind of biological reaction the nuclear membrane abnormal phenomenon promotes and manifests the aging phenomenon has just begun, and the detailed mechanism is limited. Is the current situation (see, for example, Non-Patent Document 4).
本発明は、化粧料原料（但し、医薬部外品を含む）として好適な成分、特に新たな作用機序に基づいた抗老化作用を有する成分を精度良く、かつ簡便に選別することができるスクリーニング方法を提供することを課題とする。 The present invention is a screening capable of accurately and easily selecting components suitable as cosmetic raw materials (including quasi drugs), particularly components having an anti-aging action based on a new mechanism of action. It is an object to provide a method.
本発明者らは、上記課題を解決するために鋭意研究を行った結果、被検物質を接触させた細胞と被検物質を接触させていない細胞の核膜異常状態を評価し、これらの核膜異常状態の差を指標とすることによって、抗老化作用を有する成分を精度良く、かつ簡便に選別することができることを見出し、本発明を完成させた。
即ち、本発明は、以下に示す通りである。
＜１＞ 被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態
を評価し、被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態の差を指標として、抗老化作用を有する成分を選別することを特徴とする、抗老化作用のある成分のスクリーニング方法。
＜２＞ 前記細胞が、核膜異常が生じた細胞である、＜１＞に記載のスクリーニング方法
。
＜３＞ 前記核膜異常が生じた細胞が、下記（ａ）〜（ｃ）の何れかの細胞である、＜２
＞に記載のスクリーニング方法。
（ａ）核膜異常が生じている患者から採取した細胞
（ｂ）圧力を加えることによって核膜異常を生じさせた細胞
（ｃ）アンチセンスオリゴを接触させることによって核膜異常を生じさせた細胞
＜４＞ 前記細胞が、ヒト線維芽細胞である、＜１＞〜＜３＞の何れかに記載のスクリー
ニング方法。
＜５＞ 核膜異常因子の量を測定すること及び／又は核形態を定量することによって細胞
の核膜異常状態を評価する、＜１＞〜＜４＞の何れかに記載のスクリーニング方法。
＜６＞ 前記核膜異常因子が、プロジェリン（Ｐｒｏｇｅｒｉｎ）である、＜５＞に記載
のスクリーニング方法。
＜７＞プロジェリンタンパクの量を測定することによって核膜異常因子の量を測定する、＜６＞に記載のスクリーニング方法。
＜８＞ プロジェリンｍＲＮＡの発現量を測定することによって核膜異常因子の量を測定
する、＜６＞に記載のスクリーニング方法。
＜９＞ 細胞の核染色像を画像解析することによって核形態を定量する、＜５＞〜＜６＞
の何れかに記載のスクリーニング方法。
＜１０＞ 前記核染色像が、ラミンＡタンパクの免疫染色による染色像又はＤＡＰＩによ
る染色像である、＜９＞に記載のスクリーニング方法。
＜１１＞ 被検物質を接触させていない細胞に比べて、被検物質を接触させた細胞の核膜
異常が抑制されている場合に、前記被検物質を抗老化作用を有する成分として選別する、＜１＞〜＜１０＞の何れかに記載のスクリーニング方法。
＜１２＞ 皮膚の抗老化作用を有する成分を選別するものである、＜１＞〜＜１１＞の何
れかに記載のスクリーニング方法。
＜１３＞ シワの予防作用又は改善作用を有する成分を選別するものである、＜１２＞に
記載のスクリーニング方法。
＜１４＞ ＜１＞〜＜１３＞の何れかに記載のスクリーニング方法によって選別された抗
老化作用を有する成分を含有する、抗老化用組成物。
＜１５＞ ＜１＞〜＜１３＞の何れかに記載のスクリーニング方法によって選別された抗
老化作用を有する成分を含有する、皮膚外用剤。
＜１６＞ 化粧料（但し、医薬部外品を含む）である、＜１５＞に記載の皮膚外用剤。
＜１７＞ シワの予防又は改善用である、＜１５＞又は＜１６＞に記載の皮膚外用剤。
As a result of diligent research to solve the above problems, the present inventors have evaluated the abnormal nuclear membrane state of cells that have contacted the test substance and cells that have not contacted the test substance, and these nuclei. By using the difference in the abnormal state of the membrane as an index, it has been found that components having an anti-aging action can be accurately and easily selected, and the present invention has been completed.
That is, the present invention is as follows.
<1> The nuclear membrane abnormal state of a cell contacted with the test substance and a cell not contacted with the test substance is evaluated, and the nucleus of the cell contacted with the test substance and the cell not contacted with the test substance A screening method for a component having an anti-aging action, wherein a component having an anti-aging action is selected using a difference in abnormal membrane state as an index.
<2> The screening method according to <1>, wherein the cell is a cell in which a nuclear membrane abnormality has occurred.
<3> The cell in which the nuclear membrane abnormality has occurred is any one of the following (a) to (c): <2
The screening method according to>.
(A) a cell collected from a patient having a nuclear membrane abnormality (b) a cell that has caused a nuclear membrane abnormality by applying pressure (c) a cell that has caused a nuclear membrane abnormality by contacting an antisense oligo <4> The screening method according to any one of <1> to <3>, wherein the cells are human fibroblasts.
<5> The screening method according to any one of <1> to <4>, wherein the abnormal nuclear membrane state of a cell is evaluated by measuring an amount of an abnormal nuclear membrane factor and / or quantifying a nuclear morphology.
<6> The screening method according to <5>, wherein the nuclear membrane abnormality factor is progerin.
<7> The screening method according to <6>, wherein the amount of nuclear membrane abnormality factor is measured by measuring the amount of progelin protein.
<8> The screening method according to <6>, wherein the amount of nuclear membrane abnormality factor is measured by measuring the expression level of progelin mRNA.
<9> Quantify nuclear morphology by image analysis of nuclear staining images of cells, <5> to <6>
A screening method according to any one of the above.
<10> The screening method according to <9>, wherein the nuclear staining image is a staining image by immunostaining of lamin A protein or a staining image by DAPI.
<11> When the nuclear membrane abnormality of the cells contacted with the test substance is suppressed as compared with the cells not contacted with the test substance, the test substance is selected as a component having an anti-aging action. <1>-<10> The screening method in any one of.
<12> The screening method according to any one of <1> to <11>, wherein the component having a skin anti-aging effect is selected.
<13> The screening method according to <12>, wherein a component having a wrinkle-preventing or improving effect is selected.
<14> An anti-aging composition comprising an anti-aging component selected by the screening method according to any one of <1> to <13>.
<15> A skin external preparation containing an anti-aging component selected by the screening method according to any one of <1> to <13>.
<16> The external preparation for skin according to <15>, which is a cosmetic (including quasi-drugs).
<17> The external preparation for skin according to <15> or <16>, which is used for prevention or improvement of wrinkles.
本発明によれば、抗老化作用を有する成分を精度良く、かつ簡便に判別することができることができる。 According to the present invention, it is possible to accurately and easily determine a component having an anti-aging action.
＜抗老化作用を有する成分のスクリーニング方法＞
本発明の一態様である抗老化作用を有する成分のスクリーニング方法（以下、「本発明のスクリーニング方法」と略す場合がある。）は、被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態を評価し、被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態の差を指標として、抗老化作用を有する成分を選別することを特徴とする。
本発明者らは、化粧料原料（但し、医薬部外品を含む）として好適な成分、特に新たな作用機序に基づいた抗老化作用を有する成分を選別することができるスクリーニング方法を求めて研究を重ねた結果、被検物質を接触させた細胞と被検物質を接触させていない細胞の核膜異常状態を評価し、これらの核膜異常状態の差を指標とすることによって、抗老化作用を有する成分を精度良く、かつ簡便に選別することができることを見出した。
核膜異常が関連する疾患としては、先天的遺伝子異常を原因とするハッチソン・ギルフォード・プロジェリア症候群（ＨＧＰＳ）が有名である。ＨＧＰＳは、原因遺伝子の特定により、ヒト１番染色体上にあるラミンＡ（ＬＭＮＡ）遺伝子の異常プロセッシングにより、プロジェリンと呼ばれる異常タンパクが産生され、核膜に異常が生じることにより、老化促進が引き起こされることが明らかにされている。本発明者らは、核膜異常を抑制することが老化の抑制に繋がることを確認するとともに、被検物質を接触させた細胞の核膜異常状態を評価することが抗老化作用を有する成分を選別する上で非常に有効であることを見出したのである。
<Screening method of component having anti-aging action>
The screening method for a component having an anti-aging action which is one embodiment of the present invention (hereinafter sometimes abbreviated as “the screening method of the present invention”) involves contacting a test substance with a cell and a test substance. Evaluate the abnormal state of the nuclear membrane of cells that have not yet contacted the test substance, and use the difference in the abnormal state of the nuclear membrane between the cells that have contacted the test substance and the cells that have not been in contact with the test substance as an index to select components that have anti-aging effects It is characterized by that.
The present inventors have sought a screening method capable of selecting components suitable as cosmetic raw materials (including quasi-drugs), particularly components having an anti-aging action based on a new mechanism of action. As a result of repeated research, anti-aging is evaluated by evaluating the abnormal nuclear membrane state between cells contacted with the test substance and cells not contacted with the test substance, and using the difference between these abnormal nuclear membrane states as an index. It has been found that components having an action can be accurately and easily selected.
A well-known disease associated with nuclear membrane abnormalities is Hutchison-Gilford Progeria syndrome (HGPS) caused by congenital genetic abnormalities. In HGPS, due to the identification of the causative gene, abnormal processing of the lamin A (LMNA) gene on human chromosome 1 produces an abnormal protein called progelin, which causes abnormalities in the nuclear membrane, leading to accelerated aging. It has been revealed that The present inventors have confirmed that suppressing nuclear membrane abnormality leads to suppression of aging, and evaluating a nuclear membrane abnormal state of a cell contacted with a test substance has an anti-aging effect. They found it to be very effective in sorting.
本発明のスクリーニング方法において使用する細胞は、核膜を有するものであればその具体的種類は特に限定されないが、角化細胞、線維芽細胞等が挙げられる。これらの中でも、線維芽細胞が好ましく、ヒト線維芽細胞が特に好ましい。ヒト線維芽細胞を使用することによって、特に皮膚の抗老化作用を有する成分をより精度良く選別するものと考えられる。
また、本発明のスクリーニング方法において使用する細胞は、正常な細胞のほか、核膜異常が生じた細胞であってもよい。核膜異常が生じた細胞を使用することによって、抗老化作用を有する成分をより精度良く選別することができる。
なお、「核膜異常が生じた細胞」として、下記（ａ）〜（ｃ）の何れかの細胞を挙げることができる。
（ａ）核膜異常が生じている患者から採取した細胞
核膜異常が生じている患者は、例えば後述する核膜異常状態の評価方法によって判別することができる。
（ｂ）圧力を加えることによって核膜異常を生じさせた細胞
本発明者らは、細胞に圧力を加えることによって、効率的に核膜異常を生じさせることができるという新たな知見を見出している。圧力を加える方法として、例えばシリコン製の培養容器（ＳＴＢ−ＣＨ−４Ｗ、ストレックス株式会社）等を水平方向に伸展させ、伸展状態を保持しながら細胞を培養した後に、横方向に３３％収縮して８日間培養することで横方向の圧力刺激を加えることによって核膜異常を生じさせることができる。かかる方法は、精度良く、かつ簡便に核膜異常を生じさせることができる方法であり、抗老化作用を有する成分をより精度良く、かつより簡便に選別することに繋がる。
（ｃ）アンチセンスオリゴを接触させることによって核膜異常を生じさせた細胞
アンチセンスオリゴは、核膜異常を生じさせることができるものであれば特に限定されないが、Ｆｏｎｇ Ｌ．Ｇ．らの方法（Ｈｕｍａｎ Ｍｏｌｅｃｕｌａｒ Ｇｅｎｅｔｉ
ｃｓ．１８（１３），２４６２−２４７１）により提示された、特定の配列を有するアンチセンスオリゴを使用することが好ましい。
また、本発明のスクリーニング方法は、採取等した細胞をそのまま使用するほか、培養した細胞を使用してもよい。細胞の培養は、例えば被検物質を接触させる細胞の場合、被検物質を接触させる前に行っても、被検物質を接触させた後に行っても、或いは被検物質を接触させる前及び被検物質を接触させた後の両方で行ってもよい。また、核膜異常が生じた細胞を使用する場合、核膜異常を生じさせる前に行っても、核膜異常を生じさせた後に行っても、或いは核膜異常を生じさせる前及び核膜異常を生じさせた後の両方で行ってもよい。なお、培養条件は、使用する細胞に応じて公知の培養方法を適宜採用することができる。
The cell used in the screening method of the present invention is not particularly limited as long as it has a nuclear membrane, and examples thereof include keratinocytes and fibroblasts. Among these, fibroblasts are preferable, and human fibroblasts are particularly preferable. By using human fibroblasts, it is considered that components having an anti-aging effect on the skin, in particular, are selected with higher accuracy.
Moreover, the cell used in the screening method of the present invention may be a normal cell or a cell having a nuclear membrane abnormality. By using cells in which nuclear membrane abnormality has occurred, components having an anti-aging effect can be selected more accurately.
In addition, examples of the “cell in which nuclear membrane abnormality has occurred” include any of the following cells (a) to (c).
(A) Cells collected from patients with nuclear membrane abnormalities Patients with nuclear membrane abnormalities can be identified by, for example, a nuclear membrane abnormal state evaluation method described later.
(B) Cells in which nuclear abnormalities are caused by applying pressure The present inventors have found a new finding that nuclear abnormalities can be efficiently caused by applying pressure to cells. . As a method of applying pressure, for example, a silicon culture vessel (STB-CH-4W, Strex Co., Ltd.) is expanded in the horizontal direction, and the cells are cultured while maintaining the extended state, and then contracted by 33% in the horizontal direction. Then, by culturing for 8 days, nuclear membrane abnormality can be caused by applying a lateral pressure stimulus. Such a method is a method capable of causing a nuclear membrane abnormality with high accuracy and simpleness, and leads to selection of a component having an anti-aging action with high accuracy and simpler.
(C) Cells in which nuclear membrane abnormality is caused by contacting with antisense oligo The antisense oligo is not particularly limited as long as it can cause nuclear membrane abnormality. G. Et al. (Human Molecular Geneti
cs. 18 (13), 2462-2471), preferably antisense oligos having a specific sequence.
In addition, the screening method of the present invention may use collected cells as they are, or may use cultured cells. For example, in the case of cells that are brought into contact with a test substance, cell culture is performed before the test substance is contacted, after the test substance is contacted, or before and after the test substance is contacted. It may be performed both after contacting the test substance. In addition, when using cells with nuclear membrane abnormalities, whether they are performed before the nuclear membrane abnormality is generated, after the nuclear membrane abnormality is generated, or before the nuclear membrane abnormality is generated, and the nuclear membrane abnormality It may be performed both after generating. In addition, a well-known culture method can be suitably employ | adopted for culture conditions according to the cell to be used.
本発明のスクリーニング方法は、「被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態を評価する」ことを特徴とするが、核膜異常状態を評価する具体的な評価方法は特に限定されず、光学顕微鏡等によって核膜を観察する等の公知の方法を適宜採用することができるが、核膜異常状態を定量的に表すことができる評価方法によって評価することが好ましい。具体的には、核膜異常因子の量を測定すること、及び核形態を定量することが挙げられる。
「核膜異常因子」とは、具体的には核膜異常の原因となるタンパク質を意味し、核膜異常因子の量が多いほど核膜異常が進んでいると評価することができる。なお、核膜異常因子として、例えばプロジェリン（Ｐｒｏｇｅｒｉｎ）等が挙げられる。
核膜異常因子の量を測定する方法は、特に限定されないが、タンパク発現量を測定する方法、及びｍＲＮＡ発現量を測定する方法等が挙げられる。タンパク発現量を測定する方法としては、ウェスタンブロット法（ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ）、酵素結合免疫吸着法（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）等が挙げられる。一方、ｍＲＮＡ発現量を測定する方法としては、ノーザンブロット法（ｎｏｒｔｈｅｒｎ ｂｌｏｔｔｉｎｇ）、逆転写ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ：Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）法、リアルタイムＰＣＲ（Ｒｅａｌ ｔｉｍｅ ＰＣＲ）法、さらにはリアルタイムＰＣＲ（Ｒｅａｌ ｔｉｍｉ ＰＣＲ）とＲＴ−ＰＣＲを組み合わせた方法等が挙げられる。
The screening method of the present invention is characterized by "evaluating the nuclear membrane abnormal state of a cell contacted with a test substance and a cell not contacted with a test substance". The specific evaluation method is not particularly limited, and a known method such as observing the nuclear membrane with an optical microscope or the like can be appropriately employed, but the evaluation is performed by an evaluation method capable of quantitatively expressing the nuclear membrane abnormal state. It is preferable. Specifically, the amount of nuclear membrane abnormality factor is measured, and the nuclear morphology is quantified.
The “nuclear membrane abnormality factor” specifically means a protein that causes the nuclear membrane abnormality, and it can be evaluated that the nuclear membrane abnormality progresses as the amount of the nuclear membrane abnormality factor increases. In addition, as a nuclear membrane abnormality factor, progelin (Progerin) etc. are mentioned, for example.
The method for measuring the amount of the nuclear membrane abnormality factor is not particularly limited, and examples thereof include a method for measuring the protein expression level and a method for measuring the mRNA expression level. Examples of methods for measuring the protein expression level include Western blotting, enzyme-linked immunosorbent assay, and the like. On the other hand, methods for measuring mRNA expression include Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR (Real time PCR), and real-time PCR. Examples include a method combining PCR (Real time PCR) and RT-PCR.
核形態を定量する方法は、特に限定されないが、光学顕微鏡等によって核を観察し、核の寸法、面積等の核膜異常状態を表す数値を測定する方法が挙げられる。なお、光学顕微鏡等によって核を観察する場合において、核の染色を行ってもよい。染色方法も特に限定されず、公知の方法を適宜採用することができるが、例えばラミンＡタンパクの免疫染色、ＤＡＰＩによる染色が挙げられる。また、核膜異常状態を表す数値を測定するために、画像をコンピューターに取り込み、公知の画像解析ソフトを利用して測定を行ってよい。本発明のスクリーニング方法においては、細胞の核染色像を画像解析することによって核形態を定量することが好ましく、ラミンＡタンパクの免疫染色による染色像又はＤＡＰＩによる染色像を画像解析することによって核形態を定量することが好ましい。
また、光学顕微鏡等によって核を観察して測定した核の寸法、面積等の数値を直接使用するほか、これらの数値に基づいて核のコンター比等の数値を算出してもよい。コンター比は、式（コンター比＝４π×面積／周辺長2）によって算出することができる値であり
、１に近づくほど正円であり、０に近づくほど輪郭が歪んでいることを表す。従って、核のコンター比が０に近づくほど核膜異常が進んでいると評価することができる。
The method of quantifying the nuclear morphology is not particularly limited, and examples thereof include a method of observing the nucleus with an optical microscope or the like and measuring a numerical value representing an abnormal nuclear membrane state such as the size and area of the nucleus. In addition, when nuclei are observed with an optical microscope or the like, the nuclei may be stained. The staining method is also not particularly limited, and a known method can be appropriately employed. Examples thereof include immunostaining of lamin A protein and staining with DAPI. Further, in order to measure a numerical value representing an abnormal nuclear membrane state, an image may be taken into a computer and measurement may be performed using known image analysis software. In the screening method of the present invention, it is preferable to quantify the nuclear morphology by image analysis of the nuclear staining image of the cell, and the nuclear morphology by image analysis of the staining image by immunostaining of lamin A protein or the staining image by DAPI. Is preferably quantified.
In addition to directly using numerical values such as the size and area of the nuclei measured by observing the nuclei with an optical microscope or the like, numerical values such as the contour ratio of the nuclei may be calculated based on these numerical values. The contour ratio is a value that can be calculated by the formula (contour ratio = 4π × area / peripheral length 2 ). The contour ratio is a circle as it approaches 1 and the contour is distorted as it approaches 0. Therefore, it can be evaluated that the nuclear envelope abnormality progresses as the nuclear contour ratio approaches zero.
本発明のスクリーニング方法は、「被検物質を接触させた細胞及び被検物質を接触させていない細胞の核膜異常状態の差を指標として、抗老化作用を有する成分を選別する」ことを特徴とするが、具体的には被検物質を接触させていない細胞に比べて、被検物質を接触させた細胞の核膜異常が抑制されている場合に、被検物質を抗老化作用を有する成分と
して選別するものである。
なお、抗老化作用を有する成分をより精度良く選別するため、或いは抗老化作用の強い成分を選別するために、核膜異常が抑制されているか否かの判断について独自の基準を設けて判断してもよい。
例えば、プロジェリンの量を測定することによって細胞の核膜異常状態を評価する場合、下記式によって算出されるプロジェリンの量の比率が、好ましくは９５％以下、より好ましくは８０％以下となった場合に、核膜異常が抑制されていると判断することが挙げられる。
（プロジェリンの量の比率）＝（被検物質を接触させた細胞のプロジェリンの量）／（被検物質を接触させていない細胞のプロジェリンの量）×１００
また、核のコンター比を算出することによって細胞の核膜異常状態を評価する場合、被検物質を接触させた細胞の核のコンター比が被検物質を接触させていない細胞の核のコンター比に対して増加する場合、より好ましくは統計学的に有意に増加する場合に、核膜異常が抑制されていると判断することが挙げられる。
The screening method of the present invention is characterized by “selecting a component having an anti-aging effect using as an index the difference in abnormal nuclear membrane state between cells contacted with a test substance and cells not contacted with a test substance” However, when the nuclear membrane abnormality of the cell contacted with the test substance is suppressed compared to the cells not contacted with the test substance, the test substance has an anti-aging effect. It sorts out as a component.
In addition, in order to select components with anti-aging effects with higher accuracy, or to select components with strong anti-aging effects, it is decided to determine whether or not nuclear membrane abnormalities are being suppressed by setting an original standard. May be.
For example, when evaluating the abnormal nuclear membrane state of a cell by measuring the amount of progelin, the ratio of the amount of progelin calculated by the following formula is preferably 95% or less, more preferably 80% or less. In this case, it may be judged that the nuclear membrane abnormality is suppressed.
(Progerin amount ratio) = (Amount of progelin in cells in contact with test substance) / (Amount of progelin in cells not in contact with test substance) × 100
In addition, when calculating the nuclear nuclear abnormal state by calculating the nuclear contour ratio, the nuclear contour ratio of the cell contacted with the test substance is the nucleus ratio of the cell not contacted with the test substance. When it increases with respect to, More preferably, when it increases statistically significantly, it may be judged that nuclear membrane abnormality is suppressed.
本発明のスクリーニング方法は、「抗老化作用を有する成分を選別する」ことを特徴とするが、抗老化作用を有する成分が対象とする具体的な老化現象は特に限定されず、核膜異常との関連性がある皮膚の老化、高コレステロール血症、動脈硬化の亢進、糖尿病、骨粗鬆症、老年性の白内障、白髪、脱毛等の老化現象に広く適用することができる。但し、本発明のスクリーニング方法は、皮膚の抗老化作用を有する成分を選別するものであることが好ましく、シワの予防作用又は改善作用を有する成分を選別するものであることがより好ましい。 The screening method of the present invention is characterized by “selecting a component having an anti-aging effect”, but the specific aging phenomenon targeted by the component having an anti-aging effect is not particularly limited, and a nuclear membrane abnormality It can be widely applied to aging phenomena such as skin aging, hypercholesterolemia, arteriosclerosis, diabetes, osteoporosis, senile cataract, gray hair, hair loss and the like. However, the screening method of the present invention is preferably a method for selecting a component having an anti-aging effect on the skin, and more preferably a method for selecting a component having a wrinkle-preventing or improving effect.
本発明のスクリーニング方法が選別対象とする被検物質の種類は、特に限定されず、単純な化学物質、植動植物由来の抽出物、植動植物由来の抽出物の分画精製物、及びこれらの混合組成物等に広く適用することができる。
例えば、植物由来の抽出物として、自生又は生育された植物、漢方生薬原料等として販売される日本産のものを抽出及び／又は精製したものを使用するほか、丸善製薬株式会社等の会社より販売されている市販の抽出物を購入し、使用することも挙げられる。前記抽出に用いる植物部位も特に限定されず、全草を使用するほか、植物体、地上部、根茎部、木幹部、葉部、茎部、花穂、花蕾等の部位を使用することもできる。抽出に際し、植物体などの抽出に用いる部位は、予め、粉砕或いは細切して抽出効率を向上させるように加工することが好ましい。抽出物製造においては、植物体等の抽出に用いる部位乃至はその乾燥物１質量部に対して、溶媒を１〜３０質量部加え、室温であれば数日間、沸点付近の温度であれば数時間浸漬する。浸漬後は、室温まで冷却し、所望により不溶物を除去した後、溶媒を減圧濃縮するなどにより除去することが出来る。しかる後、シリカゲルやイオン交換樹脂を充填したカラムクロマトグラフィーなどで分画精製し、所望の抽出物を得ることが出来る。
前記抽出溶媒としては、極性溶媒が好ましく、水、エタノール、イソプロピルアルコール、ブタノールなどのアルコール類、１，３−ブタンジオール、ポリプロピレングリコールなどの多価アルコール類、アセトン、メチルエチルケトンなどのケトン類、ジエチルエ−テル、テトラヒドロフランなどのエーテル類から選択される１種乃至は２種以上が好適に例示することができる。
The type of the test substance to be selected by the screening method of the present invention is not particularly limited, and a simple chemical substance, an extract derived from a plant or plant, a fraction purified product of an extract derived from a plant or plant, and a mixture thereof It can be widely applied to compositions and the like.
For example, as a plant-derived extract, a plant that has been grown or grown, or a product obtained by extracting and / or purifying Japanese products sold as a raw material for herbal medicines, etc., is sold by companies such as Maruzen Pharmaceutical Co., Ltd. It is also possible to purchase and use commercially available extracts. The plant part used for the extraction is not particularly limited, and whole plant can be used, and parts such as a plant body, an aerial part, a rhizome part, a tree trunk part, a leaf part, a stem part, a flower spike, and a flower bud can be used. At the time of extraction, it is preferable that a part used for extraction of a plant body or the like is processed in advance so as to improve extraction efficiency by crushing or chopping. In the production of extracts, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a part used for extraction of a plant or the like, or a dry product thereof. Immerse for hours. After the immersion, the solution can be cooled to room temperature, insoluble matter can be removed if desired, and then the solvent can be removed by concentration under reduced pressure. Thereafter, the desired extract can be obtained by fractional purification by column chromatography packed with silica gel or ion exchange resin.
The extraction solvent is preferably a polar solvent, alcohols such as water, ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, diethyl ether One or two or more selected from ethers such as tellurium and tetrahydrofuran can be preferably exemplified.
＜抗老化用組成物・皮膚外用剤＞
本発明のスクリーニング方法によって、抗老化作用を有する成分を精度良く、かつ簡便に選別することができることを前述したが、本発明のスクリーニング方法によって選別された抗老化作用を有する成分を含有する抗老化用組成物（以下、「本発明の抗老化用組成物」と略す場合がある。）も本発明の一態様である。
本発明の抗老化用組成物は、本発明のスクリーニング方法によって選別された抗老化作
用を有する成分を含有するものであれば、その用途は特に限定されず医薬品、化粧品、食品、飲料等として利用することができる。また、抗老化作用を有する成分以外の成分についても特に限定されず、食品、医薬品、化粧料等の用途に基づき、それぞれの製剤化で使用される任意成分を含有することができる。例えば、経口投与組成物であれば、乳糖や白糖等の賦形剤、デンプン、セルロース、アラビアゴム、ヒドロキシプロピルセルロースなどの結合剤、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロ−スカルシウム等の崩壊剤、大豆レシチン、ショ糖脂肪酸エステル等の界面活性剤、マルチトールやソルビトール等の甘味剤、クエン酸等の酸味剤、リン酸塩等の緩衝剤、シェラックやツェイン等の皮膜形成剤、タルク、ロウ類などの滑沢剤、軽質無水ケイ酸、乾燥水酸化アルミニウムゲル等の流動促進剤、生理食塩水、ブドウ糖水溶液等の希釈剤、矯味矯臭剤、着色剤、殺菌剤、防腐剤、香料等が挙げられる。経皮投与組成物であれば、スクワラン、ワセリン、マイクロクリスタリンワックス等の炭化水素類、ホホバ油、カルナウバワックス、オレイン酸オクチルドデシル等のエステル類、オリーブ油、牛脂、椰子油等のトリグリセライド類、ステアリン酸、オレイン酸、レチノイン酸等の脂肪酸、オレイルアルコール、ステアリルアルコール、オクチルドデカノール等の高級アルコール、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、アルキルベタイン塩等の両性界面活性剤類、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ソルビタン脂肪酸エステル、脂肪酸モノグリセライド、これらのポリオキシエチレン付加物、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン脂肪酸エステル等の非イオン界面活性剤類、ポリエチレングリコール、グリセリン、１，３−ブタンジオール等の多価アルコール類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等が挙げられる。
<Anti-aging composition / external preparation for skin>
As described above, the screening method of the present invention can accurately and easily select components having an anti-aging action, but the anti-aging containing components having an anti-aging action selected by the screening method of the present invention. A composition for use in the present invention (hereinafter sometimes abbreviated as “anti-aging composition of the present invention”) is also an embodiment of the present invention.
If the composition for anti-aging of this invention contains the component which has the anti-aging effect selected by the screening method of this invention, the use will not be specifically limited, It uses as a pharmaceutical, cosmetics, a foodstuff, a drink, etc. can do. Moreover, it is not specifically limited about components other than the component which has an anti-aging effect, Based on uses, such as a foodstuff, a pharmaceutical, and cosmetics, the arbitrary component used by each formulation can be contained. For example, in the case of an orally administered composition, excipients such as lactose and sucrose, binders such as starch, cellulose, gum arabic, and hydroxypropylcellulose, disintegrants such as sodium carboxymethylcellulose and carboxymethylcellulose calcium, soybeans Surfactants such as lecithin and sucrose fatty acid esters, sweeteners such as maltitol and sorbitol, sour agents such as citric acid, buffering agents such as phosphate, film forming agents such as shellac and zein, talc and waxes Lubricants, light silicic acid anhydride, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline, aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, fragrances, etc. . For transdermal administration compositions, hydrocarbons such as squalane, petrolatum and microcrystalline wax, jojoba oil, carnauba wax, esters such as octyldodecyl oleate, triglycerides such as olive oil, beef tallow and coconut oil, stearin Acids, fatty acids such as oleic acid, retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol, octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate, and amphoteric interfaces such as alkyl betaine salts Activators, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, polyoxyethylene fatty acids Nonionic surfactants such as stealth, polyhydric alcohols such as polyethylene glycol, glycerin, 1,3-butanediol, thickening / gelling agents, antioxidants, UV absorbers, colorants, preservatives, powders Examples include the body.
本発明のスクリーニング方法によって、抗老化作用を有する成分を精度良く、かつ簡便に選別することができることを前述したが、本発明のスクリーニング方法によって選別された抗老化作用を有する成分を含有する皮膚外用剤（以下、「本発明の皮膚外用剤」と略す場合がある。）も本発明の一態様である。
本発明の皮膚外用剤は、本発明のスクリーニング方法によって選別された抗老化作用を有する成分を含有するものであれば、その他については特に限定されないが、抗老化作用を有する成分の含有量は、皮膚外用剤全量に対し、通常０．００００１質量％〜１５質量％、好ましくは０．０００１質量％〜１０質量％、より好ましくは０．００１質量〜５質量％である。抗老化作用を有する成分の含有量が、少なすぎると目的とする抗老化作用が発揮されず、多すぎても効果が頭打ちになり、この系の自由度を損なう場合が存するためである。
また、本発明の皮膚外用剤は、化粧料（但し、医薬部外品を含む）であることが好ましく、シワの予防又は改善用の皮膚外用剤であることが好ましい。
As described above, the screening method of the present invention can accurately and easily select components having an anti-aging effect. However, the skin external use containing an anti-aging component selected by the screening method of the present invention has been described above. An agent (hereinafter sometimes abbreviated as “the skin external preparation of the present invention”) is also an embodiment of the present invention.
The external preparation for skin of the present invention is not particularly limited as long as it contains an anti-aging component selected by the screening method of the present invention, but the content of the anti-aging component is as follows: It is 0.00001 mass%-15 mass% normally with respect to the skin external preparation whole quantity, Preferably it is 0.0001 mass%-10 mass%, More preferably, it is 0.001 mass-5 mass%. This is because if the content of the component having anti-aging action is too small, the intended anti-aging action is not exhibited, and if it is too high, the effect reaches a peak and the degree of freedom of this system may be impaired.
The external preparation for skin of the present invention is preferably a cosmetic (including quasi-drugs), and is preferably an external preparation for skin prevention or improvement.
以下に、実施例を挙げて本発明に付いて更に詳細に説明を加えるが、本発明がかかる実施例にのみ限定を受けないことは、言うまでもない。 Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.
［実施例１］
＜試験例１：プロジェリン量の変化を指標とした核膜異常状態評価＞
正常ヒト皮膚線維芽細胞を、２４穴プレート（ＢＤ Ｆａｌｃｏｎ社製）に７００００ｃｅｌｌｓ／ｗｅｌｌになる様に播種し、１０％ＦＢＳ／ＤＭＥＭ（ＦＢＳ：ハナ・ネスコバイオ社製、ＤＭＥＭ：シグマアルドリッチ社製）で２４時間培養した。次いで核膜異常誘導のため、１００ｎＭのラミンＡアンチセンスオリゴ（ＧＴＧＡＣＧＴＧＣＡＣＴＧＡ：シグマジェノシス合成品）をＬｉｐｏｆｅｃｔａｍｉｎ２０００（ライフテクノロジーズ製）を用いてトランスフェクションした。なお、コントロールとしてスクランブルオリゴ（ＴＧＧＣＧＧＣＡＡＧＣＣＣＴＣＡ：シグマジェノシス合成品）を同手法にてトラ
ンスフェクションした細胞も用意した。その後それぞれの被検物質を所定濃度加え、７日間培養した。培養後に得られた細胞を、Ｌｙｓｉｓバッファー（２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．２）、１５０ｍＭ ＮａＣｌ、１．０％ ＮＰ−４０、１錠 Ｐｒｏｔｅｉｎａｓｅ ｉｎｈｉｂｉｔｏｒ ｃｏｃｋｔａｉｌ／５０ｍＬ超純水）にて溶解し、ＤＣプロテインアッセイ（バイオラッド製）にてタンパク量を調整後、Ｅ−ＰＡＧＥＬ（ＡＴＴＯ製）を用いて電気泳動を行った。泳動により分離されたタンパクを、Ｉｍｍｏｂｉｌｏｎ−Ｐ（ミリポア製）に電圧をかけることで転写し、４００倍希釈したヤギ抗ＬａｍｉｎＡ／Ｃ抗体（サンタクルズ製）及び内在性コントロールとしてウサギ抗β-アクチン
抗体(アブカム製)を反応させた。ＴＢＳバッファー（ミリポア製）にて洗浄後、５０００倍希釈したＨＲＰ含有抗ヤギＩｇＧ抗体（ＺｙＭａｘ製）及び抗ウサギＩｇＧ抗体（ＺｙＭａｘ製）を反応させ、洗浄した後、ＳｕｐｅｒＳｉｇｎａｌ ＷｅｓｔＦｅｍｔｏ Ｍａｘｉｍｕｍ Ｓｅｎｓｉｔｉｖｉｔｙ Ｓｕｂｓｔｒａｔｅ（サーモサイエンティフィック製）にてＨＲＰを発色させた。その後ＣｏｏｌＳａｖｅｒ（ＡＴＴＯ製）にて画像を取得し、画像解析ソフトＩｍａｇｅＪ（ＮＩＨ製）にてプロジェリン及びβ-アクチンの
バンド強度を計測した。プロジェリンのバンド強度／β-アクチンのバンド強度を求め、
さらにアンチセンスオリゴをトランスフェクションした核膜異常細胞の値を１とした時の比を算出した。また、同様に、ラミンＡアンチセンスオリゴもしくはスクランブルオリゴをトランスフェクションして７日間培養した細胞を、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いｍＲＮＡを抽出し、ＳｕｐｅｒＳｃｒｉｐｔ ＶＩＬＯ ｃＤＮＡ ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（ライフテクノロジーズ製、配列非公開）を用い、cDNAを合成し、リアルタイムＰＣＲ（ライフテクノロジーズ製）にてプロジェリンｍＲＮＡ（プライマー配列：(1)５’−ＡＣＴＧＣＡＧＣＡＧＣＴＣＧＧＧＧ−３’，(2)５’−ＴＣＴＧＧＧＧＧＣＴＣＴＧＧＧＣ−３’，プローブ配列：５’−ＦＡＭ−ＣＧＣＴＧＡＧＴＡＣＡＡＣＣＴ−ＭＧＢ−３’、ライフテクノロジーズ合成品）の発現量を測定した。スクランブルオリゴとラミンＡアンチセンスオリゴ導入細胞のプロジェリン量を比較した結果を図１に、プロジェリンｍＲＮＡ発現量を比較した結果を図２に、アンチセンスオリゴ導入細胞に対して被験物質もしくは溶媒対照を添加した際のプロジェリン量を比較した結果を表１に示す。表１の結果より、プロジェリンの産生を抑制する植物抽出物が存在することが判る。
[Example 1]
<Test Example 1: Evaluation of abnormal nuclear membrane state using change in progelin amount as an index>
Normal human dermal fibroblasts were seeded in a 24-well plate (BD Falcon) to become 70000 cells / well and 10% FBS / DMEM (FBS: Hana Nesco Bio, DMEM: Sigma Aldrich) For 24 hours. Subsequently, 100 nM Lamin A antisense oligo (GTGACGTGCACTGA: Sigma Genosys synthetic product) was transfected using Lipofectamine 2000 (manufactured by Life Technologies) for nuclear membrane abnormality induction. As a control, cells transfected with a scrambled oligo (TGGCGGCAAGCCCTCA: Sigma Genosys) were prepared in the same manner. Thereafter, each test substance was added at a predetermined concentration and cultured for 7 days. The cells obtained after the culture were dissolved in Lysis buffer (20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1.0% NP-40, 1 tablet Proteinase inhibitor cocktail / 50 mL ultrapure water), and DC protein. After adjusting the amount of protein by assay (manufactured by Bio-Rad), electrophoresis was performed using E-PAGEEL (manufactured by ATTO). Proteins separated by electrophoresis were transferred to Immobilon-P (Millipore) by applying voltage, and diluted 400-fold with goat anti-Lamin A / C antibody (Santa Cruz) and rabbit anti-β-actin antibody (internal control). (Abcam). After washing with TBS buffer (manufactured by Millipore), HRP-containing anti-goat IgG antibody (manufactured by ZyMax) and anti-rabbit IgG antibody (manufactured by ZyMax) diluted 5000 times were reacted, washed, and then SuperSignal WestFemto Maximum Sensitive Substrate HRP was developed by TIFIC. Thereafter, an image was obtained with Cool Saver (manufactured by ATTO), and band intensities of progelin and β-actin were measured with image analysis software ImageJ (manufactured by NIH). Obtain the band intensity of progelin / band intensity of β-actin,
Further, the ratio when the value of nuclear membrane abnormal cells transfected with antisense oligo was 1 was calculated. Similarly, from cells transfected with lamin A antisense oligo or scrambled oligo and cultured for 7 days, mRNA was extracted using RNeasy Mini Kit (manufactured by QIAGEN), and SuperScript VILO cDNA synthesis Kit (manufactured by Life Technologies, CDNA was synthesized using real-time PCR (manufactured by Life Technologies), and progelin mRNA (primer sequence: (1) 5′-ACTGCAGCAGCCTCGGGGG-3 ′, (2) 5′-TCTGGGGGCTCTGGGC-3 ′, The expression level of the probe sequence: 5′-FAM-CGCTGAGGATACAACCT-MGB-3 ′, Life Technologies synthetic product) was measured. FIG. 1 shows the results of comparing the progelin levels of scrambled oligo and lamin A antisense oligo-introduced cells, and FIG. 2 shows the results of comparison of progelin mRNA expression levels. Table 1 shows the result of comparison of the amount of progelin when added. From the results in Table 1, it can be seen that there is a plant extract that suppresses the production of progelin.
［実施例２］
＜試験例２：核形態の変化を指標とした核膜異常状態評価＞
正常ヒト皮膚線維芽細胞を、２４穴プレート（ＢＤ Ｆａｌｃｏｎ社製）に７００００ｃｅｌｌｓ／ｗｅｌｌになる様に播種し、１０％ＦＢＳ／ＤＭＥＭ（ＦＢＳ：ハナ・ネスコバイオ社製、ＤＭＥＭ：シグマアルドリッチ社製）で２４時間培養した。次いで核膜異常誘導のため、１００ｎＭのラミンＡアンチセンスオリゴ（ＧＴＧＡＣＧＴＧＣＡＣＴＧＡ：シグマジェノシス合成品）をＬｉｐｏｆｅｃｔａｍｉｎ２０００（ライフテクノロジーズ製）を用いてトランスフェクションした。なお、コントロールとしてスクランブルオリゴ（ＴＧＧＣＧＧＣＡＡＧＣＣＣＴＣＡ：シグマジェノシス合成品）を同手法にてトランスフェクションした細胞も用意した。その後それぞれの被検物質を所定濃度加え、７日間培養した。培養後に得られた細胞を、アセトン（和光純薬製）にて固定し、１５０倍希釈したマウス抗ＬａｍｉｎＡ／Ｃ抗体（Ａｂｃａｍ製）を４℃で一昼夜反応させた。続いてＰＢＳ（−）（和光純薬製）にて洗浄後、２００倍希釈した抗マウスＩｇＧ抗体（Ａｌｅｘａ４８８）（ライフテクノロジーズ製）を３７℃で１時間反応させた。ＰＢＳ（−）で洗浄後、１００００倍希釈したＤＡＰＩ（シグマアルドリッチ製）を室温で５分反応させた。ＰＢＳ（−）で洗浄後、フルオロマウントＧ（コスモバイオ製）にて封入し、共焦点顕微鏡ＬＭＳ５１０（カールツァイス製）にて画像を取得した。得られた画像から、画像解析ソフトＩＰ−Ｌａｂ（Ｓｃａｎａｌｙｔｉｃｓ製）を用いて１細胞核あたりの周辺長及び面積を計測し、式（４π×面積／周辺長2）にてコンター比を算出した。スクラン
ブルオリゴとラミンＡアンチセンスオリゴ導入細胞のコンター比を比較した結果を図３に、アンチセンスオリゴ導入細胞に対して被験物質もしくは溶媒対照を添加した際のコンター比を比較した結果を表２に示す。表２の結果より、核膜異常を抑制する植物抽出物が存
在することが判る。
[Example 2]
<Test Example 2: Nuclear membrane abnormal state evaluation using changes in nuclear morphology as an index>
Normal human dermal fibroblasts were seeded in a 24-well plate (BD Falcon) to become 70000 cells / well and 10% FBS / DMEM (FBS: Hana Nesco Bio, DMEM: Sigma Aldrich) For 24 hours. Subsequently, 100 nM Lamin A antisense oligo (GTGACGTGCACTGA: Sigma Genosys synthetic product) was transfected using Lipofectamine 2000 (manufactured by Life Technologies) for nuclear membrane abnormality induction. As a control, cells transfected with a scrambled oligo (TGGCGGCAAGCCCTCA: Sigma Genosys) were prepared in the same manner. Thereafter, each test substance was added at a predetermined concentration and cultured for 7 days. The cells obtained after the culture were fixed with acetone (manufactured by Wako Pure Chemical Industries, Ltd.), and a mouse anti-Lamin A / C antibody (manufactured by Abcam) diluted 150 times was reacted at 4 ° C. overnight. Subsequently, after washing with PBS (−) (manufactured by Wako Pure Chemical Industries, Ltd.), 200-fold diluted anti-mouse IgG antibody (Alexa488) (manufactured by Life Technologies) was reacted at 37 ° C. for 1 hour. After washing with PBS (−), DAPI (Sigma Aldrich) diluted 10,000 times was reacted at room temperature for 5 minutes. After washing with PBS (−), it was sealed with Fluoromount G (manufactured by Cosmo Bio), and an image was acquired with a confocal microscope LMS510 (manufactured by Carl Zeiss). From the obtained image, the peripheral length and area per cell nucleus were measured using image analysis software IP-Lab (manufactured by Scanalytics), and the contour ratio was calculated by the formula (4π × area / peripheral length 2 ). The result of comparing the contour ratio of scrambled oligo and Lamin A antisense oligo-introduced cells is shown in FIG. 3, and the result of comparison of the contour ratio when a test substance or solvent control is added to antisense oligo-introduced cells is shown in Table 2. Show. From the results in Table 2, it can be seen that there is a plant extract that suppresses nuclear membrane abnormality.
本発明は、化粧料（但し、医薬部外品を含む）等に好適な、抗老化作用を有する成分をスクリーニング方法、並びに、当該成分を含有する組成物を提供する。 The present invention provides a method for screening an ingredient having an anti-aging effect, which is suitable for cosmetics (including quasi-drugs) and the like, and a composition containing the ingredient.
Claims (17)
（ａ）核膜異常が生じている患者から採取した細胞
（ｂ）圧力を加えることによって核膜異常を生じさせた細胞
（ｃ）アンチセンスオリゴを接触させることによって核膜異常を生じさせた細胞 The screening method according to claim 2, wherein the cell in which the nuclear membrane abnormality has occurred is any one of the following (a) to (c).
(A) a cell collected from a patient having a nuclear membrane abnormality (b) a cell that has caused a nuclear membrane abnormality by applying pressure (c) a cell that has caused a nuclear membrane abnormality by contacting an antisense oligo
用を有する成分を含有する、皮膚外用剤。 The skin external preparation containing the component which has the anti-aging effect selected by the screening method of any one of Claims 1-13.
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| JP7307833B1 (en) | 2022-03-17 | 2023-07-12 | 株式会社ナリス化粧品 | nuclear morphology ameliorator |
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