JP2008245558A - Method for evaluating antiaging material and method for producing cosmetic mixed with the same - Google Patents
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Abstract
Description
本発明は、老化症状の防止や改善の為の素材の評価法、及び該評価法により得られる素材を含有した抗老化化粧料を提供する。より詳細には、デコリンの産生を促進させることによって真皮マトリックス構造を強化しシワや皮膚の弾力性低下といった老化症状の防止や改善を目的とする素材の評価法、及び該評価法により得られる素材を含有したシワや皮膚の弾力性低下といった老化症状の防止や改善の為の化粧料の製造方法に関する。 The present invention provides a method for evaluating a material for preventing or improving aging symptoms, and an anti-aging cosmetic containing a material obtained by the evaluation method. More specifically, a method for evaluating a material for the purpose of preventing or improving aging symptoms such as wrinkles and skin elasticity reduction by enhancing the production of decorin by promoting decorin production, and a material obtained by the evaluation method The present invention relates to a method for producing a cosmetic material for preventing or improving aging symptoms such as wrinkles containing fragrance and reduced skin elasticity.
近年、高齢化社会が進行するにつれて、美しく年を重ねるために、化粧料に求められる役割が大きくなってきている。ところが、肌は、加齢などの内的因子や紫外線、活性酸素などの外的因子によって、皮膚が本来維持している収縮性、柔軟性、保湿性等の機能が衰え、様々なトラブルを発生する。 In recent years, as the aging society progresses, the role required for cosmetics is increasing in order to beautifully grow older. However, due to internal factors such as aging and external factors such as ultraviolet rays and active oxygen, the skin's inherent functions such as contractility, flexibility, and moisture retention decline, causing various problems. To do.
これらのトラブルの一つである顔面や首等のシワ、たるみ等は典型的な皮膚老化の特徴であり、美容上の観点から、その予防や改善には関心が寄せられている。 One of these troubles, wrinkles on the face and neck, sagging, etc., are typical features of skin aging, and from the viewpoint of beauty, there is an interest in preventing and improving them.
シワ、たるみ等は真皮の細胞外マトリックスを産生する細胞数の減少、細胞分裂速度の衰えなどの細胞機能の老化や、コラーゲン線維の減少及び変性、皮下脂肪組織の減少等により、弾力性の損失が起こることが原因となって発生する。 Wrinkles, sagging, etc. lose elasticity due to a decrease in the number of cells producing extracellular matrix in the dermis, aging of cell functions such as a decrease in cell division rate, decrease and degeneration of collagen fibers, decrease of subcutaneous adipose tissue, etc. Occurs due to the occurrence of
従来、シワへの対処法としては、老化によって失われるコラーゲン、ヒアルロン酸等の物質を皮膚に塗布し補う組成物や、紫外線や活性酸素から皮膚を守るための防御物質を配合した間接的な老化防止剤が主流であった。（特許文献１〜３参照）また、生成したシワを根本的に改善しようとする試みとしては、レチノイン酸やグリコール酸に代表されるα -ヒドロキシ酸等がある。（特許文献４及び５参照）しかし、α -ヒドロキシ酸においては、高い配合量が必要となり、レチノイン酸においては、腫れを伴う炎症等を起こす等安全性に問題があり、長期使用に耐え得るものではなかった。 Conventionally, wrinkles are dealt with by indirect aging with a composition that supplements the skin with substances such as collagen and hyaluronic acid that are lost due to aging, and a protective substance that protects the skin from ultraviolet rays and active oxygen. Inhibitors were mainstream. (See Patent Documents 1 to 3) Further, as an attempt to fundamentally improve the generated wrinkles, there are α-hydroxy acids represented by retinoic acid and glycolic acid. (Refer to Patent Documents 4 and 5) However, in α-hydroxy acid, a high amount is required, and in retinoic acid, there is a problem in safety such as causing inflammation accompanied by swelling, and it can withstand long-term use. It wasn't.
一方、真皮構造の大部分を占めるのはコラーゲンであるが、真皮構造の維持や強化には、コラーゲン以外の成分も関与している。例えば、コラーゲンやエラスチンと並んで細胞外マトリックスを構成しているプロテオグリカンの一つに、デコリンがある。 On the other hand, collagen occupies most of the dermis structure, but components other than collagen are also involved in maintaining and strengthening the dermis structure. For example, decorin is one of the proteoglycans that constitute the extracellular matrix along with collagen and elastin.
デコリンは、分子量約４０から５０ｋDaのコアタンパク質に１本のコンドロイチン硫酸あるいはデルマタン硫酸からなるグリコサミノグリカン鎖が共有結合してできた全体の分子量が約１００ｋDaの複合生体高分子である。デコリンは、皮膚では真皮に存在し、そのコアタンパク質を介して他の細胞外マトリックス成分であるコラーゲン等と相互作用し、コラーゲンのフィブリル形成制御や線維径の制御に関わることが近年明らかになった。（非特許文献１及び２参照）デコリンの欠損はコラーゲン構造へ影響を与えることから、デコリンの産生を促進させることは、コラーゲン構造を強化しシワや皮膚の弾力性低下といった老化症状の防止や改善に有効であると考えられる。即ち、この様なデコリンの産生を促進させる物質を探し出せば、シワや皮膚の弾力性低下といった老化症状の防止や改善に対する有効手段となり得る。
本発明は、この様な状況下為されたものであり、シワや皮膚の弾力性低下といった老化症状の防止や改善の為の素材の評価法、及び該評価法により得られる素材を含有した抗老化化粧料を提供することを課題とする。 The present invention has been made under such circumstances, and is a method for evaluating a material for preventing or improving aging symptoms such as wrinkles and skin elasticity reduction, and an antibacterial agent containing the material obtained by the evaluation method. It is an object to provide an aging cosmetic.
本発明者は、係る実情に鑑み鋭意研究努力を重ねた結果、有効成分によるシワや皮膚の弾力性低下といった老化症状の防止や改善の指標として、真皮線維芽細胞によって産生されるデコリンをコードするmRNAの量を、当該mRNAの全部又は一部に対して相補的な配列を有するオリゴヌクレオチドとハイブリダイゼーションさせることでその量を測定する、又はデコリンをコードするmRNAを鋳型にしたcDNAを増幅してその量を測定することが有効であることを見出し、本発明を完成するに至った。 As a result of intensive research efforts in view of such circumstances, the present inventor encodes decorin produced by dermal fibroblasts as an indicator of prevention and improvement of aging symptoms such as wrinkles and reduced skin elasticity due to active ingredients. The amount of mRNA is measured by hybridization with an oligonucleotide having a sequence complementary to all or part of the mRNA, or a cDNA encoding decorin-encoding mRNA is used as a template to amplify. The inventors have found that measuring the amount is effective, and have completed the present invention.
即ち、本発明は、真皮線維芽細胞中のデコリンをコードするmRNAを含むRNA画分を得る工程、デコリンをコードするmRNAを鋳型にしたcDNAを作成する工程、そのcDNAを、デコリンをコードするmRNAの一部と同一又はそれに対して相補的な配列を含むオリゴヌクレオチドをプライマーとして用いて増幅する工程、及び増幅産物の量を測定する工程を含むことを特徴とする、老化症状の防止や改善を目的とする素材の評価法、及び該評価法により得られる素材を含有した抗老化化粧料を提供する。 That is, the present invention includes a step of obtaining an RNA fraction containing mRNA encoding decorin in dermal fibroblasts, a step of creating cDNA using mRNA encoding decorin as a template, and the cDNA encoding mRNA encoding decorin. A step of amplifying using an oligonucleotide containing a sequence that is the same as or complementary to a part of the primer as a primer, and a step of measuring the amount of the amplified product. Provided are a target material evaluation method and an anti-aging cosmetic containing a material obtained by the evaluation method.
本発明は、また、デコリンをコードするmRNAの一部と相補的な配列を含むオリゴヌクレオチドであって、（a）塩基配列５’-GCCCACCTGGACACAACAC-３’（ 配列番号：１）又はこの配列に対して４
個以下の塩基が、除去、付加及び／ 又は置換期より修飾されている塩基配列、あるいは( b )塩基配列５’-GACCGGGTTGCTGAAAAGAC-３’（ 配列番号：２）又はこの配列に対して４
個以下の塩基が、除去、付加及び／ 又は置換期より修飾されている塩基配列、を有するオリゴヌクレオチドを有する。好ましくは、上記オリゴヌクレオチドは、（a’）塩基配列５’-GCCCACCTGGACACAACAC-３’（
配列番号：１）、あるいは（ｂ’） 塩基配列５’-GACCGGGTTGCTGAAAAGAC-３’（ 配列番号：２）を有する。
The present invention also provides an oligonucleotide comprising a sequence complementary to a part of mRNA encoding decorin, comprising: (a) the base sequence 5′-GCCCACCTGGACACAACAC-3 ′ (SEQ ID NO: 1) or 4
A base sequence in which not more than one base is modified from the removal, addition and / or substitution period, or (b) base sequence 5′-GACCGGGTTGCTGAAAAGAC-3 ′ (SEQ ID NO: 2) or 4 for this sequence
It has an oligonucleotide having a base sequence in which no more than one base has been modified from the removal, addition and / or substitution period. Preferably, the oligonucleotide has (a ′) a base sequence 5′-GCCCACCTGGACACAACAC-3 ′ (
SEQ ID NO: 1) or (b ′) has the base sequence 5′-GACCGGGTTGCTGAAAAGAC-3 ′ (SEQ ID NO: 2).
本発明は、また、前記の塩基配列（a）又は（a’）を有するオリゴヌクレオチドと、塩基配列（ｂ）又は（ｂ’）を有するオリゴヌクレオチドとを一対として含むPCR増幅用プライマーを提供する。 The present invention also provides a PCR amplification primer comprising a pair of the oligonucleotide having the base sequence (a) or (a ′) and the oligonucleotide having the base sequence (b) or (b ′). .
本発明によれば、シワや皮膚の弾力性低下といった老化症状の防止や改善の為の素材の評価法、及び該評価法により得られる素材を含有した抗老化化粧料を提供することが可能である。 According to the present invention, it is possible to provide a method for evaluating a material for preventing and improving aging symptoms such as wrinkles and skin elasticity reduction, and an anti-aging cosmetic containing a material obtained by the evaluation method. is there.
本発明に用いられるデコリンをコードするmRNAは、真皮線維芽細胞を含む生体試料より、公知の方法、例えば、フェノール法やグアニジンチオシアネート法などにより得られる。また、市販のRNA精製キットを用いて行うこともできる。 The mRNA encoding decorin used in the present invention can be obtained from a biological sample containing dermal fibroblasts by a known method such as the phenol method or the guanidine thiocyanate method. Alternatively, a commercially available RNA purification kit can be used.
本発明に用いられるcDNAの合成方法としては、公知の方法、例えば、RNAを鋳型にして逆転写酵素を用いてcDNAを合成する方法などが挙げられる。また、市販の逆転写キットを用いて行うこともできる。 Examples of the method for synthesizing cDNA used in the present invention include known methods such as a method of synthesizing cDNA using reverse transcriptase using RNA as a template. Moreover, it can also carry out using a commercially available reverse transcription kit.
本発明に用いられるデコリンcDNA断片のPCRによる増幅の方法としては、公知の方法、例えば、デコリンの塩基配列を基に合成したオリゴヌクレオチドをプライマーとして、DNAポリメラーゼを用いて行うことができる。 A method for amplifying decorin cDNA fragments used in the present invention by PCR can be performed by a known method, for example, using an oligonucleotide synthesized based on the base sequence of decorin as a primer and a DNA polymerase.
デコリンをコードする塩基配列中の増幅すべき領域の位置は特に限定されないが、イントロン前後のエキソンに上流プライマー、下流プライマーをそれぞれ設計し増幅することが好ましい。 The position of the region to be amplified in the base sequence encoding decorin is not particularly limited, but it is preferable to design and amplify an upstream primer and a downstream primer in exons before and after the intron, respectively.
増幅される領域の長さは特に限定されないが、例えば５０〜３００塩基対程度、好ましくは８０〜１５０塩基対程度である。プライマーの塩基配列は、デコリンをコードする塩基配列中の対応する領域の塩基配列に対して完全に相補的であることが望ましいが、必ずしもその必要はなく、例えばプライマーの塩基数２０個当り４個以下、例えば３個以下、好ましくは２個以下、例えば２個又は１個の塩基が、除去、付加、若しくは他の塩基による置換により修飾されていてもよい。例えば、配列番号：１及び配列番号：２に記載の塩基配列を有する塩基数２０のプライマーにおいて、４個以下、例えば４個、３個、２個又は１個の塩基が、付加、除去又は他の塩基による置換によって修飾されていてもよい。 The length of the region to be amplified is not particularly limited, but is, for example, about 50 to 300 base pairs, preferably about 80 to 150 base pairs. The primer base sequence is preferably completely complementary to the base sequence of the corresponding region in the base sequence encoding decorin, but this is not always necessary, for example, 4 per 20 bases of the primer. Hereinafter, for example, 3 or less, preferably 2 or less, such as 2 or 1 base, may be modified by removal, addition, or substitution with another base. For example, in the primer having 20 bases having the base sequences described in SEQ ID NO: 1 and SEQ ID NO: 2, 4 or less, for example, 4, 3, 2 or 1 bases are added, removed or others May be modified by substitution with a base.
PCR反応の具体例としては、２本鎖DNAの１本鎖DNAへの熱変性、増幅を目的とする部位の両端に相補的な２種類のオリゴヌクレオチドプライマーと前述の熱変性１本鎖DNAとのアニーリング、DNAポリメラーゼによる前記オリゴヌクレオチドプライマーからのDNA鎖伸長反応、からなる増幅サイクルを繰り返すことにより、デコリン
cDNA断片を指数関数的に増幅する方法が挙げられる。
As a specific example of the PCR reaction, two kinds of oligonucleotide primers complementary to both ends of a site intended for heat denaturation and amplification of double stranded DNA to single stranded DNA and the above-mentioned heat denatured single stranded DNA By repeating the amplification cycle consisting of annealing of the DNA, and DNA strand extension reaction from the oligonucleotide primer by DNA polymerase.
A method of exponentially amplifying a cDNA fragment can be mentioned.
また、本発明における真皮線維芽細胞中デコリンの発現量の定量方法としては、上述のPCR法によって迅速に測定することができる。すなわちRNAを鋳型として逆転写酵素を用いてcDNAを合成し、引き続きDNAポリメラーゼを用いたPCR反応を行って増幅産物を定量する方法が挙げられる。 Moreover, as a method for quantifying the expression level of decorin in dermal fibroblasts in the present invention, it can be rapidly measured by the PCR method described above. That is, there is a method in which cDNA is synthesized using reverse transcriptase with RNA as a template, followed by PCR reaction using DNA polymerase to quantify the amplified product.
増幅されたDNA断片を検出又は定量するには、PCR 増幅産物を蛍光により検出するリアルタイムPCRが好適に例示される。この際、蛍光検出方法は特に限定されないが、例えば、SYBR
Green（アプライドバイオシステム社製）を使用するインターカレーター法や、TaqMan プローブ（アプライドバイオシステム社製）等を使用する蛍光標識プローブを用いる方法などが使用可能である。また、増幅されたDNAの検出又は定量方法はリアルタイムPCRに限定されるものではなく、公知の方法、例えば、増幅されたDNA断片特異的なプローブを用いた方法が挙げられる。
For detecting or quantifying the amplified DNA fragment, real-time PCR in which a PCR amplification product is detected by fluorescence is preferably exemplified. At this time, the fluorescence detection method is not particularly limited. For example, SYBR
An intercalator method using Green (Applied Biosystems) or a method using a fluorescently labeled probe using TaqMan probe (Applied Biosystems) or the like can be used. The method for detecting or quantifying the amplified DNA is not limited to real-time PCR, and includes a known method, for example, a method using an amplified DNA fragment-specific probe.
以下、実施例によって本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。
実施例１. 有効成分添加による、正常ヒト線維芽細胞のデコリンmRNAの定量
（１）細胞培養
正常ヒト線維芽細胞（HFSKF−II、RIKEN CELL BANKより入手）を、D−MEM培地に１５％ のFBSを添加したもので培養した。前記培地にて８×１０４cells／mLに調整した細胞を、滅菌プラスチック１２穴プレートに１．０mLずつ播種し、２４時間培養した。
（２）試験物質の添加
FBSを含まないD−MEM培地に交換した後、０．２μmメンブランフィルターにて濾過滅菌した対象物質を加え、一定時間培養した。
（３）RNAの抽出
正常ヒト線維芽細胞R N Aの抽出は、RNeasy MINI Kit（QIAGEN社製）を用いて行い、約１．５μgのmRNA画分を得た。
（５）逆転写反応
逆転写反応は、PrimeScript RT reagent Kit（タカラバイオ社製）を用いて行った。逆転写反応溶液を調製し、４２℃で１５分間、９５℃で２分間保持した後、４℃まで急冷して逆転写を終了した。
（６）プライマー
デコリンcDNAの増幅には、上流プライマーとして５’-GCCCACCTGGACACAACAC-３’（ 配列番号：１）を、下流プライマーとして５’-GACCGGGTTGCTGAAAAGAC-３’（
配列番号：２）を合成して用いた。内因性コントロール遺伝子のcDNAの増幅には、配列に特異的な上流プライマーと下流プライマーを合成して用いた。
（７）プライマーの増幅効率検討
プライマーの増幅効率検討には、ABI PRISM ７５００システム（アプライドバイオシステムズ社製）を用いて行った。（５）で得たcDNAを等倍希釈した系列おのおの１．０μLに、１０μMの上流及び下流プライマーをそれぞれ０．５μL、滅菌水
１０．５μL、SYBR Green PCR Master Mix（アプライドバイオシステムズ社製） １２．５μLを加え、５０℃ で２分、９５℃ で１０分保持した後、９５℃
で１５秒、６０℃ で１分の温度サイクルを４０回繰り返して増幅反応を行った。プライマーの増幅効率は、２本鎖DNAに入り込み蛍光が増幅されたSYBR Greenの蛍光強度より、立ち上がりサイクル数をCt値として算出し下記計算式にしたがって算出した。ここで、立ち上がりサイクル数とは、SYBR
Greenの蛍光強度が指数関数的に増幅されている領域のサイクル数を指す。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
Example 1. Quantification of decorin mRNA of normal human fibroblasts by addition of active ingredient (1) Cell culture Normal human fibroblasts (HFSKF-II, obtained from RIKEN CELL BANK) were added to D-MEM medium at 15%. Incubated with FBS added. Cells adjusted to 8 × 10 4 cells / mL in the medium were seeded at 1.0 mL in a sterilized plastic 12-well plate and cultured for 24 hours.
(2) Addition of test substance
After exchanging with D-MEM medium not containing FBS, the target substance filtered and sterilized with a 0.2 μm membrane filter was added and cultured for a certain period of time.
(3) RNA extraction Normal human fibroblast RNA was extracted using RNeasy MINI Kit (manufactured by QIAGEN) to obtain about 1.5 μg of mRNA fraction.
(5) Reverse transcription reaction The reverse transcription reaction was performed using PrimeScript RT reagent Kit (manufactured by Takara Bio Inc.). A reverse transcription reaction solution was prepared and held at 42 ° C. for 15 minutes and at 95 ° C. for 2 minutes, and then rapidly cooled to 4 ° C. to complete reverse transcription.
(6) Primer For decorin cDNA amplification, 5′-GCCCACCTGGACACAACAC-3 ′ (SEQ ID NO: 1) is used as an upstream primer, and 5′-GACCGGGTTGCTGAAAAGAC-3 ′ (as a downstream primer).
SEQ ID NO: 2) was synthesized and used. For amplification of the endogenous control gene cDNA, sequence-specific upstream and downstream primers were synthesized and used.
(7) Examination of primer amplification efficiency The primer amplification efficiency was examined using the ABI PRISM 7500 system (Applied Biosystems). 10 μM upstream and downstream primers 0.5 μL, sterile water 10.5 μL, SYBR Green PCR Master Mix (Applied Biosystems) Add 5 μL and hold at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes,
The amplification reaction was performed by repeating 40 times a temperature cycle of 15 seconds at 60 ° C. for 1 minute. The amplification efficiency of the primer was calculated from the fluorescence intensity of SYBR Green, which entered the double-stranded DNA and the fluorescence was amplified, with the number of rising cycles as the Ct value, and was calculated according to the following formula. Here, the number of rising cycles is SYBR
The number of cycles in the region where the green fluorescence intensity is exponentially amplified.
e＝１０-１／a−１
e：プライマーの増幅効率
a：横軸をlog[cDNA]、縦軸をCtとした時の、一次近似曲線の傾き
その結果を表１ に示す。
e = 10 <-1 > / a <-1 >
e: Primer amplification efficiency
a: The slope of the linear approximation curve when the horizontal axis is log [cDNA] and the vertical axis is Ct. The results are shown in Table 1.
表１から明らかなように、デコリンcDNA及び内因性コントロール遺伝子共に増幅され、増幅効率は同程度であることが明示された。
As is apparent from Table 1, both decorin cDNA and endogenous control gene were amplified, and the amplification efficiency was clearly shown to be comparable.
（７）リアルタイムP C R
リアルタイムPCRは、ABI PRISM ７５００システムを用いて行った。（５）で得たcDNA １．０μLに、１０μMの上流及び下流プライマーをそれぞれ０．５μL、滅菌水
１０．５μL、SYBR Green PCR Master Mix １２．５μLを加え、５０℃ で２分、９５℃ で１０分保持した後、９５℃ で１５秒、６０℃ で１分の温度サイクルを４０回繰り返して増幅反応を行った。増幅産物は、２本鎖DNAに入り込み蛍光が増幅されたSYBR
Greenの蛍光強度より、下記計算式にしたがって算出した。
(7) Real-time PCR
Real-time PCR was performed using the ABI PRISM 7500 system. Add 0.5 μL of 10 μM upstream and downstream primers, 10.5 μL of sterilized water, and 12.5 μL of SYBR Green PCR Master Mix to 1.0 μL of the cDNA obtained in (5), respectively, at 50 ° C. for 2 minutes, and at 95 ° C. After holding for 10 minutes, an amplification reaction was carried out by repeating a temperature cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute 40 times. The amplified product enters the double-stranded DNA and the fluorescence is amplified.
It calculated from the fluorescence intensity of Green according to the following formula.
Ｒ＝２-ΔΔCt
Ｒ：遺伝子発現増加率、対象物質無添加時と比較した増加率
ΔΔCt＝ΔCt1−ΔCt0
ΔCt1＝（対象物質添加時の立ち上がりサイクル数）−（対象物質添加時の内因性コントロールの立ち上がりサイクル数）
ΔCt0＝（対象物質無添加時の立ち上がりサイクル数）−（対象物質無添加時の内因性コントロールの立ち上がりサイクル数）
その結果を表２に示す。無添加時の遺伝子発現増加率は２０＝１である。
R = 2− ΔΔCt
R: Increase rate of gene expression, rate of increase compared with no addition of target substance ΔΔCt = ΔCt 1 −ΔCt 0
ΔCt 1 = (Number of rising cycles when adding the target substance)-(Number of rising cycles of intrinsic control when adding the target substance)
ΔCt 0 = (Number of rising cycles when no target substance is added) − (Number of rising cycles of intrinsic control when no target substance is added)
The results are shown in Table 2. The gene expression increase rate without addition is 2 0 = 1.
以上の結果より、被検物質１、及び３に効果があり、被検物質２には効果がないと判断した。 From the above results, it was determined that the test substances 1 and 3 were effective and the test substance 2 was not effective.
本発明により、真皮線維芽細胞中のデコリンをコードするm R N Aを測定することで、シワや皮膚の弾力性低下といった老化症状の防止や改善の為の抗老化化粧料及び素材評価法を提供が可能となるため、広く老化症状防止化粧料及び素材開発に応用が期待できる。 According to the present invention, by measuring mRNA encoding decorin in dermal fibroblasts, it is possible to provide an anti-aging cosmetic and a material evaluation method for preventing and improving aging symptoms such as wrinkles and reduced skin elasticity. Therefore, it can be widely applied to the development of cosmetics and materials for preventing aging symptoms.
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