JP5653783B2 - Evaluation method of water retention ability in skin epidermis - Google Patents
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Description
本発明は、皮膚角質細胞のｍＲＮＡ量を指標とすることにより皮膚表皮内水分保持能を評価する方法に関するものである。 The present invention relates to a method for evaluating the water retention capacity in the skin epidermis by using the amount of mRNA of skin keratinocytes as an index.
皮膚の上皮組織である、表皮は数層の細胞層からなる。このうち、最外層に存在する角質層は、基底層にある基底細胞が細胞分裂を繰り返し、その一部が基底細胞層を去って上層へ移行する過程で形成される角質細胞より構成されている。この過程を角化と呼び、角化を通して細胞は核などの細胞成分を失い主にケラチン線維の塊へと変化し、通常の細胞と異なり、遺伝子発現やタンパク質合成などの生命活動を行っていない、死んだ細胞である。一方、角質層の下層にある、顆粒層、有棘層及び基底層に存在する細胞は、生体を構成する様々な細胞と同様に、遺伝子発現、タンパク質合成及びエネルギー産生などの代謝反応を行っている生きた細胞である。 The epidermis, the epithelial tissue of the skin, consists of several cell layers. Among them, the stratum corneum existing in the outermost layer is composed of horny cells formed in the process in which the basal cells in the basal layer repeat cell division, and part of them leave the basal cell layer and move to the upper layer. . This process is called keratinization. Through keratinization, cells lose cellular components such as nuclei and change mainly into keratin fiber masses. Unlike normal cells, they do not perform life activities such as gene expression and protein synthesis. , Dead cells. On the other hand, cells existing in the granular layer, spinous layer, and basal layer under the stratum corneum undergo metabolic reactions such as gene expression, protein synthesis, and energy production in the same way as various cells that make up the living body. Is a living cell.
角質層は、バリア機能などの生理機能の他に、美醜を判断するといった官能的評価の対象として重要視されており、角質層の状態を測定する方法や角質層を正常に美しく保つための施術等は、皮膚科の臨床医だけでなく、化粧品会社の社員や美容産業に携わる多くの人々に知られている。特に、角質層の水分量は、皮膚外観や皮膚バリア機能との関連性がみられる指標であり、皮膚の状態を診断する上で、角質層の水分量を測定することは日常的に行われている（非特許文献１）。 The stratum corneum is regarded as an object of sensory evaluation, such as judging beauty, in addition to physiological functions such as the barrier function, and a method for measuring the stratum corneum state and treatment to keep the stratum corneum normal and beautiful. Are known not only by dermatologists, but also by employees of cosmetic companies and many people in the beauty industry. In particular, the water content of the stratum corneum is an indicator that is related to the appearance of the skin and the skin barrier function, and the water content of the stratum corneum is routinely measured in diagnosing skin conditions. (Non-Patent Document 1).
しかし、上述した方法による測定は、あくまで角質層の水分量に関することであり、表皮全体の水分量を表しているわけではなかった。角質層の水分量は、表皮のしっとり感、うるおい感などの官能指標に影響するが、それ以外の皮膚性状には関係なく、例えば、キメの細やかさや肌のハリ感などの指標については、角質層の水分量との関連性は認められていなかった。 However, the measurement by the above-described method is only related to the moisture content of the stratum corneum and does not represent the moisture content of the entire epidermis. The amount of moisture in the stratum corneum affects sensory indicators such as moist and moist feeling of the epidermis, but it is not related to other skin properties, for example, for the indicators such as texture fineness and skin firmness, No association with water content in the layer was observed.
一方、肌のキメやハリ感については、角質層の状態のみならず、角質層以下の生きた細胞としての表皮の状態に起因することが経験上知られている。例えば、水分を充分含んだ風呂上りの肌は、化粧水等で水分を補う以上に肌にハリが生じ、キメもふっくらしていると日常的に感じるところである。また、乾燥が原因で生じる小じわは、保湿化粧水により充分な水分を補うことで、キメが整い、小じわが改善することはよく経験される。これらの状態においては、わずか十数ミクロンしかない角質層ではなく、角質層以外の顆粒層、有棘層及び基底層の表皮細胞中の水分量が増し、その結果、実感として厚みが増加することが推測される。 On the other hand, it is known from experience that skin texture and firmness are caused not only by the state of the stratum corneum but also by the state of the epidermis as living cells below the stratum corneum. For example, the skin after bathing with plenty of water is a place where you feel daily that your skin is firmer and more textured than supplemented with lotion. It is often experienced that fine wrinkles caused by dryness are improved by supplementing with sufficient moisture with moisturizing lotion and improving fine lines. In these conditions, the amount of water in the epidermal cells of the granule layer, spinous layer, and basal layer other than the stratum corneum, not just the stratum corneum, which is only a few dozen microns, increases, resulting in an increase in thickness. Is guessed.
これまでに、測定機器を用いて表皮内の水分量を測定する方法には、近赤外分光光度計や共焦点ラマン分光装置が知られていた（特許文献１、２）。しかし、これらの機器で皮膚を測定するには、被験者を拘束、安静化される必要に加え、機器が高額で測定に時間を要するものであった。
So far, near infrared spectrophotometers and confocal Raman spectroscopic devices have been known as methods for measuring the amount of water in the epidermis using a measuring instrument (
また、他の簡便に計測する機器も無いわけではなく、中でもＭｏｉｓｔｕｒｅ Ｍｅｔｅｒ Ｄ（Ｄｅｌｆｉｎ社；フィンランド）は、非侵襲的にかつ簡便に測定できる機器として市販されている。しかしながら、皮膚の水分量を比誘電率で求めるものであるため、簡便な計測であるが、得られるデータに安定性がなく、また、センサープローブの口径により計測可能な深さが決められていることから、部位によって異なる厚さの皮膚においては、定量性に欠けていた。そのため、この装置を用いて、水分付加前後のわずかな差を見出すことは困難であった。 In addition, there is no other simple measuring device, and among these, Moisture Meter D (Delfin, Finland) is commercially available as a non-invasive and simple measuring device. However, it is a simple measurement because the moisture content of the skin is determined by the relative dielectric constant, but the obtained data is not stable, and the measurable depth is determined by the aperture of the sensor probe. Therefore, the skin with different thickness depending on the site lacked quantitativeness. For this reason, it has been difficult to find a slight difference between before and after moisture addition using this apparatus.
表皮では、水分の供給は真皮の血流より行われる。これには、主にアクアポリンという水分子輸送体、あるいは水チャンネルとも呼ばれるタンパク質が関与する。アクアポリンは、水を選択的に細胞内に取り込み、また細胞外に排出する。これまでにヒトでは１１種類のアクアポリン遺伝子（ＡＱＰ０〜ＡＱＰ１０）が発見され、このうち皮膚においては、アクアポリン３（以下、ＡＱＰ３と省略）、アクアポリン５（以下、ＡＱＰ５と省略）及びアクアポリン９（以下、ＡＱＰ９と省略）が発現しており、これが皮膚の表皮に水分を供給する役割を担っている。特に、ＡＱＰ３ついては、表皮の角質層の水分量を増加させ、皮膚にうるおいを与える作用が明らかにされているため、ＡＱＰ３の発現を促進させるなど、ＡＱＰ３を活性化させることを目的とした保湿剤の開発が盛んに行われている（特許文献３〜７、非特許文献２、３）。しかし、これらの研究は、あくまでもＡＱＰ３がグリセロールを輸送することにより、角質層における水分量に関与するという、Ｖｅｒｋｍａｎらの研究成果に基づくものであり（非特許文献４、５）、表皮全体、あるいは角質層以外の生細胞における水分量について、ＡＱＰ３の関与を対象とした研究はほとんどなかった。
In the epidermis, water is supplied from the dermis blood flow. This involves a water molecule transporter called aquaporin, or a protein called a water channel. Aquaporin selectively takes up water into the cell and excretes it out of the cell. To date, 11 types of aquaporin genes (AQP0 to AQP10) have been discovered in humans, and among these, in the skin, aquaporin 3 (hereinafter abbreviated as AQP3), aquaporin 5 (hereinafter abbreviated as AQP5) and aquaporin 9 (hereinafter abbreviated as “AQP5”). (Abbreviated as AQP9), which plays a role of supplying moisture to the epidermis of the skin. In particular, for AQP3, a moisturizing agent intended to activate AQP3, such as promoting the expression of AQP3, because it has been clarified to increase the amount of moisture in the stratum corneum of the epidermis and moisturize the skin. Has been actively developed (
皮膚表皮内水分保持能を評価するためには、水分付加前後における表皮内水分量を測定する必要がある。その機器としては、前述のごとく近赤外分光光度計や共焦点ラマン分光装置などが知られていた。しかし、これらは表皮内水分量を正確に測定可能ではあるが、被験者の拘束、安静が必要であったり、また皮膚の部位差のため、同一部位を数箇所測定する必要があったりした。さらにこれらの装置は、価格も数千万円ほどするため、機器の導入には経済的負担が大きかった。特別な機器を使用できない場合、被験者の皮膚の生検サンプルを用い、組織学的に解析することにより表皮内水分量を測定する方法もあるが、被験者への負担が大きく、医療機関以外では実施が難しいため実用的ではなかった。すなわち、本発明は、測定環境を選ばず、被験対象者の拘束、安静を必要とせず、非侵襲的かつ簡便、より正確に任意の部位について表皮内水分量を測定し、皮膚表皮内水分保持能を評価する方法を提供することを課題とする。 In order to evaluate the moisture retention capacity in the skin epidermis, it is necessary to measure the moisture content in the epidermis before and after the addition of moisture. As the equipment, as described above, a near-infrared spectrophotometer, a confocal Raman spectroscope, and the like have been known. However, although these can accurately measure the water content in the epidermis, the subject needs to be restrained and rested, and due to the difference in skin parts, several parts of the same part need to be measured. Furthermore, since these devices also cost tens of millions of yen, the introduction of the equipment was an economic burden. When special equipment cannot be used, there is a method to measure the moisture content in the epidermis by using a biopsy sample of the subject's skin and analyzing it histologically. It was not practical because it was difficult. That is, the present invention measures the moisture content in the epidermis for any site, non-invasively, simply, and more accurately, and does not require the subject's restraint and rest, regardless of the measurement environment, and retains moisture in the skin epidermis. It is an object to provide a method for evaluating performance.
以上の課題の下、本発明者らは、細胞に対して水を付加すると、細胞外が低張になり、細胞内への水の流入による細胞の体積増加が一般に観察される点と、細胞の水輸送量は、アクアポリンの発現量及び／又は機能性により規定されるという知見に基づき、簡便に表皮内水分保持能を測定する方法について鋭意検討した。その結果、アクアポリンのｍＲＮＡ量を測定することにより、細胞の水分保持能を推定できることを見出した。特に、皮膚においては、表皮外部より水を付加したときに観察される表皮の肥厚は、表皮細胞が水を細胞内に取り込んだ結果観察される現象であるが、この現象と皮膚におけるＡＱＰ３遺伝子に対応するｍＲＮＡ量との間の相関を見出したことから、ＡＱＰ３のｍＲＮＡ量を測定することにより、水を付加したときに観察される表皮の肥厚程度、即ち表皮細胞中に取り込まれた水分量である、表皮内水分保持能を測定できることを見出した。これらのｍＲＮＡ量は、皮膚の試料採取時の外的環境条件を選ばず、任意の部位について、簡便かつ安定的に実施できる。さらに、皮膚の試料採取時には機器を要しないため、大型機器を測定場所へ移動させる必要もなく、場所を選ばず実施することができる利点がある。 Under the above problems, the present inventors have found that when water is added to a cell, the outside of the cell becomes hypotonic and an increase in the volume of the cell due to the inflow of water into the cell is generally observed, Based on the knowledge that the amount of water transported is regulated by the expression level and / or functionality of aquaporins, a method for simply measuring the water retention capacity in the epidermis was studied. As a result, it was found that the water retention capacity of cells can be estimated by measuring the amount of aquaporin mRNA. In particular, in the skin, the thickening of the epidermis observed when water is added from outside the epidermis is a phenomenon observed as a result of the epidermal cells taking water into the cells, and this phenomenon and the AQP3 gene in the skin Since the correlation with the corresponding mRNA amount was found, by measuring the amount of AQP3 mRNA, the degree of thickening of the epidermis observed when water was added, that is, the amount of water taken into the epidermal cells. It was found that the water retention ability in the epidermis can be measured. These mRNA amounts can be simply and stably carried out at any site regardless of the external environmental conditions at the time of skin sample collection. Furthermore, since no equipment is required when collecting a skin sample, there is an advantage that it is not necessary to move a large equipment to a measurement place and can be carried out anywhere.
本発明は、以下に列挙する皮膚表皮内水分保持能を評価する方法を提供する。
［１］皮膚表皮内水分保持能を評価する方法であって、次の（ａ）〜（ｃ）のステップを含む方法。
（ａ）個体から皮膚の試料を得るステップ
（ｂ）皮膚の試料からアクアポリン遺伝子に対応するｍＲＮＡ量を定量化するステップ
（ｃ）ステップ（ｂ）で算出したｍＲＮＡ量を用いて皮膚表皮内水分保持能を評価するステップ
［２］アクアポリン遺伝子が、アクアポリン３であることを特徴とする前記［１］に記載の皮膚表皮内水分保持能を評価する方法。
［３］ｍＲＮＡ量を定量化するステップがポリメラーゼ連鎖反応法（ＰＣＲ法）によって行われる、前記［１］に記載の皮膚表皮内水分保持能を評価する方法。
［４］皮膚の試料が被験対象の皮膚より採取した角質細胞である、前記［１］〜［３］のいずれかに記載の皮膚表皮内水分保持能を評価する方法。
The present invention provides a method for evaluating the water retention capacity in the skin epidermis listed below.
[1] A method for evaluating water retention ability in the skin epidermis, comprising the following steps (a) to (c).
(A) Obtaining a skin sample from an individual (b) Quantifying the amount of mRNA corresponding to the aquaporin gene from the skin sample (c) Retaining moisture in the skin epidermis using the amount of mRNA calculated in step (b) Step [2] The method for evaluating water retention capacity in the skin epidermis according to [1], wherein the aquaporin gene is
[3] The method for evaluating the water retention capacity in the skin epidermis according to [1], wherein the step of quantifying the amount of mRNA is performed by a polymerase chain reaction method (PCR method).
[4] The method for evaluating the water retention capacity in the skin epidermis according to any one of the above [1] to [3], wherein the skin sample is keratinocytes collected from the skin of the test subject.
本発明の方法により、皮膚のＡＱＰ３遺伝子に対応するｍＲＮＡ量を指標にして、表皮内水分保持能を評価する。皮膚の中で最外層に存在する角質細胞をｍＲＮＡ量測定の試料として用いることから、本発明の皮膚表皮内水分保持能評価法は、皮膚の任意の部位について非侵襲的に実施可能なものとなる。また、角質細胞中のｍＲＮＡ量は、皮膚の試料採取時の心理的、物理的作用による一過性の変化に影響されないため、環境条件を選ばず、皮膚の任意の部位について、簡便かつ安定的に実施できる。さらに、皮膚の試料採取時には高価な機器を必要とせず、またそのような大型機器を測定場所へ移動させる必要もなく、場所を選ばず実施することができる。 By the method of the present invention, the water retention capacity in the epidermis is evaluated using the amount of mRNA corresponding to the AQP3 gene in the skin as an index. Since the horny cells present in the outermost layer in the skin are used as a sample for measuring the amount of mRNA, the method for evaluating water retention capacity in the skin epidermis of the present invention can be performed noninvasively on any part of the skin. Become. In addition, the amount of mRNA in the keratinocytes is not affected by transient changes caused by psychological and physical effects at the time of skin sampling, so it is easy and stable for any part of the skin regardless of environmental conditions. Can be implemented. Further, when collecting a sample of skin, it is not necessary to use an expensive device, and it is not necessary to move such a large device to a measurement place, and it can be carried out anywhere.
本発明は、皮膚のＡＱＰ３遺伝子に対応するｍＲＮＡ量を指標にして皮膚表皮内水分保持能を評価する方法を提供する。ここで言う皮膚表皮内水分保持能とは、皮膚表皮を構成する全ての細胞における水分保持能であり、角質層を構成する細胞だけでなく、基底層、有棘層、顆粒層を構成する細胞中に存在する水分を維持する能力である。表皮の角質層以外の細胞は生きた細胞であり、角質層細胞中よりも圧倒的に多い水分を細胞内に溜め込む能力、すなわち皮膚表皮内水分保持能を有する。また、皮膚表皮内水分保持能は、水、化粧水などの水溶液状の液体を皮膚に付加したときに観察される表皮肥厚の原因となる性質である。そのため、皮膚表皮内水分保持能は、水溶液などを皮膚に付加して観察される表皮肥厚又は表皮厚の変化量を指標にして測定することができる。また、そのような水溶液の付加により、皮膚柔軟性の改善、キメの改善などがみられることから、ここでいう皮膚表皮内水分保持能がこれら皮膚性状に関与していると考えられ、この指標を測定することが美容科学上重要であると考えられる。本発明の皮膚表皮内水分保持能評価法では、被験対象の皮膚より採取した試料を用いてアクアポリン遺伝子に対応するｍＲＮＡの定量化を行い、その結果を基に皮膚表皮内水分保持能を評価する。このアクアポリン遺伝子は、ＡＱＰ３、ＡＱＰ５及びＡＱＰ９より選択される一つ又はそれ以上の遺伝子であることが好ましく、さらにＡＱＰ３であることがより好ましい。さらに、皮膚細胞より採取した試料として角質細胞を用いることにより、非侵襲的な評価が可能になる。以下に各Ｇｅｎｅ ＳｙｍｂｏｌのＧｅｎｅ ＮａｍｅとＧｅｎｂａｎｋ Ａｃｃｅｓｓｉｏｎ Ｎｕｍｂｅｒを示す（表１）。 The present invention provides a method for evaluating the water retention capacity in the skin epidermis using the amount of mRNA corresponding to the AQP3 gene in the skin as an index. The water retention capacity in the skin epidermis here refers to the water retention capacity of all cells that make up the skin epidermis, and not only the cells that make up the stratum corneum, but also the cells that make up the basal, spinous, and granule layers. The ability to maintain the moisture present in it. Cells other than the stratum corneum of the epidermis are living cells, and have the ability to accumulate much more water in the cells than in the stratum corneum cells, that is, the ability to retain moisture in the skin epidermis. In addition, the water retention capacity in the skin epidermis is a property that causes epidermal thickening observed when an aqueous liquid such as water or skin lotion is added to the skin. Therefore, the water retention capacity in the skin epidermis can be measured using as an index the epidermis thickening or the change in the skin thickness observed by adding an aqueous solution or the like to the skin. In addition, since the addition of such an aqueous solution improves skin softness and texture, it is considered that the water retention capacity in the skin epidermis here is involved in these skin properties. It is considered that it is important in cosmetic science to measure. In the method for evaluating water retention capacity in the skin epidermis of the present invention, mRNA corresponding to the aquaporin gene is quantified using a sample collected from the skin of the test subject, and the water retention capacity in the skin epidermis is evaluated based on the result. . The aquaporin gene is preferably one or more genes selected from AQP3, AQP5 and AQP9, and more preferably AQP3. Furthermore, non-invasive evaluation is possible by using keratinocytes as a sample collected from skin cells. The Gene Name and Genbank Accession Number of each Gene Symbol are shown below (Table 1).
皮膚からの角質細胞の採取の方法は特に限定されないが、可能な限り非侵襲的な採取法を採用することが好ましい。すなわち、角質細胞の採取の際、顆粒層や有棘層等の角質層以外の部分を、可能な限り傷つけないことが望まれる。非侵襲的な採取法としては、例えば、粘着性のテープや接着剤等を皮膚へ付着させた後に剥がす方法や、粗面の材料（例えば、不織布等）で皮膚表面を擦る方法によって角質細胞を採取することができる。非常に簡便に実施できる点において、前者の方法は特に好ましい。なお、以降に実施される遺伝子発現の定量化に影響しないものである限り、粘着性のテープや接着剤に用いられる材料（接着成分）は特に限定されない。 The method for collecting keratinocytes from the skin is not particularly limited, but it is preferable to employ a noninvasive collection method as much as possible. In other words, it is desired that when the corneocytes are collected, portions other than the stratum corneum such as the granule layer and the spiny layer are not damaged as much as possible. Non-invasive sampling methods include keratinocytes by, for example, a method in which an adhesive tape or adhesive is attached to the skin and then peeled off, or a method in which the skin surface is rubbed with a rough surface material (for example, non-woven fabric). Can be collected. The former method is particularly preferable because it can be carried out very simply. In addition, as long as it does not affect the quantification of gene expression implemented after that, the material (adhesive component) used for an adhesive tape or an adhesive agent is not specifically limited.
本発明における「ｍＲＮＡの定量化」とは、当該遺伝子の転写産物であるｍＲＮＡの量を測定することである。又は、ｍＲＮＡの情報を基に合成される蛋白質の量を測定しても良い。 The “quantification of mRNA” in the present invention is to measure the amount of mRNA that is a transcription product of the gene. Or you may measure the quantity of the protein synthesize | combined based on the information of mRNA.
ｍＲＮＡ量の測定には、マイクロアレイ解析や、逆転写反応を行った後にＰＣＲ法にて行う方法等を用いることができる。これらの方法は、常法に従って実施すれば良い。各方法について様々なプロトコールが報告されており、当業者であれば公知のプロトコールに従い、又は公知のプロトコールを適宜修正・変更したプロトコールによって、適切な測定系を構築し実施することができる。なお、ｍＲＮＡの定量化の詳細については後述する。 For the measurement of the amount of mRNA, microarray analysis, a method performed by PCR after reverse transcription reaction, or the like can be used. These methods may be carried out according to conventional methods. Various protocols have been reported for each method, and those skilled in the art can construct and implement an appropriate measurement system according to a known protocol or according to a protocol obtained by appropriately modifying or changing a known protocol. Details of mRNA quantification will be described later.
一方、蛋白質量の測定であれば、例えば、蛍光物質、色素、酵素等を利用する免疫染色法、ウエスタンブロット法、免疫測定法（例えば、ＥＬＩＳＡ法やＥＩＡ法等）等を用いることができる。これらの方法についても、常法に従って実施すれば良い。各方法について様々なプロトコールが報告されており、当業者であれば公知のプロトコールに従い、又は公知のプロトコールを適宜修正・変更したプロトコールによって、適切な測定系を構築し実施することができる。 On the other hand, for the measurement of protein mass, for example, an immunostaining method using a fluorescent substance, a dye, an enzyme or the like, a Western blot method, an immunoassay method (for example, ELISA method, EIA method, etc.) and the like can be used. These methods may be carried out according to a conventional method. Various protocols have been reported for each method, and those skilled in the art can construct and implement an appropriate measurement system according to a known protocol or according to a protocol obtained by appropriately modifying or changing a known protocol.
本発明の皮膚表皮内水分保持能評価法は、好ましくは以下の一連のステップ（１）〜（３）によって実施される。
（１）被験対象の皮膚より採取した角質細胞を用意するステップ
（２）前記角質細胞から抽出したＲＮＡを試料としてｍＲＮＡの定量化を行い、アクアポリン３遺伝子に対するｍＲＮＡ量を算出するステップ
（３）ステップ（２）で算出したｍＲＮＡ量を用いて、皮膚表皮内水分保持能を評価するステップ
The skin epidermis moisture retention evaluation method of the present invention is preferably carried out by the following series of steps (1) to (3).
(1) Step of preparing keratinocytes collected from the skin of the test subject (2) Step of quantifying mRNA using the RNA extracted from the keratinocytes as a sample and calculating the amount of mRNA for the
ステップ（１）では、被験対象の皮膚より採取した角質細胞を用意する。以下に、角質細胞の採取法及びＲＮＡ抽出法の具体例を示す。
（ａ）顆粒層や有棘層等を傷つけないように、最外層である角質層の一部を剥離する。例えば、粘着性のテープを皮膚へ付着させた後に剥がし（必要に応じて、数回繰り返す）、角質細胞を採取する。
（ｂ）採取した角質細胞を、ＳＬＳ、β−メルカプトエタノール、グアニジンイソチオシアネート等を含む溶解性緩衝液に浸した後、蛋白質分解酵素を加え反応させる。
（ｃ）反応終了後、例えば、超音波破砕機等の物理的な力によって角質細胞を破砕する。
（ｄ）角質細胞破砕物を含む溶液から、周知の核酸抽出法に準じた方法や市販されたキットを用いた方法により、ＲＮＡを抽出する。例えば、グアニジンイソチオシアネート、フェノール又はクロロホルムを用いたＲＮＡ抽出法や、ニッポンジーン社のＩＳＯＧＥＮ、インビトロジェン社のＴｒｉｚｏｌ Ｒｅａｇｅｎｔ、あるいはＱＩＡＧＥＮ社のＲＮｅａｓｙ ＫｉｔやＡｍｂｉｏｎ社のＲＮＡｑｕｅｏｕｓ−４ＰＣＲ Ｋｉｔ等を用いる方法によってＲＮＡを抽出する。
（ｅ）必要に応じて、ＤＮＡ分解酵素を反応させＤＮＡを除去する。
（ｆ）必要に応じて、エタノール沈殿等の核酸濃縮法を用いてＲＮＡを濃縮する。
In step (1), keratinocytes collected from the skin of the test subject are prepared. Specific examples of the corneocyte collection method and RNA extraction method are shown below.
(A) A part of the stratum corneum that is the outermost layer is peeled off so as not to damage the granular layer, the spiny layer, and the like. For example, an adhesive tape is attached to the skin, and then peeled off (repeated several times as necessary) to collect keratinocytes.
(B) The soaked keratinocytes are immersed in a soluble buffer containing SLS, β-mercaptoethanol, guanidine isothiocyanate, etc., and then reacted with a protease.
(C) After completion of the reaction, the keratinocytes are crushed by physical force such as an ultrasonic crusher.
(D) RNA is extracted from a solution containing a keratinocyte disruption product by a method according to a well-known nucleic acid extraction method or a method using a commercially available kit. For example, RNA extraction using guanidine isothiocyanate, phenol or chloroform, RNA extraction using Nippon Gene's ISOGEN, Invitrogen's Trizol Reagent, QIAGEN's RNeasy Kit, Ambion's RNAqueous-4PCR Kit, etc. To do.
(E) If necessary, a DNA decomposing enzyme is reacted to remove DNA.
(F) If necessary, RNA is concentrated using a nucleic acid concentration method such as ethanol precipitation.
ステップ（２）では、採取した角質細胞から抽出したＲＮＡを試料としてｍＲＮＡの定量化を行う。そして、当該遺伝子に対するｍＲＮＡ量を算出する。このように、本発明の一態様では、角質細胞中に残存する当該遺伝子に対応するｍＲＮＡの定量化を行う。なお、ｍＲＮＡの定量化では、測定値は絶対値又は相対値（比較対象又は標準のｍＲＮＡ量との比率や差等）として算出される。 In step (2), mRNA is quantified using RNA extracted from the collected corneocytes as a sample. Then, the amount of mRNA for the gene is calculated. Thus, in one embodiment of the present invention, mRNA corresponding to the gene remaining in the corneocytes is quantified. In the quantification of mRNA, the measured value is calculated as an absolute value or a relative value (a ratio or a difference from the comparison target or standard mRNA amount).
ここで、角質細胞から抽出したｍＲＮＡの定量化法の具体例を以下に示す。 Here, a specific example of a method for quantifying mRNA extracted from keratinocytes is shown below.
ＲＴ−ＰＣＲを用いた方法
（ａ）抽出したＲＮＡを鋳型に、逆転写酵素を用いてｃＤＮＡを合成する。
（ｂ）ｃＤＮＡを鋳型に、ターゲットとなる遺伝子に対応するプライマーを用いてＰＣＲ反応を行い、当該遺伝子に対応するＤＮＡ断片を得る。
（ｃ）内部標準となるような遺伝子（β−アクチン、ＧＡＰＤＨ、ケラチン６Ｂ、ＣＴＳＬ２等）の反応も並行して行う。
（ｄ）得られたＤＮＡ断片の電気泳動を行った後、エチジウムブロマイド等で染色してバンドの強度を測定し、その遺伝子に対応するｍＲＮＡ量とする。
（ｅ）必要に応じて、ＳＹＢＲ ｇｒｅｅｎ等の蛍光色素やＴａｑｍａｎ ｐｒｏｂｅ等の蛍光プローブを用いて定量ＰＣＲ反応を行い、ｍＲＮＡを定量する。
（ｆ）内部標準となる遺伝子に対応するｍＲＮＡ量に対する、当該遺伝子に対応するｍＲＮＡ量の比率を算出する。
Method using RT-PCR (a) Using the extracted RNA as a template, cDNA is synthesized using reverse transcriptase.
(B) A PCR reaction is performed using cDNA as a template and a primer corresponding to the target gene to obtain a DNA fragment corresponding to the gene.
(C) Reactions of genes (β-actin, GAPDH, keratin 6B, CTSL2, etc.) that serve as internal standards are also performed in parallel.
(D) After electrophoresis of the obtained DNA fragment, the intensity of the band is measured by staining with ethidium bromide or the like to obtain the amount of mRNA corresponding to the gene.
(E) If necessary, quantitative PCR reaction is performed using a fluorescent dye such as SYBR green or a fluorescent probe such as Taqman probe to quantify mRNA.
(F) The ratio of the amount of mRNA corresponding to the gene to the amount of mRNA corresponding to the gene serving as the internal standard is calculated.
ステップ（３）では、ステップ（２）で算出したｍＲＮＡ量を用いて、皮膚表皮内水分保持能を評価する。例えば、算出したｍＲＮＡ量を予め設定した基準ｍＲＮＡ量と比較して、その比率を計算する。そして、その比率が、皮膚表皮内水分保持能の評価が関連付けられた複数の区分の中のいずれに該当するのかを調べる。区分の設定に関する具体例を以下に示す。
（例１）皮膚表皮内水分保持能（低い）：比率＜ａ、（中程度）：ａ≦比率＜ｂ、（高い）：ｂ≦比率
In step (3), the water retention capacity in the skin epidermis is evaluated using the mRNA amount calculated in step (2). For example, the calculated mRNA amount is compared with a preset reference mRNA amount, and the ratio is calculated. Then, it is examined which of the plurality of categories that the ratio of the water retention ability in the skin epidermis is associated with. Specific examples regarding the setting of categories are shown below.
(Example 1) Moisture retention capacity in skin epidermis (low): ratio <a, (medium): a ≦ ratio <b, (high): b ≦ ratio
（例１）では区分数を３としているが、区分数は特に限定されるものではない。例えば、区分数を２〜１０のいずれかにすることができる。区分数及び各区分に関連付けられる基準発現量の値の範囲は、予備実験の結果を基に任意に設定可能である。 In (Example 1), the number of divisions is 3, but the number of divisions is not particularly limited. For example, the number of sections can be any of 2-10. The range of the number of sections and the value of the reference expression level associated with each section can be arbitrarily set based on the results of preliminary experiments.
以下、本発明を効果的に説明するために、実験例を挙げる。なお、本発明はこれにより限定されるものではない。 In order to effectively explain the present invention, experimental examples are given below. In addition, this invention is not limited by this.
実験例１ 皮膚表皮の厚さの測定と角質細胞採取
健常女性被験者６名（平均年齢３５．３歳）を対象として、頬部に化粧水を含浸させたコットンを１０分間貼付し、貼付する前後における表皮厚の変化を測定した。表皮厚の測定には、共焦点レーザー顕微鏡ＶｉｖａＳｃｏｐｅ３０００（Ｌｕｃｉｄ社製）を用い、レーザー強度１．５ｍＷにて深さ１μｍ毎１００枚垂直方向の連続画像を得、顆粒層から真皮内のコラーゲンの線維構造が見え始める画像までの枚数により表皮の厚さを求めた。
Experimental Example 1 Measurement of skin epidermis thickness and corneocyte collection For 6 healthy female subjects (average age 35.3 years old), a cotton impregnated with lotion was applied to the cheek for 10 minutes, before and after application The change in skin thickness was measured. For the measurement of the epidermis thickness, a confocal laser microscope VivaScope 3000 (manufactured by Lucid) was used to obtain 100 continuous images in a vertical direction at a depth of 1 μm at a laser intensity of 1.5 mW, and collagen fibers in the dermis from the granular layer The thickness of the epidermis was determined from the number of images until the structure began to appear.
被験者６名のコットンを貼付する前の表皮厚の平均は３７．４μｍであったが、コットン貼付後は４５．１μｍとなり、表皮厚の有意な増加がみられた（ｐ＝０．０００３、図１）。この表皮厚の変化は、表皮細胞が水を取り込むことにより体積が増加し表皮の厚さが増加した結果であると考えられる。 The average skin thickness of the 6 subjects before applying the cotton was 37.4 μm, but after applying the cotton, it became 45.1 μm, and a significant increase in the skin thickness was observed (p = 0.0003, FIG. 1). This change in epidermal thickness is considered to be a result of the increase in volume and the increase in epidermal thickness due to the uptake of water by epidermal cells.
実験例２ 頬部の皮膚からの角質細胞採取と角質細胞採取近傍部における表皮厚の測定
健常女性被験者１０名（平均年齢３４．７歳）を対象として、額部より２ｃｍ×２ｃｍの角質層テープストリップを２枚採取した。さらに、その近傍部にて、化粧水を含浸させたコットン貼付前後における表皮厚の変化を実験例１と同様に測定した。
Experimental Example 2 Measurement of stratum corneum from skin of cheek and measurement of epidermis thickness in the vicinity of corneocyte collection For 10 healthy female subjects (average age of 34.7 years), 2 cm × 2 cm stratum corneum tape from the forehead Two strips were collected. Further, in the vicinity thereof, the change in the skin thickness before and after sticking the cotton impregnated with the skin lotion was measured in the same manner as in Experimental Example 1.
実験例３ 皮膚角質細胞に含まれるＲＮＡの抽出
実験例２にて得られた角質細胞に含まれるＲＮＡを、ＲＮＡ抽出キット（Ａｍｂｉｏｎ社、ＲＮＡｑｕｅｏｕｓ−４ＰＣＲ Ｋｉｔ）を用いて抽出した。すなわち、角質細胞の付着したテープ２枚を、テープごとＬｙｓｉｓ／Ｂｉｎｄｉｎｇ Ｓｏｌｕｔｉｏｎに入れ、ｐｒｏｔｅｉｎａｓｅ Ｋ（Ｉｎｖｉｔｒｏｇｅｎ社）を添加し、５６℃でインキュベートした。その後、超音波破砕機を用いて分散させた後、６４％エタノールを加えて攪拌してテープを取り除き、角質細胞抽出液を得た。付属のＦｉｌｔｅｒ ＣａｒｔｒｉｄｇｅにＲＮＡを吸着させた後、溶出液でＲＮＡを溶出した。得られた溶出液にＤＮａｓｅＩを加え、残存しているＤＮＡを完全に除去した後、エタノールを用いてＲＮＡを沈殿させて濃縮した。得られたＲＮＡのペレットを必要量の水に溶解し、角質細胞に含まれるＲＮＡを得た。
Experimental Example 3 Extraction of RNA contained in skin keratinocytes RNA contained in the keratinocytes obtained in Experimental Example 2 was extracted using an RNA extraction kit (Ambion, RNAqueous-4PCR Kit). That is, two tapes with keratinocytes attached were placed in the Lysis / Binding Solution together with the tape, proteinase K (Invitrogen) was added, and incubated at 56 ° C. Then, after making it disperse | distribute using an ultrasonic crusher, 64% ethanol was added and stirred, the tape was removed, and the keratinocyte extract was obtained. After adsorbing RNA to the attached Filter Cartridge, the RNA was eluted with the eluate. DNase I was added to the obtained eluate to completely remove the remaining DNA, and then RNA was precipitated using ethanol and concentrated. The obtained RNA pellet was dissolved in a necessary amount of water to obtain RNA contained in keratinocytes.
実験例４ 角質細胞に含まれるＲＮＡを用いたＡＱＰ３遺伝子のＰＣＲ解析
皮膚の角質細胞より、実験例３の方法で得たＲＮＡを、ＳｕｐｅｒＳｃｒｉｐｔ ＩＩＩ Ｏｎｅ−Ｓｔｅｐ ＲＴ−ＰＣＲ Ｓｙｓｔｅｍ ｗｉｔｈ Ｐｌａｔｉｎｕｍ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（Ｉｎｖｉｔｒｏｇｅｎ社）を用いたＲＴ−ＰＣＲ反応に供し、ＡＱＰ３及びＡＱＰ９に対応するｍＲＮＡに対するｃＤＮＡを合成した。ＲＴ−ＰＣＲ反応後、リアルタイムＰＣＲ反応により、合成したｃＤＮＡ量を測定し、これを各遺伝子に対応するｍＲＮＡ量とした。また、同時に、内部標準として、ＧＡＰＤＨ遺伝子のｍＲＮＡ量について、同様の方法にて解析し、各遺伝子の数値をＧＡＰＤＨの数値にて除したものを、相対ｍＲＮＡ量とした。なお、ＧＡＰＤＨ及びアクアポリンのｍＲＮＡに対するＲＴ−ＰＣＲ反応に使用したプライマーは、次の通りである。
Experimental Example 4 PCR analysis of AQP3 gene using RNA contained in keratinocytes RNA obtained by the method of Experimental Example 3 from skin keratinocytes was converted into SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) And a cDNA for mRNA corresponding to AQP3 and AQP9 was synthesized. After RT-PCR reaction, the amount of synthesized cDNA was measured by real-time PCR reaction, and this was defined as the amount of mRNA corresponding to each gene. At the same time, as an internal standard, the amount of GAPDH gene mRNA was analyzed by the same method, and the value obtained by dividing the value of each gene by the value of GAPDH was used as the relative mRNA amount. The primers used in the RT-PCR reaction for GAPDH and aquaporin mRNA are as follows.
ＧＡＰＤＨ用のプライマー
ＴＧＡＡＣＧＧＧＡＡＧＣＴＣＡＣＴＧＧ（配列番号１）
ＴＣＣＡＣＣＡＣＣＣＴＧＴＴＧＣＴＧＴＡ（配列番号２）
ＡＱＰ３用のプライマー
ＴＧＧＧＴＧＣＴＧＧＡＡＴＡＧＴＴＴＴＴＧＧ（配列番号３）
ＧＣＴＧＧＴＴＧＴＣＧＧＣＧＡＡＧＴ（配列番号４）
ＡＱＰ５用のプライマー
ＣＣＣＣＡＧＡＧＣＴＣＣＴＴＡＧＧＡＡＧＡ（配列番号５）
ＣＡＴＧＣＡＴＣＣＴＣＡＣＣＡＧＡＡＡＴＡＡＡＴ（配列番号６）
ＡＱＰ９用のプライマー
ＣＧＣＴＧＡＣＴＧＴＣＣＣＣＴＧＡＡＡＣ（配列番号７）
ＣＡＡＧＡＡＴＴＣＣＣＡＴＣＧＡＧＧＡＴＧＡ（配列番号８）
Primer for GAPDH TGAACGGGAAGCTCACTGG (SEQ ID NO: 1)
TCCACCACCCCTGTGCTGTA (SEQ ID NO: 2)
Primer for AQP3 TGGGTGCTGGAATAGTTTTTGG (SEQ ID NO: 3)
GCTGGTTGTCGCGCAAGT (SEQ ID NO: 4)
Primer for AQP5 CCCCAGAGCTCCCTTAGGAAGA (SEQ ID NO: 5)
CATGCATCCTCACCAGAAATAAAT (SEQ ID NO: 6)
Primer for AQP9 CGCTGACTGTCCCCCTGAAAC (SEQ ID NO: 7)
CAAGAATTCCCATCGAGGATGA (SEQ ID NO: 8)
ＡＱＰ３、ＡＱＰ５及びＡＱＰ９に対応するｍＲＮＡ量の相対値と化粧水付加による表皮厚変化量との相関係数は、それぞれＲ２＝０．４９２、ｐ＜０．０５、Ｒ２＝０．３４９、ｐ＜０．１、Ｒ２＝０．３７６、ｐ＜０．１であり、相関が認められた（図２〜４）。この結果から、ＡＱＰ３、ＡＱＰ５及びＡＱＰ９遺伝子に対応するｍＲＮＡ量の相対値は、化粧水などの水溶液を付加したときに観察される表皮厚の変化を反映していることが示された。この場合の表皮厚の変化量の大きさは、細胞内に存在する水の体積に依存していると考えられるため、表皮厚の変化量が表皮細胞の水分保持能を表していると考えられる。そして、表皮厚の変化がアクアポリン遺伝子に対応するｍＲＮＡ量の相対値と相関が認められたため、このｍＲＮＡ量は表皮細胞の水分保持能を表していると考えられた。これらの結果から、特に、ＡＱＰ３と表皮厚変化量とは有意な相関が認められたことから、ＡＱＰ３に対応するｍＲＮＡの量を表皮細胞内の水分保持能に対する指標として用いることが好ましいと考えられた。 The correlation coefficient between the relative value of the amount of mRNA corresponding to AQP3, AQP5 and AQP9 and the amount of change in skin thickness due to the addition of lotion is R 2 = 0.492, p <0.05, R 2 = 0.349, p <0.1, R 2 = 0.376, p <0.1, and a correlation was observed (FIGS. 2 to 4). From this result, it was shown that the relative value of the amount of mRNA corresponding to the AQP3, AQP5 and AQP9 genes reflects changes in the skin thickness observed when an aqueous solution such as lotion is added. In this case, the amount of change in the epidermis thickness is thought to depend on the volume of water present in the cells, so the amount of change in the epidermis thickness is considered to represent the water retention capacity of the epidermis cells. . And since the change of the epidermal thickness was correlated with the relative value of the mRNA amount corresponding to the aquaporin gene, this mRNA amount was considered to represent the water retention ability of the epidermal cells. From these results, it was considered that it is preferable to use the amount of mRNA corresponding to AQP3 as an index for the water retention ability in epidermal cells, since a significant correlation was observed between AQP3 and the change in epidermal thickness. It was.
実験例４ 化粧水付加における表皮厚の変化と皮膚性状に関する官能評価との関連性
実験例２に示した被験者に対して、皮膚性状に関するアンケート調査を行い、化粧水付加における表皮厚の変化とＡＱＰ３の相対発現量との関連性を検討するために、相関解析を行った。
Experimental Example 4 Relevance between Skin Thickness Change with Skin Lotion and Sensory Evaluation on Skin Properties For the subjects shown in Experimental Example 2, a questionnaire survey on skin properties was conducted, and skin thickness changes with skin lotion and AQP3 In order to examine the relationship with the relative expression level, correlation analysis was performed.
９項目の質問に対して、１０段階で評価させ、表皮厚変化との相関を解析したところ、「キメがふっくらしていると感じる」、「肌がみずみずしいと感じる」、「ハリがあると感じる」という３つの質問項目のスコアと、化粧水付加による表皮厚の変化との間に有意な相関が観察された（図５）。これらの結果は、皮膚表皮内水分保持能が高いほど、肌に対する官能的評価が高いことを示しており、特に、皮膚のキメ、みずみずしさ、ハリ感についての実感が高いと考えられた。また、ＡＱＰ３に対応するｍＲＮＡ量に対して、前記３つの質問項目との相関係数は、それぞれＲ２＝０．６３８、ｐ＜０．００６、Ｒ２＝０．３９４、ｐ＜０．０３５、Ｒ２＝０．７２６、ｐ＜０．００２となった。このように、ＡＱＰ３に対応するｍＲＮＡ量に対する相関性は、化粧水付加による表皮厚の変化に対するものと同等以上であったことから、ＡＱＰ３に対応するｍＲＮＡの量を皮膚表皮内水分保持能の指標として用いることが可能であると考えられた。 Nine questions were evaluated on a 10-point scale, and the correlation with changes in skin thickness was analyzed. As a result, "I feel that my skin is fluffy", "I feel that my skin is fresh", and "I feel that my skin is full" A significant correlation was observed between the scores of the three question items “and the change in skin thickness due to the addition of lotion (FIG. 5). These results indicate that the higher the moisture retention capacity in the skin epidermis, the higher the sensory evaluation for the skin, and in particular, it was considered that the feeling of skin texture, freshness and firmness was higher. Further, the correlation coefficients with the three question items with respect to the amount of mRNA corresponding to AQP3 are R 2 = 0.638, p <0.006, R 2 = 0.394, p <0.035, respectively. , R 2 = 0.726, p <0.002. Thus, since the correlation with the amount of mRNA corresponding to AQP3 was equal to or greater than that with respect to the change in skin thickness due to the addition of skin lotion, the amount of mRNA corresponding to AQP3 was used as an index of the ability to retain water in the skin epidermis. It was thought that it could be used as
なお、角質細胞は表皮ケラチノサイトに由来する死滅した細胞であり、前記実験例にて検出されたｍＲＮＡ量の変化は、表皮を構成し角質層の下層に位置する顆粒細胞、有棘細胞及び基底細胞といった生細胞において生じた生体変化を表していると考えられる。 The keratinocytes are dead cells derived from epidermal keratinocytes, and the change in the amount of mRNA detected in the experimental examples is the granule cells, spinous cells, and basal cells that constitute the epidermis and are located below the stratum corneum. It is thought that it represents a biological change that occurs in a living cell.
本発明の皮膚表皮内水分保持能評価法によれば、角質細胞中に存在する当該遺伝子に対するｍＲＮＡを定量的に測定するため、非侵襲的に客観性の高い評価結果を得ることができる。また、心理的、物理的作用による一過性の変化を除外できることに加えて、測定場所を選ばず、非侵襲的かつ簡易に採取可能な任意の皮膚部位について、簡便かつ安定的に皮膚表皮内水分保持能評価が可能である。さらに、高価な測定機器を購入する必要がなく、特別な大型測定機器を測定場所まで移動させる必要がないという利点もある。本発明は、化粧品及び皮膚外用剤やその使用法、あるいは皮膚に対する施術法などの評価に応用が可能である。 According to the method for evaluating the water retention ability in the skin epidermis of the present invention, mRNA for the gene present in the keratinocytes is quantitatively measured, so that a highly objective evaluation result can be obtained non-invasively. In addition to being able to exclude transient changes due to psychological and physical effects, any skin site that can be collected non-invasively and easily, regardless of measurement location, can be easily and stably applied to the skin epidermis. Moisture retention ability can be evaluated. Furthermore, there is an advantage that it is not necessary to purchase an expensive measuring device and it is not necessary to move a special large measuring device to a measuring place. The present invention can be applied to the evaluation of cosmetics and external preparations for skin, methods for using them, and methods for treating skin.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (1)
（ａ）個体から得られた皮膚の角質細胞からアクアポリン３遺伝子に対応するｍＲＮＡ量を定量化するステップ
（ｂ）ステップ（ａ）で算出したｍＲＮＡ量を用いて皮膚表皮内顆粒層より下層における水分保持能を評価するステップ A method for evaluating water retention ability in a layer lower than a granular layer in the skin epidermis, comprising the following steps (a) and (b) .
(A) quantifying the amount of mRNA corresponding the horny skin cells obtained from an individual to aquaporin 3 gene step (b) step (a) using the calculated amount of mRNA in the water in the lower layer than the skin epidermal granular layer Steps to assess retention
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