JP5731678B1 - Blood flow evaluation or indirect measurement method - Google Patents
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- 108010082786 Interleukin-1alpha Proteins 0.000 claims abstract description 21
- 102000004125 Interleukin-1alpha Human genes 0.000 claims abstract description 21
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/02—Instruments for taking cell samples or for biopsy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Abstract
【課題】外科的に皮膚を摘出したりして皮膚に刺激を与える等のない方法であって、血流の状態を簡便かつ確実に測定又は評価することができる、生化学的指標を用いた新規な測定又は評価方法を提供することを課題とする。【解決手段】皮膚角層中のインターロイキン−１レセプターアンタゴニスト及び／又はインターロイキン−１αの発現量を指標とすることを特徴とする皮膚の血流量の間接測定又は評価方法。【選択図】図１[PROBLEMS] To use a biochemical index, which is a method that does not cause irritation to the skin by surgically removing the skin and can measure or evaluate a blood flow state easily and reliably. It is an object to provide a new measurement or evaluation method. A method for indirectly measuring or evaluating skin blood flow characterized by using an expression level of an interleukin-1 receptor antagonist and / or interleukin-1α in the stratum corneum as an index. [Selection] Figure 1
Description
本発明は、肌の血流量のレベル評価又は間接測定方法に関する。 The present invention relates to a level evaluation or indirect measurement method for skin blood flow.
肌の老化現象の原因の一つとして、血行不良による肌色の赤みの低下を挙げることができる。この肌の赤みの低下現象が、端的には「肌のくすみ」として現れ、化粧によるカバーリングが難しい現象とされている。そして肌の血流を改善することにより、透明性及び明るさが向上し、皮膚組織の代謝活性の促進、細胞賦活が期待でき、その結果としてくすみの原因を始め肌の各種の老化現象の原因に対処できるといわれている。
肌組織は、血流量の増加により代謝が促進され、老廃物が除去される。例えば、化学発光により近赤外線を含む光を照射することで、肌の血流を改善する技術が開発されている（特許文献１：ＷＯ２００５／０７７４５８号公報）。特許文献１では肌の血流量の変化はレーザードップラー血流計を用いて測定している。
このように、肌の健康状態を評価するために、皮膚の血流量を測定することが必要である。前記のレーザードップラー式血流計と同様に、レーザー光を用いるマルチチャンネル式の血流計（特許文献２：特許第3783491号公報）や加圧式血流計（特許文献３：特開平１１−２８５４７６号公報）が肌の血流測定に用いられている。しかしながら、測定に当たっては被験者を直接、血流量計で測定する必要があり、遠隔地の被験者の肌の血流量を測定するためには、測定装置を被験者の住居近くに設置するか、あるいは被験者が直接、測定装置の設置場所に出向かない限り測定することは不可能である。
One of the causes of the skin aging phenomenon is a decrease in redness of the skin color due to poor blood circulation. This phenomenon of redness of the skin appears as “skin dullness” in short, making it difficult to cover with makeup. And by improving the blood flow of the skin, transparency and brightness are improved, and the metabolic activity of the skin tissue and cell activation can be expected. As a result, the cause of various skin aging phenomena including dullness It is said that it can cope with.
Skin tissue is promoted for metabolism by increasing blood flow, and waste products are removed. For example, a technique for improving skin blood flow by irradiating light including near infrared rays by chemiluminescence has been developed (Patent Document 1: WO 2005/077458). In Patent Document 1, the change in the blood flow rate of the skin is measured using a laser Doppler blood flow meter.
Thus, it is necessary to measure the blood flow in the skin in order to evaluate the health condition of the skin. Similar to the laser Doppler blood flow meter, a multi-channel blood flow meter using a laser beam (Patent Document 2: Japanese Patent No. 3783491) and a pressure blood flow meter (Patent Document 3: Japanese Patent Laid-Open No. 11-285476). No.) is used for measuring blood flow in the skin. However, when measuring, it is necessary to measure the subject directly with a blood flow meter, and in order to measure the blood flow of the skin of the subject in a remote place, a measuring device is installed near the subject's residence, or the subject It is impossible to measure without directly going to the place where the measuring device is installed.
上述したように、遠隔地の被験者の血流の状態を間接的に評価することを可能とするために、被験者から採取した角層を用いて、血流量の間接的に測定又は評価することが望まれている。
本発明は、外科的に皮膚を摘出したりして皮膚に刺激を与える等のない方法であって、血流の状態を簡便かつ確実に測定又は評価することができる、生化学的指標を用いた新規な測定又は評価方法を提供することを課題とする。
As described above, in order to be able to indirectly evaluate the blood flow state of a subject at a remote location, it is possible to indirectly measure or evaluate blood flow using the stratum corneum collected from the subject. It is desired.
The present invention is a method that does not cause skin irritation by surgically removing the skin, and uses a biochemical index that can easily and reliably measure or evaluate the state of blood flow. It is an object to provide a novel measurement or evaluation method.
本発明は以下の構成である。
（１）皮膚角層中のインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を指標とすることを特徴とする皮膚の血流量の間接測定又は評価方法。
（２）測定又は評価方法に用いる皮膚角層がテープストリッピング法により採取されたものである（１）に記載の方法。
（３）血流量と皮膚角層中のインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量の相関関係に基づいてあらかじめ作成された相関式から、インターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を測定することで皮膚の血流量を間接的に測定する方法。
（４）角層を採取する採取工程と、
前記採取工程で採取された角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を測定する測定工程と、
前記測定工程で測定されたインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を角層のインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量と血流量の分布からあらかじめ求めた回帰式により皮膚血流量に換算する工程、
とからなる、皮膚血流量の間接測定法。
（５）血流量と皮膚角層中のインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量の相関関係に基づいてあらかじめ作成した散布図と、測定したインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量とを対比して評価する、皮膚の血流量の評価方法。
（６）角層を採取する採取工程と、
前記採取工程で採取された角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を測定する測定工程と、
前記測定工程で測定されたインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を、あらかじめ測定しておいた集団の、角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量と血流量に基づいて、あらかじめ作成した分布図と比較する比較工程、
とから構成される、皮膚血流の評価方法。
The present invention has the following configuration.
(1) Interleukin-1 receptor-antagonizing varnish DOO skin stratum layer, or indirect measurement or evaluation methods in blood flow in the skin, characterized in that as an index the expression of interleukin-1 alpha.
(2) The method according to (1), wherein the skin stratum corneum used in the measurement or evaluation method is collected by a tape stripping method.
(3) blood flow and interleukin-1 receptor-antagonizing varnish DOO skin stratum layer, or a correlation equation that has been previously created based on the correlation between the expression of interleukin-1 alpha, interleukin-1 receptor antagonist, Alternatively, a method of indirectly measuring blood flow in the skin by measuring the expression level of interleukin-1α .
(4) a sampling process for sampling the stratum corneum;
A measuring step of measuring the expression level of interleukin-1 receptor antagonist or interleukin-1α in the stratum corneum collected in the collecting step;
Determined in advance from the measurement step interleukin-1 receptor antagonist was measured at, or interleukin-interleukin 1 receptor antagonist expression level stratum corneum of the 1 alpha, or interleukin expression level and blood flow distribution of 1 alpha step of converting the skin blood flow by the regression formula,
An indirect method for measuring skin blood flow.
(5) Blood Flow and interleukin of stratum corneum in interleukin-1 receptor-antagonizing varnish DOO, or a scatter diagram created in advance based on the correlation between the expression of interleukin-1 alpha, measured interleukin-1 receptor antagonist or interleukin versus assessing the expression level of 1 alpha, the evaluation method of the blood flow in the skin.
(6) a sampling process for sampling the stratum corneum;
A measuring step of measuring the expression level of interleukin-1 receptor antagonist or interleukin-1α in the stratum corneum collected in the collecting step;
Said measuring step interleukin-1 receptor antagonist was measured in, or the expression level of interleukin 1 alpha, populations advance measurement, interleukin-1 receptor antagonist in the horny layer, or interleukin-1 alpha A comparison process to compare with a distribution map created in advance based on the expression level and blood flow volume,
A method for evaluating skin blood flow.
本発明の方法によれば、皮膚の血流の測定又は評価を角層におけるインターロイキン−１レセプターアンタゴニスト又はインターロイキン−１αの発現量を測定することで間接的に測定又は評価することができる。
また、本発明の方法は、テープストリッピング法など簡便な操作方法で評価試料を採取するため、被験者の負担が少なく、だれでも簡単に評価することが可能となる。
特に遠隔地の被験者から採取したサンプル、例えば、テープストリッピングによる角層を用いて、簡便に肌の血流量あるいは血流の状態を間接的に評価することができるため被験者が直接試験場所に来る必要がなくなる。そうすれば、血流量測定の対象とする適用範囲が広がり、簡便に皮膚の状態を診断又は評価できる。
さらに血流量計で直接被験者を測定する必要がなく、遠隔地の被験者であっても角層さえ入手できれば、簡便に皮膚血流量の測定又は評価が可能となる。
さらにまた、本発明の方法によって肌の状態や化粧料などの効果を客観的に評価することが可能となる。
According to the method of the present invention, skin blood flow can be measured or evaluated indirectly by measuring the expression level of interleukin-1 receptor antagonist or interleukin-1α in the stratum corneum.
Moreover, since the evaluation sample is collected by a simple operation method such as a tape stripping method in the method of the present invention, the burden on the subject is small, and anyone can easily evaluate.
In particular, samples taken from a remote subject, such as the stratum corneum by tape stripping, can be used to easily evaluate the blood flow or blood flow of the skin, so the subject needs to come directly to the test site. Disappears. By doing so, the application range for blood flow measurement is expanded, and the skin condition can be easily diagnosed or evaluated.
Furthermore, it is not necessary to directly measure a subject with a blood flow meter, and even a remote subject can easily measure or evaluate skin blood flow as long as the stratum corneum is available.
Furthermore, the method of the present invention makes it possible to objectively evaluate the skin condition and cosmetic effects.
本発明は、皮膚角層中のインターロイキン−１レセプターアンタゴニスト及び／又はインターロイキン−１αの発現量を指標とすることを特徴とする皮膚の血流量の間接測定又は評価方法に係る発明である。
以下、本発明について詳細に説明する。
本発明の血流量の測定又は評価方法は、皮膚角層におけるインターロイキン−１レセプターアンタゴニスト（以下、ＩＬ−１ＲＡとよぶ。）、又は、インターロイキン−１α（以下、ＩＬ−１αとよぶ。）の発現量を指標とすることを特徴としている。
本発明における血流量とは、レーザードップラー血流量計で測定される皮膚の毛細血管内血流量である。レーザードップラー血流量計で測定された血流量と、皮膚角層におけるＩＬ−１ＲＡ又はＩＬ−１αの発現量が相関することが見出された。したがってＩＬ−１ＲＡ又はＩＬ−１αの皮膚角層における発現量を測定することで、間接的に皮膚血流量又は皮膚血流状態の評価を行うことができることが可能となる。
The present invention relates to an indirect measurement or evaluation method for skin blood flow characterized by using the expression level of interleukin-1 receptor antagonist and / or interleukin-1α in the stratum corneum as an index.
Hereinafter, the present invention will be described in detail.
The method for measuring or evaluating blood flow according to the present invention uses an interleukin-1 receptor antagonist (hereinafter referred to as IL-1RA) or interleukin-1α (hereinafter referred to as IL-1α) in the stratum corneum. It is characterized by using the expression level as an index.
The blood flow in the present invention is the blood flow in the capillary of the skin measured with a laser Doppler blood flow meter. It was found that the blood flow measured with a laser Doppler blood flow meter correlates with the expression level of IL-1RA or IL-1α in the skin stratum corneum. Therefore, by measuring the expression level of IL-1RA or IL-1α in the skin stratum corneum, it is possible to indirectly evaluate the skin blood flow rate or the skin blood flow state.
ＩＬ−１ＲＡは単球やマクロファージ等で産生される抗炎症性のサイトカインであり、炎症時に増加し、皮膚を正常化させる。ＩＬ−１ＲＡはインターロイキン−１の働きを拮抗的に阻害する。細胞レベルでＩＬ−１ＲＡが増加すると、コラーゲンを分解し、断片化する酵素であるマトリックスメタロプロテアーゼ−１が減少する。 IL-1RA is an anti-inflammatory cytokine produced by monocytes, macrophages, etc., and increases during inflammation to normalize the skin. IL-1RA antagonistically inhibits the function of interleukin-1. When IL-1RA increases at the cellular level, matrix metalloproteinase-1, an enzyme that degrades and fragments collagen, decreases.
ＩＬ−１αは単球やマクロファージ等で産生される炎症性のサイトカインであり、リンパ球やマクロファージを活性化し、炎症を引き起こす。ＩＬ−１ＲＡはインターロイキン−１の働きを拮抗的に阻害する。ＩＬ−１αが増加すると、コラーゲンを分解し、断片化する酵素であるマトリックスメタロプロテアーゼ−１が増加することが知られている。 IL-1α is an inflammatory cytokine produced by monocytes, macrophages and the like, and activates lymphocytes and macrophages to cause inflammation. IL-1RA antagonistically inhibits the function of interleukin-1. It is known that when IL-1α increases, matrix metalloproteinase-1, which is an enzyme that degrades and fragments collagen, increases.
角層は、皮膚の一番上にある組織であり、体の外からの異物や刺激から皮膚を守る働きを有している。
本発明における測定又は評価対象部位は、角層が入手できる部分であれば、いかなる部位をも包含しうるが、主な部位としては顔面、頚部、上腕部を挙げることができる。従来の方法に従い、これらの部位の皮膚由来の角層を得ることができる。しかし、前述のように、外科的に皮膚を摘出する等の方法は、ユーザーに負担を与えるため、テープストリッピング、擦過等の簡便に角層を得られる方法が好ましい。特にテープストリッピングが好ましい。
The stratum corneum is the tissue on the top of the skin and has a function of protecting the skin from foreign substances and irritation from outside the body.
The site to be measured or evaluated in the present invention may include any site as long as the stratum corneum is available, but examples of the main site include the face, neck, and upper arm. According to the conventional method, the stratum corneum derived from the skin at these sites can be obtained. However, as described above, the method of surgically removing the skin places a burden on the user, and therefore, a method that can easily obtain the stratum corneum, such as tape stripping and rubbing, is preferable. Tape stripping is particularly preferable.
こうして用意した各試料におけるＩＬ−１ＲＡ又はＩＬ−１αの発現量は、従来から知られている方法で測定することができる。例えば、ＩＬ−１ＲＡ又はＩＬ−１αに対する抗体との反応に基づくエンザイムイムノアッセイ、ラジオイムノアッセイ、ウエスタンブロッティング等の方法を用いることができる。このような測定手段はすでに市販されており、代表的なＩＬ−１ＲＡのエンザイムイムノアッセイキットとしてR&D Systems製のHuman IL-1ＲＡ/IL-1F3 Quantikine ELISA KitやLife Technologies社製のIL-1ＲＡ Human ELISA Kitを例示することができる。
また代表的なＩＬ−１αのエンザイムイムノアッセイキットとしてR&D Systems製のHuman IL-1α/IL-1F1 Quantikine ELISA KitやThermo Scientific社製のHuman IL-1 alpha ELISA Kitを例示することができる。
各試料からＩＬ−１ＲＡ、ＩＬ−１αをそれ自体既知の生化学的方法、たとえば凍結融解法、超音波破砕法、ホモジュネート法等により可溶性画分として抽出する。抽出後、速やかに測定する。
The expression level of IL-1RA or IL-1α in each sample thus prepared can be measured by a conventionally known method. For example, methods such as enzyme immunoassay, radioimmunoassay, Western blotting and the like based on a reaction with an antibody against IL-1RA or IL-1α can be used. Such measuring means are already commercially available, and as representative IL-1RA enzyme immunoassay kits, Human IL-1RA / IL-1F3 Quantikine ELISA Kit manufactured by R & D Systems and IL-1RA Human ELISA Kit manufactured by Life Technologies Can be illustrated.
Examples of typical IL-1α enzyme immunoassay kits include Human IL-1α / IL-1F1 Quantikine ELISA Kit manufactured by R & D Systems and Human IL-1 alpha ELISA Kit manufactured by Thermo Scientific.
From each sample, IL-1RA and IL-1α are extracted as a soluble fraction by a biochemical method known per se, for example, a freeze-thaw method, an ultrasonic disruption method, a homodulate method, or the like. Measure immediately after extraction.
皮膚の血流量が多い人の角層におけるＩＬ−１ＲＡの発現量が皮膚の血流量が少ない人の角層におけるＩＬ−１ＲＡの発現量と比べて高いため、角層におけるＩＬ−１ＲＡの発現量の多寡を指標として、簡便に皮膚の血流量の多寡を測定又は評価できる。
また、皮膚の血流量の多い人の角層におけるＩＬ−１αの発現量が皮膚の血流量が少ない人の角層におけるＩＬ−１αの発現量と比べて低いため、角層におけるＩＬ−１αの発現量の多寡を指標として、簡便に皮膚の血流量の多寡を評価できる。
本発明に用いる角層は、テープストリッピング法といった角層の表層部分のみを角質テープで採取する簡単な方法で採取することができる。このため、被験者に負担を与えることなく、被験者の皮膚の血流量の測定又は皮膚の血流の状態を評価することができる。
The expression level of IL-1RA in the stratum corneum is higher because the expression level of IL-1RA in the stratum corneum of a person with high skin blood flow is higher than the expression level of IL-1RA in the stratum corneum of a person with low skin blood flow. The amount of blood flow in the skin can be easily measured or evaluated using the amount of blood as an index.
In addition, since the expression level of IL-1α in the stratum corneum of a person with high skin blood flow is lower than the expression level of IL-1α in the stratum corneum of a person with low skin blood flow, IL-1α in the stratum corneum The amount of blood flow in the skin can be easily evaluated using the amount of expression as an index.
The stratum corneum used in the present invention can be collected by a simple method of collecting only the surface layer portion of the stratum corneum with a keratin tape, such as a tape stripping method. For this reason, it is possible to measure the blood flow rate of the subject's skin or evaluate the blood flow state of the skin without imposing a burden on the subject.
本発明の皮膚血流量の測定方法は、角層を採取する採取工程と、前記採取工程で採取された角層におけるＩＬ−１ＲＡ、ＩＬ−１αの発現量を測定する測定工程と、前記測定工程で測定されたＩＬ−１ＲＡ、ＩＬ−１αの発現量を予め測定しておいた集団の角層におけるＩＬ−１ＲＡ、ＩＬ−１αの発現量分布から求めた血流量の相関を求めた回帰式により皮膚血流量に換算する工程からなる。 The method for measuring skin blood flow according to the present invention includes a sampling step of sampling the stratum corneum, a measuring step of measuring expression levels of IL-1RA and IL-1α in the stratum corneum sampled in the sampling step, and the measuring step The regression equation which calculated | required the correlation of the blood flow rate calculated | required from the expression level distribution of IL-1RA and IL-1 alpha in the stratum corneum of the group which measured the expression level of IL-1RA and IL-1 alpha measured in advance. It consists of the process of converting into skin blood flow.
本発明の皮膚血流量の評価方法は、角層を採取する採取工程と、前記採取工程で採取された角層におけるＩＬ−１ＲＡ、ＩＬ−１αの発現量を測定する測定工程と、前記測定工程で測定されたＩＬ−１ＲＡ、ＩＬ−１αの発現量を予め測定しておいた集団の角層におけるＩＬ−１ＲＡ、ＩＬ−１αの発現量分布と比較する比較工程から構成される。被験者のＩＬ−１ＲＡの発現量が予め測定しておいた集団の角層におけるＩＬ−１ＲＡの発現量分布と比べて、発現量が多ければ、皮膚の血流量が大きいと判定され、発現量が少なければ、皮膚の血流量が小さいと判定される。被験者のＩＬ−１αの発現量が予め測定しておいた集団の角層におけるＩＬ−１αの発現量分布と比べて、発現量が多ければ、皮膚の血流量が小さいと判定され、発現量が少なければ、皮膚の血流量が大きいと判定される。 The method for evaluating skin blood flow according to the present invention includes a collecting step of collecting a stratum corneum, a measuring step of measuring expression levels of IL-1RA and IL-1α in the stratum corneum collected in the collecting step, and the measuring step. The expression level of IL-1RA and IL-1α measured in (1) is compared with the expression level distribution of IL-1RA and IL-1α in the stratum corneum of the population that has been measured in advance. If the expression level is large compared to the IL-1RA expression level distribution in the stratum corneum of the population in which the expression level of the IL-1RA of the subject has been measured in advance, the skin blood flow is determined to be large, and the expression level is If it is less, it is determined that the blood flow in the skin is small. If the expression level is large compared to the IL-1α expression level distribution in the stratum corneum of the population in which the expression level of IL-1α of the subject has been measured in advance, it is determined that the skin blood flow is small, and the expression level is If it is less, it is determined that the blood flow in the skin is large.
以下、具体的な実施例について説明するが、本発明は下記の実施例に限定されるものではない。 Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.
試験例１
被験集団の皮膚の血流量の測定結果とＩＬ−１ＲＡの発現量測定結果の分布と相関
＜被験者＞
健常人の女性７５名の頬の角層をテープストリッピング法により採取した。
＜角層サンプル抽出方法、タンパク質定量＞
被験者の頬に角層チェッカー(アサヒバイオメッド社製)を貼り付け、角層を採取した。角層チェッカーからビーズ法（容器に検体と直径約2mmのガラスビーズと抽出液を入れて振盪抽出する方法）にてT-PER抽出バッファー（Tissue Protein ExtRAction Reagent Thermo Scientific製（product# 78510）））５００μｌを用いて角層タンパク質の抽出を行った。各サンプルのタンパク質量はPierce BCA protein Assay Kit (Thermo Scientific #23225)で測定した。測定には前記角層タンパク質抽出液10μlにPierce BCA protein Assay Kitの反応試薬液２００μｌを加え、６０℃３０分でインキュベーションしたのち、５６２ｎｍの吸光度を測定した。同時にＢＳＡ（ウシ血清アルブミン）で検量線をひき、吸光度の値からタンパク質量を算出した。
Test example 1
Distribution and correlation of measurement results of skin blood flow of test population and measurement results of expression level of IL-1RA <subject>
The stratum corneum of the cheeks of 75 healthy women was collected by the tape stripping method.
<Extracting method of stratum corneum sample, protein quantification>
A stratum corneum checker (manufactured by Asahi Biomed) was attached to the cheek of the subject, and the stratum corneum was collected. T-PER Extraction Buffer (Tissue Protein ExtRAction Reagent Thermo Scientific (product # 78510)) using the stratum corneum checker with the bead method (a method in which a specimen, a glass bead with a diameter of about 2 mm and an extract are placed in a container and shake extracted) The stratum corneum protein was extracted using 500 μl. The amount of protein in each sample was measured with Pierce BCA protein Assay Kit (Thermo Scientific # 23225). For the measurement, 200 μl of the reaction reagent solution of Pierce BCA protein Assay Kit was added to 10 μl of the stratum corneum protein extract, incubated at 60 ° C. for 30 minutes, and then the absorbance at 562 nm was measured. At the same time, a calibration curve was drawn with BSA (bovine serum albumin), and the amount of protein was calculated from the absorbance value.
＜角層中のＩＬ−１ＲＡの発現量測定＞
同様にテープから抽出した角層タンパク質抽出液中のＩＬ−１ＲＡの発現量をR&D SystemsのELISAキット［Human IL-1ＲＡ/IL-1F3 Quantikine ELISA Kit］を用いて測定した。まず、costar製の９６wellプレート（#3590）に1次抗体となる固相化抗体をキット指定の濃度で20℃、overnightで固相化した。次に１％ＢＳＡ、３７℃１時間でプレートをブロッキングし、０．０１％ Tween-PBSでプレートを洗浄したのち、検量線となるスタンダードおよびサンプルを100μlずつアプライし、２５℃で２時間インキュベーションした。０．０１％ Tween-PBSでプレートを洗浄したのち、２次抗体をキット指定濃度で２５℃、２時間インキュベーションした。さらに、０．０１％ Tween-PBSでプレートを洗浄したのち、Streptavidin-HRPをキット指定濃度で２５℃、３０分インキュベーションした。最後に、０．０１％ Tween-PBSでプレートを洗浄したのち、発色基質であるTMB solution (Promega, G7431))を１００μｌ／ｗｅｌｌで加え、キット指定の時間の後、stop solution (０．５Ｍ 硫酸)を１００μｌ加え、４５０ｎｍの吸光度を測定し、ＩＬ−１ＲＡの発現量を算出した。
<Measurement of expression level of IL-1RA in stratum corneum>
Similarly, the expression level of IL-1RA in the stratum corneum protein extract extracted from the tape was measured using R & D Systems ELISA kit [Human IL-1RA / IL-1F3 Quantikine ELISA Kit]. First, a solid phased antibody to be a primary antibody was immobilized on a costar 96-well plate (# 3590) at 20 ° C. and overnight at a concentration specified by the kit. Next, the plate was blocked with 1% BSA at 37 ° C. for 1 hour, and the plate was washed with 0.01% Tween-PBS. Then, 100 μl each of the standard and sample used as a calibration curve were applied and incubated at 25 ° C. for 2 hours. . After the plate was washed with 0.01% Tween-PBS, the secondary antibody was incubated at a designated concentration of the kit at 25 ° C. for 2 hours. Furthermore, after the plate was washed with 0.01% Tween-PBS, Streptavidin-HRP was incubated at 25 ° C. for 30 minutes at a kit-specified concentration. Finally, after the plate was washed with 0.01% Tween-PBS, the chromogenic substrate TMB solution (Promega, G7431)) was added at 100 μl / well, and after the time specified in the kit, stop solution (0.5 M sulfuric acid) was added. ) Was added, the absorbance at 450 nm was measured, and the expression level of IL-1RA was calculated.
＜皮膚の血流量の測定＞
レーザードップラー血流量計（Lisca Ab製PIMII Laser Doppler Perfusion Imager）を用いて、被験者の頬の血流量に比例する相対出力量（Ｖ）を測定した。
<Measurement of blood flow in skin>
Using a laser Doppler blood flow meter (PIMII Laser Doppler Perfusion Imager manufactured by Lisca Ab), the relative output (V) proportional to the blood flow of the subject's cheek was measured.
＜結果＞
測定結果を、Ｘ軸をＩＬ−１ＲＡ発現量、Ｙ軸を血流量に比例する相対出力量（Ｖ）として散布図を作成し、この撒布データをもとに直線回帰式を求めた。散布図及び回帰直線、相関係数、相関式を図１に示す。
Ｘ軸をＩＬ−１ＲＡ値、Ｙ軸を血流量に比例する相対出力量（Ｖ）としたとき、回帰式はＹ ＝ ０．００５１１０ ＋ ０．４４３４、Ｒ2＝ ０．０７０３、相関係数０．２７の直線回帰を示した。
図１に示す回帰直線又は回帰式から、測定したＩＬ−１ＲＡを血流量に比例する相対出力量（Ｖ）に換算することができた。また散布図から標準的な皮膚血流量に比例する相対出力量（Ｖ）の分布が判明した。この散布図及び回帰式に基づけば、皮膚角層中のＩＬ−１ＲＡの発現量を測定することで皮膚の血流量が明らかとなり、また分布から被験者の血流量が正常か否かを評価できた。
<Result>
A scatter diagram was created with the measurement results as the IL-1RA expression level on the X axis and the relative output level (V) proportional to the blood flow on the Y axis, and a linear regression equation was obtained based on this distribution data. A scatter diagram, regression line, correlation coefficient, and correlation equation are shown in FIG.
The X-axis IL-1RA value, when the Y-axis and the relative output amount proportional to the blood flow (V), the regression equation Y = 0.005110 + 0.4434, R 2 = 0.0703, the correlation coefficient 0 A linear regression of .27 was shown.
From the regression line or regression equation shown in FIG. 1, the measured IL-1RA could be converted into a relative output (V) proportional to the blood flow. In addition, the distribution of relative output (V) proportional to the standard skin blood flow was found from the scatter plot. Based on this scatter diagram and regression equation, the blood flow volume of the skin was clarified by measuring the expression level of IL-1RA in the horny layer of the skin, and the blood flow volume of the subject could be evaluated from the distribution. .
試験例２．
被験集団の皮膚の血流量に比例する相対出力量（Ｖ）の測定結果とＩＬ−１αの発現量測定結果の分布と相関
＜被験者＞
健常人の女性７３名の頬の角層をテープストリッピング法により採取した。
＜角層サンプル抽出方法、タンパク質定量＞
ＩＬ−１ＲＡの測定と同様に測定した。
Test Example 2
Correlation between measurement results of relative output (V) proportional to skin blood flow of test population and distribution of IL-1α expression measurement results <Subject>
The stratum corneum of the cheeks of 73 healthy women was collected by a tape stripping method.
<Extracting method of stratum corneum sample, protein quantification>
It measured similarly to the measurement of IL-1RA.
＜角層中のＩＬ−１αの測定＞
試験例１同様にテープから抽出した角層タンパク質抽出液中のＩＬ−１αの発現量をR&D SystemsのＥＬＩＳＡキット［Human IL-1α/IL-1F1 Quantikine ELISA Kit］を用いて測定した。まず、costar製の９６ｗｅｌｌプレート（#3590）に1次抗体となる固相化抗体をキット指定の濃度で20℃、overnightで固相化した。次に１％ＢＳＡで３７℃１時間でプレートをブロッキングし、0.01% Tween-PBSでプレートを洗浄したのち、検量線となるスタンダードおよびサンプルを１００μｌずつアプライし、２５℃で２時間インキュベーションした。０．０１％ Tween-PBSでプレートを洗浄したのち、２次抗体をキット指定濃度で２５℃、２時間インキュベーションした。さらに、０．０１％ Ｔｗｅｅｎ−ＰＢＳでプレートを洗浄した後、Streptavidin-HRPをキット指定濃度で25℃、30分インキュベーションした。最後に、0.01% Tween-PBSでプレートを洗浄したのち、発色基質であるTMB solution (Promega, G7431))を１００μｌ／ｗｅｌｌで加え、キット指定の時間の後、stop solution (０．５Ｍ 硫酸）を１００μｌ加え、４５０ｎｍの吸光度を測定し、発現量を算出した。
<Measurement of IL-1α in stratum corneum>
In the same manner as in Test Example 1, the expression level of IL-1α in the stratum corneum protein extract extracted from the tape was measured using an ELISA kit [Human IL-1α / IL-1F1 Quantikine ELISA Kit] of R & D Systems. First, a solid phased antibody to be a primary antibody was immobilized on a costar 96-well plate (# 3590) at 20 ° C. and overnight at a concentration specified by the kit. Next, the plate was blocked with 1% BSA at 37 ° C. for 1 hour, and after washing the plate with 0.01% Tween-PBS, 100 μl each of the standard and sample to be a calibration curve was applied and incubated at 25 ° C. for 2 hours. After the plate was washed with 0.01% Tween-PBS, the secondary antibody was incubated at a designated concentration of the kit at 25 ° C. for 2 hours. Furthermore, after washing the plate with 0.01% Tween-PBS, Streptavidin-HRP was incubated at 25 ° C. for 30 minutes at a kit-specified concentration. Finally, after the plate was washed with 0.01% Tween-PBS, TMB solution (Promega, G7431)) as a chromogenic substrate was added at 100 μl / well, and after the time specified in the kit, stop solution (0.5 M sulfuric acid) was added. 100 μl was added, the absorbance at 450 nm was measured, and the expression level was calculated.
＜皮膚の血流量の測定＞
試験例１と同様にレーザードップラー血流量計（Lisca Ab製PIMII Laser Doppler Perfusion Imager）を用いて、被験者の頬の血流量に比例する相対出力量（Ｖ）を測定した。
<Measurement of blood flow in skin>
In the same manner as in Test Example 1, a laser Doppler blood flow meter (PIMII Laser Doppler Perfusion Imager manufactured by Lisca Ab) was used to measure the relative output (V) proportional to the blood flow of the subject's cheek.
＜結果＞
測定結果を、Ｘ軸をＩＬ−１α発現量、Ｙ軸を血流量として散布図を作成し、この撒布データをもとに直線回帰式を求めた。散布図及び回帰直線、相関係数、相関式を図２に示す。
Ｘ軸をＩＬ−１α値、Ｙ軸を血流量に比例する相対出力量（Ｖ）としたとき、回帰式はＹ ＝ −０．１８７３Ｘ ＋ ０．５６８１、Ｒ2 ＝ ０．０９１２、相関係数０．３０の負の直線回帰を示した。
図２に示す回帰直線又は回帰式から、測定したＩＬ−１αを血流量に換算することができた。また散布図から標準的な皮膚血流量の分布が判明した。この散布図及び回帰式に基づけば、皮膚角層中のＩＬ−１αの発現量を測定することで皮膚の血流量が明らかとなり、また分布から被験者の血流量が正常か否かを評価できた。
<Result>
The measurement results, an X-axis IL- 1 alpha expression levels, the Y-axis creates a scatter plot as blood flow was determined linear regression equation the sprayed data based. A scatter diagram, a regression line, a correlation coefficient, and a correlation equation are shown in FIG.
The X-axis IL- 1 alpha value, when the Y-axis and the relative output amount proportional to the blood flow (V), the regression equation Y = -0.1873X + 0.5681, R 2 = 0.0912, correlation A negative linear regression of number 0.30 was shown.
From the regression line or regression equation shown in FIG. 2 , the measured IL-1α could be converted into a blood flow rate. The distribution of standard skin blood flow was also found from the scatter plot. Based on this scatter diagram and regression equation, measuring the expression level of IL-1α in the stratum corneum revealed the blood flow in the skin, and the distribution was able to evaluate whether the blood flow of the subject was normal or not. .
Claims (6)
前記採取工程で採取された角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を測定する測定工程と、
前記測定工程で測定されたインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を角層のインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量と血流量の分布からあらかじめ求めた回帰式により皮膚血流量に換算する工程、
とからなる、皮膚血流量の間接測定法。 A sampling process for sampling the stratum corneum;
A measuring step of measuring the expression level of interleukin-1 receptor antagonist or interleukin-1α in the stratum corneum collected in the collecting step;
The interleukin-1 receptor antagonist was measured by the measuring step, or interleukin interleukin-1 receptor antagonist expression levels corneum Kin 1 alpha, or advance the expression levels and blood flow distribution of interleukin 1 alpha step of converting the skin blood flow by the determined regression equation,
An indirect method for measuring skin blood flow.
前記採取工程で採取された角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を測定する測定工程と、
前記測定工程で測定されたインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量を、あらかじめ測定しておいた集団の、角層におけるインターロイキン−１レセプターアンタゴニスト、又はインターロイキン−１αの発現量と血流量に基づいて、あらかじめ作成した分布図と比較する比較工程、
とから構成される、皮膚血流の評価方法。 A sampling process for sampling the stratum corneum;
A measuring step of measuring the expression level of interleukin-1 receptor antagonist or interleukin-1α in the stratum corneum collected in the collecting step;
Said measuring step interleukin-1 receptor antagonist was measured in, or the expression level of interleukin 1 alpha, populations advance measurement, interleukin-1 receptor antagonist in the horny layer, or interleukin-1 alpha A comparison process to compare with a distribution map created in advance based on the expression level and blood flow volume,
A method for evaluating skin blood flow.
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| JP2014002663A JP5731678B1 (en) | 2014-01-09 | 2014-01-09 | Blood flow evaluation or indirect measurement method |
| CN201410563850.2A CN104777306B (en) | 2014-01-09 | 2014-10-21 | The evaluation of blood flow or indirect determination method |
| TW103138015A TWI638990B (en) | 2014-01-09 | 2014-11-03 | Evaluating method or indirect measuring method for blood flow |
| HK15110139.4A HK1209483A1 (en) | 2014-01-09 | 2015-10-15 | Evaluating method or indirect measuring method for blood flow |
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| JP2014002663A JP5731678B1 (en) | 2014-01-09 | 2014-01-09 | Blood flow evaluation or indirect measurement method |
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| JP (1) | JP5731678B1 (en) |
| CN (1) | CN104777306B (en) |
| HK (1) | HK1209483A1 (en) |
| TW (1) | TWI638990B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10216106A (en) * | 1997-02-07 | 1998-08-18 | Shiseido Co Ltd | Method for evaluating skin texture |
| JP2001112742A (en) * | 1999-10-21 | 2001-04-24 | Kao Corp | Method and apparatus for evaluating skin condition |
| WO2005077458A1 (en) * | 2004-02-13 | 2005-08-25 | Fancl Corporation | Method of chemiluminescence-utilizing makeup and beautification, luminant for skin irradiation beautification and makeup/ beautification equipment |
| WO2011087523A1 (en) * | 2010-01-17 | 2011-07-21 | The Procter & Gamble Company | Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin |
| JP2012024027A (en) * | 2010-07-23 | 2012-02-09 | Nippon Menaade Keshohin Kk | Method for evaluating cutaneous blood vessel function |
| JP2014041043A (en) * | 2012-08-22 | 2014-03-06 | Rohto Pharmaceut Co Ltd | Skin condition evaluation method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851544A (en) * | 1997-12-18 | 1998-12-22 | Chesebrough-Pond's Usa Co., Division Of Conopco, Inc. | Cosmetic skin or hair care compositions containing fluorocarbons infused with carbon dioxide |
| JPH11285476A (en) * | 1998-04-01 | 1999-10-19 | Advance Co Ltd | Method of measuring blood flow and rheometer |
| US20140057955A1 (en) * | 2011-03-30 | 2014-02-27 | The Johns Hopkins University | Novel, protective, anti-inflammatory receptor and its use in preservation of mitochondrial function, wound healing and repair |
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- 2014-01-09 JP JP2014002663A patent/JP5731678B1/en active Active
- 2014-10-21 CN CN201410563850.2A patent/CN104777306B/en not_active Expired - Fee Related
- 2014-11-03 TW TW103138015A patent/TWI638990B/en not_active IP Right Cessation
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2015
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10216106A (en) * | 1997-02-07 | 1998-08-18 | Shiseido Co Ltd | Method for evaluating skin texture |
| JP2001112742A (en) * | 1999-10-21 | 2001-04-24 | Kao Corp | Method and apparatus for evaluating skin condition |
| WO2005077458A1 (en) * | 2004-02-13 | 2005-08-25 | Fancl Corporation | Method of chemiluminescence-utilizing makeup and beautification, luminant for skin irradiation beautification and makeup/ beautification equipment |
| WO2011087523A1 (en) * | 2010-01-17 | 2011-07-21 | The Procter & Gamble Company | Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin |
| JP2012024027A (en) * | 2010-07-23 | 2012-02-09 | Nippon Menaade Keshohin Kk | Method for evaluating cutaneous blood vessel function |
| JP2014041043A (en) * | 2012-08-22 | 2014-03-06 | Rohto Pharmaceut Co Ltd | Skin condition evaluation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104777306A (en) | 2015-07-15 |
| JP2015132482A (en) | 2015-07-23 |
| TWI638990B (en) | 2018-10-21 |
| TW201527737A (en) | 2015-07-16 |
| HK1209483A1 (en) | 2016-04-01 |
| CN104777306B (en) | 2018-05-01 |
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