JP2014163715A - Method for evaluating resistance to ultraviolet damage - Google Patents
Method for evaluating resistance to ultraviolet damage Download PDFInfo
- Publication number
- JP2014163715A JP2014163715A JP2013032745A JP2013032745A JP2014163715A JP 2014163715 A JP2014163715 A JP 2014163715A JP 2013032745 A JP2013032745 A JP 2013032745A JP 2013032745 A JP2013032745 A JP 2013032745A JP 2014163715 A JP2014163715 A JP 2014163715A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- stratum corneum
- skin
- expression level
- ultraviolet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
本発明は、紫外線傷害に対する抵抗性の評価方法に関する。 The present invention relates to a method for evaluating resistance to ultraviolet damage.
紫外線傷害は照射された紫外線がメラニンの保護能力を超える時に起こる。紫外線傷害の現象には２種類あり、紫外線にあたった直後には発症せず、２〜６時間後皮膚が赤くなり、６〜４８時間後に痛みが最もひどくなるサンバーン（紅斑：ｓｕｎｂｕｒｎ）と、２４〜７２時間の間、色素沈着が進行するサンタン(ｓｕｎｔａｎ)がある。サンバーンはＵＶＢが表皮を透過し、真皮乳頭体まで達した結果、乳頭体内の毛細血管が炎症反応として充血を起こし、皮膚の色が赤くなった状態（紅斑）を指す。紫外線量がメラニン色素の防御反応を超えると、細胞組織が損傷を受け、発熱や水泡、痛みが起きる。これを日光皮膚炎という。 UV injury occurs when irradiated UV rays exceed the protective ability of melanin. There are two types of UV injury phenomenon, sunburn (erythema: sunburn) that does not develop immediately after exposure to UV rays, the skin becomes red after 2 to 6 hours, and the pain is most severe after 6 to 48 hours. There is a suntan where the pigmentation proceeds for ~ 72 hours. Sunburn refers to a state (erythema) in which the color of the skin becomes red as a result of UVB penetrating the epidermis and reaching the dermal papillae, causing the capillaries in the papillary body to become engorged as an inflammatory reaction. When the amount of ultraviolet light exceeds the protective response of melanin, cellular tissue is damaged, causing fever, blisters, and pain. This is called sunlight dermatitis.
また、サンタンはＵＶＡがメラノサイトに働きかけ、メラニン色素の生成を促す。ＵＶＡは発赤や炎症を伴うことは無いが、皮膚の新陳代謝を遅らせるためシミを発生する。
これら紫外線傷害の元となっている紫外線には皮膚に対する発ガン作用、白内障等の眼疾患への影響、免疫機能への影響など多くの健康への影響が懸念されている。皮膚においても紫外線にさらされると、様々な紫外線傷害を引き起こす。紫外線は皮膚に炎症を起こしたり、皮膚の不飽和脂質を酸化して脂質ラジカルを発生させ、過酸化脂質を生成する。この様な紫外線による炎症や酸化は組織、細胞、ＤＮＡの傷害を引き起こす。皮膚の紫外線曝露による現象としては一般的に、シワ、シミ、皮膚癌等が挙げられる。
Suntan also causes UVA to act on melanocytes and promote the production of melanin pigments. UVA does not cause redness or inflammation, but it causes spots to delay the metabolism of the skin.
There are concerns about many health effects such as carcinogenic effects on the skin, effects on eye diseases such as cataracts, effects on immune functions, and the like, which are the sources of these UV injuries. Exposure to UV rays in the skin also causes various UV injuries. Ultraviolet rays cause inflammation in the skin, or oxidize unsaturated lipids in the skin to generate lipid radicals, thereby producing lipid peroxides. Such inflammation and oxidation caused by ultraviolet rays cause tissue, cell and DNA damage. Phenomena caused by UV exposure of skin generally include wrinkles, spots, skin cancer and the like.
近年、疫学的なデータとして紫外線に対する感受性の違い（肌の色、スキンタイプ等）により皮膚癌のリスクが高まるとの報告もある。さらに皮膚癌の一種である悪性黒色腫（メラノーマ）の危険リスク度はｍｅｌａｎｏｃｏｒｔｉｎ１ ｒｅｃｅｐｔｏｅｒの１塩基置換の遺伝子変異によりリスクが数倍高くなることも明らかとなっている。紫外線に対する感受性はスキンタイプ（紫外線傷害を受けた際のサンバーン、サンタンの状態の違いにより分類）によって大きく異なり、６段階に分類するタイプ別にわけたり、赤み（紅斑）を生じる最小紫外線エネルギー（最小紅斑量：ＭＥＤ)を目安に行われたりするが、前者は主観的観測、また後者は紫外線を当てなくては測定できないというリスクが伴う。オゾン層破壊などにより以前にも増して紫外線増加が問題になっており、個人に対する紫外線への感受性リスク評価も適切に行われる必要がある。 In recent years, as epidemiological data, there is a report that the risk of skin cancer increases due to differences in sensitivity to ultraviolet rays (skin color, skin type, etc.). Furthermore, it has also been clarified that the risk level of malignant melanoma (melanoma), which is a type of skin cancer, is several times higher due to a single base substitution gene mutation of melanocortin1 receptor. Sensitivity to ultraviolet rays varies greatly depending on the skin type (classified according to the state of sunburn and suntan at the time of UV injury), divided into 6 types, and the minimum ultraviolet energy that causes redness (erythema) (minimum erythema) Amount: MED) is used as a guide, but there is a risk that the former cannot be measured without subjective observation, and the latter with ultraviolet light. The increase in ultraviolet rays has become a problem more than before due to ozone layer destruction, etc., and it is necessary to appropriately evaluate the sensitivity risk to individuals for ultraviolet rays.
一方、一般的にシミと言われている色素沈着ができる初期段階においては、皮膚表面に顕在化する前に様々な刺激により表皮角化細胞がα−ＭＳＨ、ｂＦＧＦ、ＣＧＲＰ、エンドセリンなどの情報伝達物質を生成し、メラノサイトに伝達される。メラノサイトに伝達された後、細胞内においてメラニン色素が大量に作られる。さらに紫外線によってターンオーバーが異常となり、大量に作られたメラニン色素が排出できないため色素性病変へと変化する。ｉｎ ｖｉｔｒｏ系を用いた実験においてもメラノサイトにＵＶを照射した場合、細胞の増殖因子マーカーであるｐ７３、Ｎｕｐ８８、ｐ２７、Ｉｄ１、ＰＣＮＡが、またアポトーシス関連蛋白質であるｂｃｌ−２の発現が増減したという報告がある（非特許文献１）。 On the other hand, in the initial stage where pigmentation, generally referred to as a stain, is possible, epidermal keratinocytes transmit information such as α-MSH, bFGF, CGRP, endothelin, etc. by various stimuli before appearing on the skin surface. A substance is produced and transmitted to melanocytes. After being transmitted to melanocytes, a large amount of melanin is made in the cell. Furthermore, the turnover becomes abnormal due to ultraviolet rays, and the melanin pigment produced in large quantities cannot be excreted, which changes to pigmented lesions. In experiments using an in vitro system, when melanocytes were irradiated with UV, the expression of cell growth factor markers p73, Nup88, p27, Id1, and PCNA and the expression of bcl-2, an apoptosis-related protein, increased or decreased. There is a report (Non-Patent Document 1).
これまでに、バイオプシーによる皮膚組織を用いて侵襲的にシミ部と正常部を比較し、シミ部においてはＮＴ−３、ＡＤＡＭ９、ＨＢ−ＥＧＦといった蛋白質の発現が顕著に増加したという報告がある（特許文献１）。さらに、皮膚のシミ形成を予知する検査方法として、バイオプシーによってＭＬＳＴＤ１、ＭＯＧＡＴ１、Ｍｃｐ９、Ｋｒｔ２−６ｂ等のｍＲＮＡ量を測定し、しみ形成のリスクを判断できるという報告がある（特許文献２、３、４）。また、非侵襲的な方法としてテープストリッピングもしくは擦過を介して採取した皮膚に由来する角層を試料として、インターロイキン１（ＩＬ−１）とインターロイキン１レセプターアンタゴニスト（ＩＬ−１ｒａ）の存在量分析をし、肌質の評価をする方法が特許文献５に開示されている。 So far, there has been a report that the skin part by biopsy is used to compare the spot part and the normal part invasively, and the expression of proteins such as NT-3, ADAM9, HB-EGF is remarkably increased in the spot part ( Patent Document 1). Furthermore, as a test method for predicting skin spot formation, there is a report that the amount of mRNA such as MLSTD1, MOGAT1, Mcp9, Krt2-6b, etc. can be measured by biopsy to determine the risk of spot formation (Patent Documents 2, 3, 4). In addition, as a non-invasive method, the abundance analysis of interleukin 1 (IL-1) and interleukin 1 receptor antagonist (IL-1ra) is performed using a stratum corneum derived from the skin collected through tape stripping or abrasion as a sample. A method for evaluating skin quality is disclosed in Patent Document 5.
また本出願人は、非侵襲的な手段を用いて採取した皮膚角層からアネキシンII、ブレオマイシン ハイドロラーゼ、カテプシンＤ、アルギナーゼ−１、ＳＣＣＡ２を検出し、皮膚サンバーンに対する抵抗性の指標とする技術を特許出願した（特許文献６）。また、非侵襲的な手段を用いて採取した皮膚角層からブレオマイシン ハイドロラーゼ、 アネキシンII、カテプシンＤ、アルギナーゼ−１、β−アクチン、ケラチン１４、ケラチン１、ＳＣＣＡ２を検出し、色素沈着傾向の指標とする技術を特許出願した（特許文献７）。 The present applicant also has a technique for detecting annexin II, bleomycin hydrolase, cathepsin D, arginase-1, and SCCA2 from the stratum corneum collected using non-invasive means and using it as an index of resistance to skin sunburn. A patent application was filed (Patent Document 6). In addition, bleomycin hydrolase, annexin II, cathepsin D, arginase-1, β-actin, keratin 14, keratin 1 and SCCA2 are detected from the stratum corneum collected using non-invasive means, and an index of pigmentation tendency A patent application was filed for this technology (Patent Document 7).
さらにまた、本出願人は、抗酸化作用を有するＤＪ−１タンパク質（別名ＰＡＲＫ-７タンパク質）がアトピー性皮膚炎マーカーとして有用であることを見出して特許出願した（特許文献８）。加えて、近年このＤＪ−１タンパク質が各種疾患のマーカーとして利用可能であることが徐々に明らかになってきている。特許文献９には酸化型ＤＪ−１タンパク質を特異的に認識する抗体が開示されている。そして、この抗体を用いて酸化型ＤＪ−１を測定することでアルツハイマー病やパーキンソン病を診断する方法が提案されている。また特許文献１０には、脳脊髄液や血液などのＤＪ−１タンパク質を測定することで脳傷害性の疾患を診断する方法が開示されている。また特許文献１１には、ＤＪ−１タンパク質を測定することで糖尿病性網膜症を診断する方法と試薬が提案されている。 Furthermore, the present applicant has found that DJ-1 protein (also known as PARK-7 protein) having an antioxidant action is useful as a marker for atopic dermatitis, and has filed a patent application (Patent Document 8). In addition, in recent years, it has gradually become clear that this DJ-1 protein can be used as a marker for various diseases. Patent Document 9 discloses an antibody that specifically recognizes oxidized DJ-1 protein. And the method of diagnosing Alzheimer's disease and Parkinson's disease by measuring oxidized DJ-1 using this antibody is proposed. Patent Document 10 discloses a method for diagnosing a brain-damaging disease by measuring DJ-1 proteins such as cerebrospinal fluid and blood. Patent Document 11 proposes a method and a reagent for diagnosing diabetic retinopathy by measuring DJ-1 protein.
本発明は、非侵襲的な手段を用いて採取した皮膚角層を用いて、紫外線傷害に対する抵抗性の評価指標を提供することを課題とする。尚、本発明で言う紫外線傷害に対する抵抗性とは、紫外線曝露による発赤が１５日以内に回復し、また、皮膚の明度も併せて回復することである。 This invention makes it a subject to provide the evaluation parameter | index of the resistance with respect to an ultraviolet-ray injury using the skin stratum corneum extract | collected using the noninvasive means. The term “resistance to UV injury” as used in the present invention means that redness due to UV exposure is recovered within 15 days, and the brightness of the skin is also recovered.
本願発明者らは、ヒトの日焼けを研究する過程で、非侵襲的な手法を用いて得られたヒト皮膚角層のＤＪ−１タンパク質を測定することで紫外線傷害に対する抵抗性を評価できることを見出した。本発明はこの研究成果に基づくものである。
（１）皮膚角層のＤＪ−１タンパク質の発現量を測定することを特徴とする、紫外線傷害に対する抵抗性の評価方法。
（２）被験者の評価対象部位の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ＤＪ−１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ＤＪ−１タンパク質の発現量を、標本集団の前記評価対象部位の角層のＤＪ−１の発現量分布と比較する比較工程と、を備えたことを特徴とする（１）記載の評価方法。
（３）皮膚の角層を採取する工程がテープストリッピング法によるものである（２）に記載の評価方法。
In the process of studying human sunburn, the present inventors have found that the resistance to ultraviolet ray injury can be evaluated by measuring the DJ-1 protein of the human skin horny layer obtained using a non-invasive technique. It was. The present invention is based on this research result.
(1) A method for evaluating resistance to UV injury, characterized by measuring the expression level of DJ-1 protein in the stratum corneum.
(2) A sampling step of sampling the stratum corneum of the subject's evaluation target, a measuring step of measuring the expression level of DJ-1 in the stratum corneum sampled in the sampling step, and DJ- measured in the measuring step The evaluation method according to (1), comprising a comparison step of comparing the expression level of 1 protein with the expression level distribution of DJ-1 in the stratum corneum of the evaluation target site of the sample population.
(3) The evaluation method according to (2), wherein the step of collecting the stratum corneum of the skin is based on a tape stripping method.
本発明により、紫外線を実際に照射することなく、ヒトの紫外線傷害に対する抵抗性を評価することができる。またバイオプシーなど人体に影響を及ぼす手法ではなく、非侵襲的なテープストリッピング手段を用いて採取した皮膚角層を用いて評価することができるので安全であり、かつ簡便である。
さらにまた本発明により、紫外線による炎症（紅斑）の回復力を評価することが可能となる。
また、紫外線による炎症の回復力が低いと、紫外線曝露による皮膚老化、シワ、シミ、さらには皮膚癌のリスクが高いと推測されるので、紫外線傷害に対する抵抗性の弱い人には、あらかじめ強力なサンスクリーン剤の使用を薦める等の美容カウンセリングを的確に行うことが可能となる。
According to the present invention, it is possible to evaluate human resistance to ultraviolet rays without actually irradiating ultraviolet rays. Moreover, it is safe and simple because it can be evaluated using a skin stratum corneum collected using a non-invasive tape stripping means rather than a technique that affects the human body such as biopsy.
Furthermore, the present invention makes it possible to evaluate the resilience of inflammation (erythema) caused by ultraviolet rays.
In addition, if the ability to recover inflammation due to ultraviolet rays is low, it is presumed that there is a high risk of skin aging, wrinkles, spots, and skin cancer due to exposure to ultraviolet rays. Beauty counseling such as recommending the use of sunscreen agents can be performed accurately.
以下、本発明の実施の形態についてより詳細に説明する。
本発明の評価方法は、皮膚角層におけるＤＪ−１タンパク質の存在量を指標とすることを特徴としている。
ＤＪ−１タンパク質とはＰＡＲＫ７とも呼ばれ、分子量１９，８９１Ｄａの細胞内タンパク質である。ＤＪ−１タンパク質はパーキンソン病に関係したタンパク質として同定されていたが、その後皮膚の創傷治癒過程での再上皮化（ｒｅ−ｅｐｉｔｈｅｌｉａｌｉｚａｔｉｏｎ）に関与することが明らかになった。また、その立体構造からプロテアーゼとしても働いているという可能性が示唆されている。
皮膚角層は、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。
Hereinafter, embodiments of the present invention will be described in more detail.
The evaluation method of the present invention is characterized by using the abundance of DJ-1 protein in the stratum corneum as an index.
DJ-1 protein is also called PARK7 and is an intracellular protein having a molecular weight of 19,891 Da. The DJ-1 protein has been identified as a protein associated with Parkinson's disease, but has subsequently been shown to be involved in re-epithelialization during the skin wound healing process. In addition, the three-dimensional structure suggests the possibility of acting as a protease.
The stratum corneum is a tissue on the top of the skin and has a function of protecting the skin from foreign substances and irritation from outside the body.
本発明における評価対象部位は、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。なお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、本発明の実施にあたっては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を採用することが好ましい。特にテープストリッピング法が好ましい。 The evaluation target site in the present invention can 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. It should be noted that a method such as surgically removing the skin places a burden on the user, and therefore it is preferable to adopt a method that can easily obtain the stratum corneum, such as tape stripping or rubbing, in the practice of the present invention. The tape stripping method is particularly preferable.
各試料のＤＪ−１タンパク質の発現量は、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、ＤＪ−１タンパク質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。 The expression level of the DJ-1 protein in each sample can be measured by a conventionally known method. For example, methods such as enzyme immunoassay, radioimmunoassay, Western blotting based on reaction with an antibody against DJ-1 protein can be used.
採取した試料からＤＪ−１タンパク質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、ホモジュネート法を用いて可溶性画分を調製する。可溶性画分を抽出後、速やかにＤＪ−１を測定する。
そしてＤＪ−１タンパク質の発現量が多い人は、紫外線傷害に対する抵抗性が強いものと評価する。
またＤＪ−１の測定結果に基づき、日焼けに注意が必要な者に屋外活動を注意するよう指導することができる。あるいは、ＤＪ−１の測定結果から、紫外線傷害の抵抗性が低いことが判明した場合は、紫外線遮蔽効果の強い化粧料を選択することなどの対処を一人一人が採ることができる。
そして、従来あいまいな基準で行われていた化粧料選択のカウンセリングを科学的な根拠をもって正確に行うことができる。
A soluble fraction is prepared from the collected sample by using a biochemical method known per se, such as freeze-thaw method, ultrasonic disruption method, and homogenate method. DJ-1 is measured immediately after extracting the soluble fraction.
And the person with much expression level of DJ-1 protein evaluates that the resistance with respect to an ultraviolet-ray injury is strong.
In addition, based on the measurement result of DJ-1, it is possible to instruct people who need attention to sunburn to be careful about outdoor activities. Alternatively, if it is found from the DJ-1 measurement results that the resistance to UV damage is low, each person can take measures such as selecting a cosmetic with a strong UV shielding effect.
And the counseling of the cosmetics selection conventionally performed by the ambiguous standard can be accurately performed with a scientific basis.
本願発明は、テープストリッピング等の非侵襲的な方法によって安全に、かつ容易に皮膚角層を採取して、その皮膚角層を用いて短時間に特定のタンパク質の発現量を測定することができる。このため、化粧品売り場等で簡単に測定して、その場で化粧料選択の情報として活用することができる。この結果に基づいて、適した化粧料を表示することもできる。 The present invention can safely and easily collect a skin stratum corneum by a noninvasive method such as tape stripping and measure the expression level of a specific protein in a short time using the skin stratum corneum. . For this reason, it can measure easily in cosmetics departments etc., and it can utilize as information on cosmetics selection on the spot. Based on this result, a suitable cosmetic can be displayed.
本発明を実施するための、皮膚角層の採取とタンパク質の抽出、ＤＪ−１タンパク質発現量の測定は次の操作により実施することができる。
（１）テープストリッピング法による角層の採取
テープストリッピング法とは皮膚に粘着テープを貼り付けて、粘着テープを剥がすことにより、粘着面に付着した角層を採取する方法である。
市販の粘着テープを用いて、例えばヒト頬部に粘着テープを押し付けることにより、角層を粘着テープに付着させて採取することができる。市販の粘着テープとしてはアサヒバイオメッド社製角質チェッカー、モリテックス社製角層シール、ＰＲＯＭＯＴＯＯＬ社製角質チェッカー（ディスクタイプＷ、ディスクタイプＧ、ＰＲＯタイプ）、Ｉｎｔｅｇｒａｌ社製Ｃｏｒｎｅｏｆｉｘ、３Ｍ社製透明テープ、３Ｍ社製透明両面テープ等が挙げられる。
The collection of the skin stratum corneum, the extraction of the protein, and the measurement of the expression level of DJ-1 protein for carrying out the present invention can be carried out by the following operations.
(1) Collection of stratum corneum by tape stripping method The tape stripping method is a method of collecting a stratum corneum adhering to the adhesive surface by applying an adhesive tape to the skin and peeling off the adhesive tape.
By using a commercially available adhesive tape, for example, by pressing the adhesive tape on the human cheek, the stratum corneum can be attached to the adhesive tape and collected. Commercially available adhesive tapes include Asahi Biomed's horny checker, Moritex's horny layer seal, PROMOTOOL's horny checker (disk type W, disk type G, PRO type), Integral Corneofix, 3M transparent tape, Examples thereof include a transparent double-sided tape manufactured by 3M.
（２）角層からのタンパク質抽出用バッファー
粘着テープに付着した角層から角層タンパク質を抽出するために抽出バッファーを用いる。抽出バッファーとして例えばPBS（０．１％SDS含有）などを例示できる。
(2) Buffer for protein extraction from stratum corneum An extraction buffer is used to extract stratum corneum protein from the stratum corneum attached to the adhesive tape. Examples of the extraction buffer include PBS (containing 0.1% SDS).
（３）角層タンパク質の抽出
角層の付着した粘着テープを円筒容器に入れて、高速回転するホモジナイゼーション用のペッスル（以下、「ペッスル」と呼ぶ）を用いて粘着面を擦ることにより迅速に角層タンパク質の抽出が可能である。粘着テープは粘着面を円筒容器内側に向けて、粘着面の反対面を円筒容器の内側に沿わせるようにして、円筒容器に入れる。粘着テープの粘着面とは反対側の面を円筒容器の内側に沿わせて入れることにより、ペッスルで容易に粘着面を擦ることができる。
あるいは角層の付着した粘着テープを抽出バッファーに浸してスクレーパーで擦って角層タンパク質を抽出しても良く、振盪法や超音波法を用いても良い。
あるいはまた、ガラスビーズを用いてボルテックスミキサーなどの装置によって振盪しながら破砕し、抽出しても良い。
(3) Extraction of stratum corneum protein Put the adhesive tape with the stratum corneum in a cylindrical container and quickly rub the adhesive surface with a high-speed rotating homogenization pestle (hereinafter referred to as “pestle”). It is possible to extract stratum corneum proteins. The adhesive tape is placed in the cylindrical container, with the adhesive surface facing the inside of the cylindrical container and the opposite surface of the adhesive surface along the inside of the cylindrical container. By putting the surface opposite to the adhesive surface of the adhesive tape along the inside of the cylindrical container, the adhesive surface can be easily rubbed with the pestle.
Alternatively, the stratum corneum protein may be extracted by immersing an adhesive tape with a stratum corneum in an extraction buffer and rubbing with a scraper, or using a shaking method or an ultrasonic method.
Alternatively, the glass beads may be crushed and extracted by shaking with a device such as a vortex mixer.
（４）角層タンパク質抽出液の回収
角層タンパク質抽出液を約１分間静置し、泡を沈静化し、その後、円筒容器から角層タンパク質抽出液を吸引して回収する。
(4) Recovery of stratum corneum protein extract The stratum corneum protein extract is allowed to stand for about 1 minute to calm the foam, and then the stratum corneum protein extract is sucked and collected from the cylindrical container.
（５）角層タンパク質中のＤＪ−１タンパク質の発現量の分析
得られた角層タンパク質抽出液中のＤＪ−１タンパク質は、免疫ブロッティング法、ELISA法、抗体チップ法、FRET法など公知の方法により定量分析することができる。
以下に実施例を示し本発明をさらに説明する。
(5) Analysis of expression level of DJ-1 protein in stratum corneum protein DJ-1 protein in the obtained stratum corneum protein extract is a known method such as immunoblotting method, ELISA method, antibody chip method, FRET method, etc. Can be quantitatively analyzed.
The following examples further illustrate the present invention.
１．紫外線照射に対する抵抗性と皮膚角層のＤＪ−１タンパク質量の変化測定
男女１９名の被験者の紫外線照射による皮膚赤みの変化、照射後の回復の程度とＤＪ−１タンパク質の関係を調べた。
＜紫外線照射及び照射前後の皮膚状態の観察＞
健常肌を有する男女19名の上腕内側部にデルマレイ（テルモ・クリニカルサプライ社製）を用いて60mJ/cm2照射（1cm×1cm）紫外線を照射した。紫外線照射前および1、5、8、15日後に分光測色計（SM-500、コニカミノルタ社製）で皮膚色を測定し、皮膚状態を観察した。
また、紫外線照射前および照射8日後に角層チェッカー（アサヒバイオメッド社）を用いてテープストリッピング法により角層を採取した。
1. Measurement of changes in resistance to ultraviolet irradiation and the amount of DJ-1 protein in the stratum corneum The relationship between DJ-1 protein and changes in skin redness due to ultraviolet irradiation, the degree of recovery after irradiation in 19 male and female subjects were examined.
<Ultraviolet irradiation and observation of skin condition before and after irradiation>
The inner side of the upper arm of 19 men and women with healthy skin was irradiated with 60 mJ / cm 2 (1 cm × 1 cm) ultraviolet rays using Delmaray (Terumo Clinical Supply). The skin color was measured with a spectrocolorimeter (SM-500, manufactured by Konica Minolta) before UV irradiation and 1, 5, 8, and 15 days later, and the skin condition was observed.
In addition, the stratum corneum was collected by a tape stripping method using a stratum corneum checker (Asahi Biomed) before ultraviolet irradiation and 8 days after the irradiation.
＜角層中のＤＪ−１タンパク質発現量の測定＞
ガラスビーズとT-PERバッファー（Thermo scientific）500μlの入ったチューブに角層を採取した角層チェッカーを入れ、25分ボルテックスミキサーにて振盪し、角層タンパク質を抽出した。各サンプルのタンパク量はBCA protein Assay Kit (Thermo Scientific)で測定した。角層抽出液中に含まれるＤＪ−１タンパク質量はHuman Park7/ＤＪ−１タンパク質 DuoSet (R&D systems社製)を用いて定量した。タンパク質測定、ＤＪ−１タンパク質の測定はキットの測定マニュアルに従った。ＤＪ−１タンパク質量はタンパク質１μｇ当たりのＤＪ−１量（ｐｇ/μｇ Protein）で表した。
<Measurement of expression level of DJ-1 protein in stratum corneum>
The stratum corneum checker which collected the stratum corneum was put into a tube containing glass beads and 500 μl of T-PER buffer (Thermo scientific), and shaken with a vortex mixer for 25 minutes to extract stratum corneum protein. The amount of protein in each sample was measured with BCA protein Assay Kit (Thermo Scientific). The amount of DJ-1 protein contained in the stratum corneum extract was quantified using Human Park7 / DJ-1 protein DuoSet (manufactured by R & D systems). Protein measurement and DJ-1 protein measurement were performed according to the kit measurement manual. The amount of DJ-1 protein was expressed as the amount of DJ-1 per 1 μg of protein (pg / μg Protein).
＜測定結果の解析＞
（１）紫外線照射に伴う赤み（発赤）変化とＤＪ−１タンパク質量の関係解析
紫外線照射15日目の赤みの指標であるΔa*値より以下の2群に分けた。
「赤みが戻らない」：15日目にΔa*値が１以上の被験者( 7名)
「赤みが戻る」 ：15日目にΔa*値が1未満の被験者(12名)
なお「赤みが戻る」と評価した被験者は紫外線抵抗性があるといえる。
<Analysis of measurement results>
(1) Analysis of relationship between redness (redness) change associated with UV irradiation and DJ-1 protein amount UVA irradiation was divided into the following two groups based on Δa * value which is an index of redness on the 15th day.
“Redness does not return”: Subjects with Δa * value of 1 or more on day 15 (7 subjects)
"Redness returns": Subjects with Δa * value less than 1 on day 15 (12 subjects)
In addition, it can be said that the test subject who evaluated "redness returns" has ultraviolet resistance.
「赤みが戻らない」群のＤＪ−１タンパク質の測定結果：
ＤＪ−１タンパク質中央値：1.60pg/μg protein、平均値：1.52 pg/μg protein
「赤みが戻る」群のＤＪ−１タンパク質の測定結果：
ＤＪ−１タンパク質中央値：2.59pg/μg protein、平均値：2.66 pg/μg protein
二群間の有意差検定を、マン・ホイットニーＵ検定法を用いて行った。
二群のＤＪ−１タンパク質の測定値には有意差があり、「赤みが戻る」群の被験者のＤＪ−１タンパク質は「赤みが戻らない」群の被験者に比較して高い値を示した。その境界値は２ｐｇ/μg protein付近であった。二群のＤＪ−１タンパク質量の比較と検定結果を図１に示した。
Measurement results for the DJ-1 protein of the group “redness does not return”:
DJ-1 protein median: 1.60 pg / μg protein, average: 1.52 pg / μg protein
Measurement results of DJ-1 protein of “redness returns” group:
DJ-1 protein median: 2.59 pg / μg protein, average: 2.66 pg / μg protein
A significant difference test between the two groups was performed using the Mann-Whitney U test.
There was a significant difference in the measured values of the two groups of DJ-1 proteins, and the DJ-1 protein of the subjects in the “redness return” group showed a higher value than the subjects in the “no redness return” group. The boundary value was around 2 pg / μg protein. The comparison of the amount of DJ-1 protein in the two groups and the test results are shown in FIG.
（２）紫外線照射後の「白さ」の回復程度による解析
15日目のΔL*値より以下の2群に分けた。
紫外線照射前より「白さが戻らない」：15日目にΔL*値が-1未満の被験者( 6名)
紫外線照射前の「白さが戻る」 ：15日目にΔL*値が-1以上の被験者(13名)
なお「白さが戻る」とした被験者は紫外線抵抗性があるといえる。
(2) Analysis by degree of recovery of “whiteness” after UV irradiation
Based on ΔL * values on day 15, they were divided into the following two groups.
“Whiteness does not return” from before UV irradiation: Subjects with ΔL * value less than -1 on day 15 (6 subjects)
“Whiteness returns” before UV irradiation: Subjects with ΔL * value of -1 or more on day 15 (13 subjects)
In addition, it can be said that the test subject who "whiteness returns" has ultraviolet-ray resistance.
「白さが戻らない」：ＤＪ−１タンパク質中央値：1.59pg/μg protein、平均値：1.46 pg/μg protein
「白さが戻る」：ＤＪ−１タンパク質中央値：2.11pg/μg protein、平均値：2.60 pg/μg protein
二群間の有意差検定を、マン・ホイットニーＵ検定法を用いて行った。
二群のＤＪ−１タンパク質の測定値には有意差があり、「白さが戻る」群の被験者のＤＪ−１タンパク質は「白さが戻らない」群の被験者に比較して高い値を示した。その境界値は２ｐｇ/μg protein付近であった。二群のＤＪ−１タンパク質量の比較と検定結果を図２に示した。
以上の１．２．の解析結果から、ＤＪ−１タンパク質の発現量は日焼けからの回復能力の指標となりうることが明らかとなった。
“White does not return”: DJ-1 protein median: 1.59 pg / μg protein, average: 1.46 pg / μg protein
"Whiteness returns": DJ-1 protein median: 2.11 pg / μg protein, average: 2.60 pg / μg protein
A significant difference test between the two groups was performed using the Mann-Whitney U test.
There is a significant difference in the measured values of the two groups of DJ-1 protein, and the DJ-1 protein of the subjects in the “whiteness returns” group shows a higher value than the subjects in the “whiteness does not return” group It was. The boundary value was around 2 pg / μg protein. The comparison of the amount of DJ-1 protein of the two groups and the test results are shown in FIG.
Above 1.2. From the analysis results, it was revealed that the expression level of DJ-1 protein can be an index of the ability to recover from sunburn.
（３）ＤＪ−１タンパク質初期値による「赤み」、「白さ」の解析
紫外線照射0日目のＤＪ−１タンパク質量（初期値）の上位30%、下位30%の被験者に分け、「赤み」と「白さ」の状態を比較解析した。「ＤＪ−１タンパク質少」に分類した被験者6名のＤＪ−１タンパク質初期値は0.65-1.59 pg/μg proteinに分布していた。
一方「ＤＪ−１タンパク質多」に分類した被験者６名のＤＪ−１タンパク質初期値は、2.87-4.85 pg/μg protein に分布していた。
紫外線照射後の皮膚の「赤み」を示すΔa*の変化を図３に示した。紫外線照射15日後には、ＤＪ−１の初期値の高い群は、「赤み」を示すΔa*値が０まで低下し、発赤から回復したことが確認された。一方、ＤＪ−１の初期値の低い群は紫外線照射１５日後もΔa*値は殆ど低下しなかった。紫外線照射１５日目のＤＪ１の高い群と低い群の赤みの差は、マン・ホイットニーのＵ検定で５％の危険率で有意であった。このことから、ＤＪ−１の初期値の高い群は、紫外線傷害からの回復力を有することが分かった。
(3) Analysis of “redness” and “whiteness” based on the initial value of DJ-1 protein The subjects were divided into the top 30% and the lowest 30% of the amount of DJ-1 protein (initial value) on the 0th day of ultraviolet irradiation. "And" whiteness "were compared and analyzed. The initial values of DJ-1 protein of 6 subjects classified as “low DJ-1 protein” were distributed in 0.65-1.59 pg / μg protein.
On the other hand, the initial value of DJ-1 protein of 6 subjects classified as “DJ-1 protein multiple” was distributed in 2.87-4.85 pg / μg protein.
The change in Δa * indicating “redness” of the skin after ultraviolet irradiation is shown in FIG. After 15 days of UV irradiation, it was confirmed that the group with a high initial value of DJ-1 had a Δa * value indicating “redness” decreased to 0 and recovered from redness. On the other hand, in the group with a low initial value of DJ-1, the Δa * value hardly decreased even 15 days after the ultraviolet irradiation. The difference in redness between the high and low DJ1 groups on the 15th day of UV irradiation was significant at a 5% risk by Mann-Whitney U test. From this, it was found that the group with a high initial value of DJ-1 has a resilience from UV damage.
紫外線照射後の皮膚の「白さ」を示すΔL*の変化を図４に示した。図４に示すとおり紫外線照射２４時間後の皮膚の「白さ」の程度はＤＪ−１の初期値の高い群がＤＪ−１の初期値の低い群に比べて高い値を示した。このことからＤＪ−１の高い群は、ＤＪ−１の低い群より紫外線抵抗性を有していることが分かった。 FIG. 4 shows the change in ΔL * indicating the “whiteness” of the skin after ultraviolet irradiation. As shown in FIG. 4, the degree of “whiteness” of the skin 24 hours after UV irradiation was higher in the group with a high initial value of DJ-1 than in the group with a low initial value of DJ-1. From this, it was found that the group with high DJ-1 has ultraviolet resistance than the group with low DJ-1.
また紫外線照射15日後には、ＤＪ−１の初期値の高い群は、「白さ」を示すΔL*値が上昇しほぼ初期値まで回復したことが確認された。一方、ＤＪ−１の初期値の低い群は紫外線照射１５日後もΔL*値は殆ど変化しなかった。紫外線照射１５日目のＤＪ−１の高い群と低い群のΔL*の差は、マン・ホイットニーのＵ検定で５％の危険率で有意であった。このことから、ＤＪ−１の初期値の高い群は、紫外線傷害からの回復力を有することが分かった。 Further, after 15 days of ultraviolet irradiation, it was confirmed that the group with a high initial value of DJ-1 increased in ΔL * value indicating “whiteness” and almost recovered to the initial value. On the other hand, in the group with a low initial value of DJ-1, the ΔL * value hardly changed even after 15 days of ultraviolet irradiation. The difference in ΔL * between the high and low DJ-1 groups on the 15th day of UV irradiation was significant at a 5% risk by Mann-Whitney U test. From this, it was found that the group with a high initial value of DJ-1 has a resilience from UV damage.
以上の紫外線照射後の皮膚の「赤み」変化、「白さ」変化とＤＪ−１初期値の解析結果から、ＤＪ−１を指標として、紫外線傷害に対する皮膚の抵抗性と回復性を評価することが可能であることが分かった。またその抵抗性の境界はＤＪ−１を指標とした場合２ｐｇ/μg protein付近にあるものと予想される。 Evaluation of skin resistance and recoverability to UV injury using DJ-1 as an index from the analysis results of skin redness, whiteness change and DJ-1 initial value after UV irradiation. Was found to be possible. The resistance boundary is expected to be in the vicinity of 2 pg / μg protein when DJ-1 is used as an index.
２．無作為選抜した205名のＤＪ−１タンパク質量の分布と紫外線傷害抵抗性予測
（１）試験試料の採取
無作為に選抜した女性205名を対象として、頬よりテープストリッピング法によって角層サンプルを採取した。サンプルは1部位より1枚採取した。
2. Distribution of the amount of DJ-1 protein and prediction of UV damage resistance of 205 randomly selected (1) Collection of test samples Sampled stratum corneum samples from 205 cheeks by tape stripping method for 205 women did. One sample was collected from one site.
（２）角層中ＤＪ−１測定
ガラスビーズとT-PERバッファー（Thermo scientific社）500μlの入ったチューブに角層を採取した角層チェッカーを入れ、25分ボルテックスミキサーにて振盪し、角層タンパク質を抽出した。各サンプルのタンパク量はBCA protein Assay Kit (Thermo Scientific社)で測定した。測定には角層サンプルを10μlに reagentA: reagentB=50:1で混和した液200μlを加え、60℃30分でインキュベーションしたのち、562nmの吸光度で測定した。同時にウシ血清アルブミン（BSA）で検量線を作成した。この検量線と吸光度の値からタンパク量を算出した。
角層抽出液中に含まれるＤＪ−１量はHuman Park7/ＤＪ−１ DuoSet (R&D systems社)を用いて定量した。測定結果を０．５ｐｇ/μｇ ｐroteinきざみの人数及び累計率として図５に示した。
(2) DJ-1 measurement in stratum corneum The stratum corneum checker which collected the stratum corneum was put into a tube containing 500 μl of glass beads and T-PER buffer (Thermo scientific), and shaken with a vortex mixer for 25 minutes. Protein was extracted. The amount of protein in each sample was measured with BCA protein Assay Kit (Thermo Scientific). For the measurement, 200 μl of a stratum corneum sample mixed with 10 μl of reagentA: reagentB = 50: 1 was added, incubated at 60 ° C. for 30 minutes, and then measured by absorbance at 562 nm. At the same time, a calibration curve was prepared with bovine serum albumin (BSA). The protein amount was calculated from the calibration curve and the absorbance value.
The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7 / DJ-1 DuoSet (R & D systems). The measurement results are shown in FIG. 5 as the number of 0.5 pg / μg protein increments and the cumulative rate.
図５に示したとおり、無作為に抽出した２０５名のＤＪ−１タンパク質の角層内の発現量は、0.29pg/μg proteinから8.8ｐｇ/μｇ ｐroteinまでの広い範囲で分布していた。平均値は2.6pg/μg proteinであった。
１の試験で予測した２ｐｇ/μｇ ｐroteinを紫外線傷害抵抗性の境界値とすると、約４０％の被験者は紫外線傷害の抵抗性が低いと予測した。
As shown in FIG. 5, the expression level of 205 randomly extracted DJ-1 proteins in the stratum corneum was distributed in a wide range from 0.29 pg / μg protein to 8.8 pg / μg protein. The average value was 2.6 pg / μg protein.
Assuming that 2 pg / μg protein predicted in the first test is the threshold value for UV damage resistance, about 40% of the subjects were predicted to have low UV damage resistance.
Claims (3)
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013032745A JP5775538B2 (en) | 2013-02-22 | 2013-02-22 | Evaluation method of resistance to redness (redness) generation and reduction of whiteness induced by UV exposure |
| CN201410008695.8A CN104007056B (en) | 2013-02-22 | 2014-01-08 | To the evaluation method of the repellence of ultraviolet injury |
| TW103105065A TWI612307B (en) | 2013-02-22 | 2014-02-17 | Method for evaluating resistance to uv damage |
| HK14111911A HK1198451A1 (en) | 2013-02-22 | 2014-11-25 | Method for evaluating resistance to ultraviolet damage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013032745A JP5775538B2 (en) | 2013-02-22 | 2013-02-22 | Evaluation method of resistance to redness (redness) generation and reduction of whiteness induced by UV exposure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014163715A true JP2014163715A (en) | 2014-09-08 |
| JP5775538B2 JP5775538B2 (en) | 2015-09-09 |
Family
ID=51367813
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013032745A Active JP5775538B2 (en) | 2013-02-22 | 2013-02-22 | Evaluation method of resistance to redness (redness) generation and reduction of whiteness induced by UV exposure |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP5775538B2 (en) |
| CN (1) | CN104007056B (en) |
| HK (1) | HK1198451A1 (en) |
| TW (1) | TWI612307B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017009426A (en) * | 2015-06-22 | 2017-01-12 | 株式会社ファンケル | Method of evaluating hair growth condition and compressive stress on scalp |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109839507A (en) * | 2017-11-27 | 2019-06-04 | 江苏坤辉生物科技有限公司 | A kind of biomarker detecting ultraviolet light injury and its application |
| CN109833479A (en) * | 2017-11-27 | 2019-06-04 | 上海交通大学 | It is a kind of to treat or prevent the drug and its application that Keratin 1 is degraded |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007040027A1 (en) * | 2005-10-05 | 2007-04-12 | Ezaki Glico Co., Ltd. | Pharmaceutical preparation for external application to skin comprising phosphorylated sugar |
| JP2009040753A (en) * | 2007-08-10 | 2009-02-26 | Advangen Inc | Skin repair composition and screening method thereof |
| WO2011118812A1 (en) * | 2010-03-26 | 2011-09-29 | 国立大学法人北海道大学 | Neurodegenerative disease therapeutic agent |
| US20120114628A1 (en) * | 2008-11-21 | 2012-05-10 | L'oreal | Cosmetic and therapeutic use of proteins of dj-1 type for treating skin dryness |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2007014304A (en) * | 2005-05-20 | 2008-02-08 | Premd Inc | Direct assay of skin protein in skin samples removed by tape stripping. |
| CN102498127B (en) * | 2009-09-17 | 2015-02-25 | 雷蒙特亚特特拉维夫大学有限公司 | Peptides for the treatment of oxidative stress related disorders |
-
2013
- 2013-02-22 JP JP2013032745A patent/JP5775538B2/en active Active
-
2014
- 2014-01-08 CN CN201410008695.8A patent/CN104007056B/en active Active
- 2014-02-17 TW TW103105065A patent/TWI612307B/en active
- 2014-11-25 HK HK14111911A patent/HK1198451A1/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007040027A1 (en) * | 2005-10-05 | 2007-04-12 | Ezaki Glico Co., Ltd. | Pharmaceutical preparation for external application to skin comprising phosphorylated sugar |
| JP2009040753A (en) * | 2007-08-10 | 2009-02-26 | Advangen Inc | Skin repair composition and screening method thereof |
| US20120114628A1 (en) * | 2008-11-21 | 2012-05-10 | L'oreal | Cosmetic and therapeutic use of proteins of dj-1 type for treating skin dryness |
| WO2011118812A1 (en) * | 2010-03-26 | 2011-09-29 | 国立大学法人北海道大学 | Neurodegenerative disease therapeutic agent |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017009426A (en) * | 2015-06-22 | 2017-01-12 | 株式会社ファンケル | Method of evaluating hair growth condition and compressive stress on scalp |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201439538A (en) | 2014-10-16 |
| CN104007056B (en) | 2018-01-02 |
| TWI612307B (en) | 2018-01-21 |
| CN104007056A (en) | 2014-08-27 |
| HK1198451A1 (en) | 2015-04-24 |
| JP5775538B2 (en) | 2015-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kikuchi et al. | Improvement of Mild Inflammatory Changes of the Facial Skin Induced by Winter Environment with Daily Applications of a Moisturizing CreamA Half-Side Test of Biophysical Skin Parameters, Cytokine Expression Pattern and the Formation of Cornified Envelope | |
| Naik et al. | Influence of ethnicities and skin color variations in different populations: a review | |
| TWI580964B (en) | Method for evaluating stress accumulation degree of skin | |
| JP5775538B2 (en) | Evaluation method of resistance to redness (redness) generation and reduction of whiteness induced by UV exposure | |
| JP5065237B2 (en) | Noninvasive evaluation method of local pathology of atopic dermatitis | |
| JP2024026167A (en) | Minimally invasive skin condition diagnostic kit including microneedle patch | |
| JP4469762B2 (en) | Sensitive skin evaluation method and evaluation kit | |
| TWI600904B (en) | Method of evaluating degree of skin stress accumulation | |
| Evans et al. | The impact of different hair‐removal behaviours on the biophysical and biochemical characteristics of female axillary skin | |
| JP4446010B1 (en) | Evaluation method of resistance to skin sunburn by ultraviolet rays. | |
| JP4473926B1 (en) | Evaluation method of skin darkening tendency by ultraviolet rays | |
| JP6397733B2 (en) | Acne Skin Evaluation Method | |
| TWI541506B (en) | Method for evaluating skin stress accumulation | |
| Raimonds | Melanoma | |
| JP2016216436A (en) | Skin viscoelasticity improver | |
| KR20210009981A (en) | Method for evaluating resistance to skin sunburn by ultraviolet light | |
| Harvey et al. | Integrating skin color assessments into clinical practice and research: A review of current approaches | |
| JP2012039970A (en) | Method for examining degree of skin damage | |
| KR20130098211A (en) | Analysis method for skin stress accumulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20140815 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20140815 |
|
| A975 | Report on accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A971005 Effective date: 20141014 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20141021 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20141209 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150224 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150401 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20150630 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150703 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5775538 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |