JP4896827B2 - How to differentiate stratum corneum AGEs - Google Patents
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Description
本発明は、角層の鑑別方法に関する。さらに詳しくは、免疫組織染色法を用い終末糖化産物(AGEs:advanced glycation endproducts)を検出することを特徴とする、角層の鑑別方法に関する。
The present invention relates to a method for distinguishing stratum corneum . More specifically, the present invention relates to a method for distinguishing stratum corneum, characterized by detecting advanced glycation endproducts (AGEs) using an immunohistochemical staining method .
タンパク質糖化反応(メイラード反応)は、アミノ酸、ペプチド、タンパク質のアミノ基と、ケトンやアルデヒド(特に還元糖)との非酵素的反応であり、前期段階と後期段階の2つの反応に分けられる。後期段階の反応は不可逆反応であり、前期段階で生成されたアマドリ化合物が、さらに転移や縮合などの複雑な反応過程を経て、後期糖化反応生成物(AGEs)と呼ばれる安定な物質を形成する反応である。前記AGEsとしては、例えば、カルボキシメチルリジン、ペントシジン、ピラリン、クロスリン等が知られているが、生体内には構造が明らかにされていない未知のAGEsが多種存在していると考えられている。 The protein saccharification reaction (Maillard reaction) is a non-enzymatic reaction between an amino group of an amino acid, a peptide, or a protein and a ketone or an aldehyde (especially a reducing sugar), and can be divided into two reactions, an early stage and a late stage. The reaction at the later stage is an irreversible reaction, and the Amadori compound produced at the earlier stage undergoes a complex reaction process such as transfer or condensation to form a stable substance called late saccharification reaction products (AGEs). It is. As the AGEs, for example, carboxymethyllysine, pentosidine, pyralin, croslin, and the like are known, but it is considered that there are various unknown AGEs whose structures are not clarified in the living body.
近年、前記メイラード反応の生成物は、医学領域で注目されており、様々な病気、疾患、老化との関連性が発表されている。中でも、公知のAGEsは、タンパク質の機能を低下させるのみならず、細胞障害や炎症反応を誘発する事が知られており、さらに、AGEsの形成によって、タンパク質が凝集、不溶化して組織中に異常蓄積し、これにより組織
を変性させているとも考えられている。また、糖尿病患者におけるヘモグロビンA1C(前期反応のアマドリ化合物)の上昇、糖尿病性腎症や慢性腎不全の腎臓や、動脈硬化病変部におけるAGEsの蓄積は、生体内におけるタンパク糖化反応の代表的な例として知られ、AGEsへの関心がさらに高まっていた。
In recent years, the product of the Maillard reaction has attracted attention in the medical field, and its relevance to various diseases, diseases, and aging has been announced. Above all, known AGEs not only reduce the function of the protein, are known to induce cell failure or inflammatory response, In addition, the formation of AGEs, protein aggregation, insolubilization to the tissue It is also thought that abnormal accumulation has caused tissue degeneration. In addition, the increase of hemoglobin A1C (Amadori compound of the early reaction) in diabetic patients, accumulation of AGEs in diabetic nephropathy and chronic renal failure, and atherosclerotic lesions are typical examples of in vivo protein glycation reactions. The interest in AGEs was further increased.
かようなAGEsの測定には、その褐色変化や蛍光特性を利用した吸光光度計や蛍光分光光度計による比色定量法、生体組織を加水分解してHPLCやGC/MSによる分析等が知られている(例えば、特許文献1、特許文献2、特許文献3)。また、簡便且つ特殊な分析機器を必要としない等の理由により、免疫学的検出方法、具体的には、免疫組織学的方法、酵素免疫学的測定方法等が、医学分野における研究や臨床診断で広く使われており、このため、糖化タンパク質やAGEsに特異的に反応する抗体が各種作製され利用されている(例えば、特許文献4、特許文献5、特許文献6、非特許文献1)。 For the measurement of such AGEs, a colorimetric method using an absorptiometer or a fluorescence spectrophotometer utilizing the brown color change or fluorescence characteristics, an analysis by HPLC or GC / MS by hydrolyzing a living tissue, and the like are known. (For example, Patent Document 1, Patent Document 2, and Patent Document 3). In addition, immunological detection methods, specifically, immunohistological methods, enzyme immunological measurement methods, etc. are used in research and clinical diagnosis in the medical field because they do not require a simple and special analytical instrument. Therefore, various antibodies that specifically react with glycated proteins and AGEs are produced and used (for example, Patent Document 4, Patent Document 5, Patent Document 6, and Non-Patent Document 1).
このような状況下において、肌の美しさにとって重要である表皮、特に角層中におけるAGEsの存在やその意味については全く明らかでなかった。これは比色定量法、HPLCやGC/MS等においてはAGEsの組織中の局在性を測定するのに適さず、また、特異的抗体による生体組織中のAGEs測定においては、表皮より下層を対象としており、表皮や角層中におけるAGEsの存在の有無やその局在性について全く知られていなかった。即ち、角層中におけるAGEsの存在やその意味を明らかにできる非侵襲的且つ客観的に測定する手段が切望されていた。 Under such circumstances, the existence and meaning of AGEs in the epidermis, particularly the stratum corneum, which is important for the beauty of the skin, was not clear at all. This is not suitable for measuring the localization of AGEs in tissues by colorimetry, HPLC, GC / MS, etc., and in the measurement of AGEs in biological tissues by specific antibodies, the layer below the epidermis is used. There was no knowledge of the presence or location of AGEs in the epidermis or stratum corneum. That is, there has been a strong demand for a noninvasive and objective measurement means that can clarify the existence and meaning of AGEs in the stratum corneum.
本発明はこのような状況下で為されたものであり、角層の鑑別方法に関する。さらに詳細には、免疫組織染色法を用いた、角層中における、AGEs(AGEs:advancedglycation endproducts)の非侵襲的且つ定量的方法を提供することを課題とする。
The present invention has been made under such circumstances, and relates to a method for distinguishing stratum corneum . More specifically, it is an object of the present invention to provide a noninvasive and quantitative method for AGEs (AGEs: advanced glycation endproducts) in the stratum corneum using immunohistochemical staining.
このような状況を鑑みて、本発明者らは、鋭意研究努力を重ねた結果、免疫組織染色された角層の拡大イメージ画像を画像処理してAGEsを定量し、角層を鑑別できることを見出し、発明を完成させるに至った。即ち、本発明は以下に示す技術に関するものである。
In view of such a situation, the present inventors have quantified the AGEs result of extensive research efforts, the enlarged image of immune tissue stained horny layer image processed to be able to distinguish corneum The present invention has been completed. That is, the present invention relates to the following technique.
(1)免疫組織染色法を用いて角層中におけるAGEsを検出することを特徴とするAGEsの定量方法。
(2)免疫組織染色された角層の拡大イメージ画像を画像処理して定量化することを特徴とする、(1)に記載のAGEsの定量方法。
(3)(1)又は(2)に記載の定量法を用いてAGEsを定量し、AGEsの存在割合が多いほど紫外線被爆による角層のダメージ度合いが大であると判断することを特徴とする、紫外線による角層のダメージ度合いの鑑別方法。
(4)(1)もしくは(2)、又は(3)に記載の鑑別方法を用い、経時的な角層の変化を時系列的に捉えていくことを特徴とする、角層の変化のモニタリング方法。
(1) A method for quantifying AGEs, comprising detecting AGEs in the stratum corneum using an immunohistochemical staining method.
(2) The method for quantifying AGEs according to (1), wherein an enlarged image image of the stratum corneum stained with immune tissue is image-processed and quantified.
(3) AGEs are quantified by using the quantification method described in (1) or (2), and it is judged that the degree of damage to the stratum corneum due to ultraviolet exposure increases as the AGEs content increases. , A method for distinguishing the degree of damage to the stratum corneum by ultraviolet rays .
(4) Monitoring of stratum corneum changes, characterized in that changes in the stratum corneum over time are captured in time series using the identification method described in (1), (2), or (3). Method.
本発明によって、角層中におけるAGEsを非侵襲的且つ定量的に測定する方法を提供することができ、角層のダメージ度合の判定や経時的な角層の変化をモニタリングに貢献できる。 According to the present invention, it is possible to provide a method for noninvasively and quantitatively measuring AGEs in the stratum corneum, and it is possible to contribute to monitoring the degree of damage of the stratum corneum and the change of the stratum corneum over time.
本発明は、免疫組織染色された角層の拡大イメージ画像を画像処理してAGEsを定量
化することを特徴とする。
The present invention quantifies AGEs by processing an enlarged image of the stratum corneum stained with immunohistochemistry.
It is characterized by becoming .
皮膚は、身体の表面を覆い、外界との境界機能を果たす器官であり、表皮、真皮及び皮下組織より構成されている。表皮はさらに、角層、顆粒層、有棘層及び基底層より構成されている。最外層の角層は、様々な外部からの刺激を直接受けながら皮膚の防御機能を担い、また人の目に触れるところでありため、角層は皮膚生理学的にも美容上でも極めて重要な役割を果たしている。かような角層においてAGEsが存するならば、公知のAGEs知見より、タンパク質の機能を低下や、さらに、タンパク質の凝集や不溶化による組織中での異常蓄積や組織変性の可能性が考えられ、AGEsの存在の有無やそれによる角層機能の変化を知ることが重要である。
The skin is an organ that covers the surface of the body and performs a boundary function with the outside world, and is composed of the epidermis, dermis, and subcutaneous tissue. The epidermis is further composed of a stratum corneum, a granular layer, a spiny layer and a basal layer. The outermost stratum corneum plays a vital role in both skin physiology and cosmetics because it is responsible for the skin's defense functions while directly receiving various external stimuli and is also in contact with human eyes. Plays. If AGEs resides in such a stratum corneum, Ri by known AGEs knowledge, and decrease the function of the protein, furthermore, the possibility of abnormal accumulation and tissue degeneration in the tissue due to the aggregation and insolubilization of proteins thought It is important to know the presence or absence of AGEs and the change in stratum corneum function.
角層中のAGEsは次のようにして検出できる。皮膚より市販の粘着テープ等を利用してテープストリッピングにより角層採取を行い、溶剤で半日処理して粘着テープ部分を除去する。得られた角層に対して免疫組織染色を行い、染色された角層標本を顕微鏡下で観察する。前記溶剤としては、有機溶媒、特にキシレンが好ましく例示できる。免疫組織染色の条件として、一次抗体(anti AGE monocronal antibody(mouse) TransGenic KH001)、ヒ゛オチン化2 次抗体(Biotin-Rabbit Anti Mouse IGg conjugate ZYMED 81-6740)、ABC 試薬(R.T.U VECTASTAIN Elite ABC REAGENT BECTOR PK-7100)、AEC基質(ENVISION kit/HRP(AEC) Dako K3464)が例示できる。 AGEs in the stratum corneum can be detected as follows. The stratum corneum is collected from the skin by tape stripping using a commercially available adhesive tape or the like, and the adhesive tape portion is removed by treatment with a solvent for half a day. The obtained stratum corneum is subjected to immunohistochemical staining, and the stained stratum corneum specimen is observed under a microscope. Preferred examples of the solvent include organic solvents, particularly xylene. Conditions for immunohistochemical staining include primary antibody (anti AGE monocronal antibody (mouse) TransGenic KH001), biotinylated secondary antibody (Biotin-Rabbit Anti Mouse IGg conjugate ZYMED 81-6740), ABC reagent (RTU VECTASTAIN Elite ABC REAGENT BECTOR PK -7100) and AEC substrates (ENVISION kit / HRP (AEC) Dako K3464).
前記染色された角層標本において、赤〜赤橙色に染色された部分がAGEsである。顕微鏡で20〜50倍に観察されたこの染色されたAGEsである拡大イメージ画像をデジタル式のマイクロスコープ等を利用して、コンピュータに取り込み、汎用的画像解析のソフトウェアを用いて、手動又は自動処理を行い、AGEsを定量化できる。デジタル式のマイクロスコープとしては、例えば、(株)モリテックスのコスメティック用マイクロスコープ、汎用的画像解析のソフトウェアとしては、例えば、三谷商事(株)のWinROOF(登録商標)が例示できる。画像処理は、一般的な手法によって行えばよく、例えば、AGEsの拡大イメージ画像であるカラー画像をモノクロ濃淡画像に変換後、該モノクロ画像に必要に応じてフィルター処理後二値化処理を行い、統計処理によってAGEsの存在割合に相関する染色部総面積を算出すればよい。 In the stained stratum corneum specimen, the portion stained red to red-orange is AGEs. This stained image of AGEs observed 20 to 50 times with a microscope is taken into a computer using a digital microscope or the like, and manually or automatically processed using general-purpose image analysis software. AGEs can be quantified. Examples of the digital microscope include a cosmetic microscope manufactured by Moritex Co., Ltd., and an example of general purpose image analysis software is WinROOF (registered trademark) manufactured by Mitani Corporation. The image processing may be performed by a general method. For example, after converting a color image that is an enlarged image image of AGEs into a monochrome grayscale image, the monochrome image is subjected to binarization processing after filtering as necessary, What is necessary is just to calculate the dyeing | staining part total area correlated with the abundance ratio of AGEs by statistical processing.
前述のようにして画像処理によって得られた染色部総面積はAGEsの存在割合を示す。また、後述する実施例の如く、染色部総面積が紫外線暴露部位(量)や年齢との間に正の相関関係を有することが分かる。これよりAGEsの存在割合が紫外線暴露や加齢によって高くなることが分かる。紫外線暴露や加齢によって、バリアー機能やドライスキンの増加等、角層のダメージ度合いが高いことはよく知られており、これより、AGEsの存在割合が多いほど、角層のダメージ度合いが大であると判断することができる。 The total stained area obtained by image processing as described above indicates the presence ratio of AGEs. Further, as shown in the examples described later, it can be seen that the total area of the stained portion has a positive correlation with the ultraviolet ray exposure site (amount) and age. From this, it can be seen that the abundance of AGEs increases with exposure to ultraviolet rays and aging. It is well known that the degree of damage to the stratum corneum is high due to exposure to UV rays and aging, such as an increase in barrier function and dry skin. From this, the more AGEs are present, the greater the degree of damage to the stratum corneum. It can be judged that there is.
かような非侵襲的なAGEsの定量方法を用いて、経時的な角層の変化を時系列的に捉えていくモニタリング方法は汎用性が非常に高い。例えば、抗老化化粧料や紫外線防御化粧料等を使用することによる1週〜数ヶ月の時系列モニタリングを行い、AGEsの存在割合の変化によりその効果を判断することが例示できる。 Using a quantitative method of such non-invasive AGEs, spider Nitaringu methods have chronologically capture the change with time in the stratum corneum is versatile very high. For example, it is possible to exemplify performing time-series monitoring for one week to several months by using anti-aging cosmetics, UV protective cosmetics, etc., and judging the effect by changing the abundance of AGEs.
以下に実施例を挙げて、本発明について更に詳細に説明を加えるが、本発明がこれら実施例にのみ限定されないのは言うまでもない。 Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited to these examples.
角層中におけるAGEsの存在を検討した。健常人男性(30才)の上腕内側部及び前腕外側部の角層を市販の粘着テープを利用してテープストリッピングにより角層採取を行い、キシレンで半日処理して粘着テープ部分を除去する。得られた角層に対して免疫組織染色(条件は段落番号0013参照)を行い、染色された角層標本を顕微鏡下で観察した。ポジコンとして、同角層を250mMグルコース溶液に浸漬させ、37℃条件下で1週間放置したものを用いた。結果を図1に示す。 The presence of AGEs in the stratum corneum was examined. The stratum corneum of the inner part of the upper arm and the outer part of the forearm is collected by tape stripping using a commercially available adhesive tape and treated with xylene for half a day to remove the adhesive tape part. The obtained stratum corneum was subjected to immunohistochemical staining (see paragraph No. 0013 for the conditions), and the stained stratum corneum specimen was observed under a microscope. As a positive control, the same horny layer was immersed in a 250 mM glucose solution and allowed to stand at 37 ° C. for 1 week. The results are shown in FIG.
図1の染色されたAGEsのイメージ画像(×40)は、左側が男性の角層、右側にポジコン、上段に上腕内側部、下段に前腕外側部を示している。角層にポジコンと同様に赤〜赤橙色のAGEsが観察されることから、角層にAGEsが存することが分かる。 The stained AGEs image (× 40) in FIG. 1 shows the male stratum corneum on the left, the positive control on the right, the inner upper arm on the upper side, and the outer forearm on the lower side. Since red to red-orange AGEs are observed in the stratum corneum as in the positive control, it can be seen that AGEs exist in the stratum corneum.
実施例1と同様の手法を用い、健常人男性20人(20代〜50代、各年代5名ずつ)を対象に、上腕内側部及び前腕外側部の角層中におけるAGEsの存在割合を検討した。即ち、(株)モリテックスのコスメティック用マイクロスコープを用いて、AGEsの拡大イメージ画像をコンピュータに取り込んだ後、三谷商事(株)のWinROOF(登録商標)を用いてモノクロ濃淡画像に変換し、フィルター処理及び二値化処理を行って、AGEsの存在割合である染色部総面積を算出した。結果を図2及び図3に示す。 Using the same method as in Example 1, the proportion of AGEs in the stratum corneum of the inner upper arm and outer forearm was examined for 20 healthy males (20 to 50s, 5 each). did. In other words, after using the Moritex Cosmic Microscope to capture an enlarged image of AGEs into a computer, it is converted into a monochrome grayscale image using WinROOF (registered trademark) of Mitani Corporation, and filtered. And binarization processing was performed, and the dyeing | stained part total area which is an abundance ratio of AGEs was computed. The results are shown in FIGS.
図2は上腕内側部における加齢に伴う角層中のAGEsの存在割合を示す1例で、加齢と共にAGEsが微増の傾向を示す。図3は、30代の5名の男性の中の2名(A及びB)の上腕内側部及び前腕外側部のAGEsの存在割合を示し、屋外活動の多いAは前腕外側部のAGEsの存在割合が非常に高いことを示す。上腕内側部及び前腕外側部において、年齢との相関分析を行った結果、上腕内側部において正の相関関係を確認したが(r=0.501、P<0.05)、前腕外側部では有意な相関関係を示さなかった。 FIG. 2 is an example showing the proportion of AGEs in the stratum corneum accompanying aging in the inner side of the upper arm, and shows a tendency for AGEs to slightly increase with aging. FIG. 3 shows the proportion of AGEs in the inner and outer forearms of two of the five men in their 30s (A and B), and A with a lot of outdoor activities is the presence of AGEs in the outer forearm Indicates that the rate is very high. As a result of correlation analysis with age in the inner side of the upper arm and the outer side of the forearm, a positive correlation was confirmed in the inner side of the upper arm (r = 0.501, P <0.05), but significant in the outer side of the forearm No significant correlation.
これより、非露出部位である上腕内側部では年齢と共にゆるやかに角層中のAGEsの存在割合が増加することが分かる。一方、紫外線露出部位である前腕外側部においては、個人差が非常に大きく、生活スタイル(屋外活動)等により紫外線暴露されている人ではAGEsの存在割合が非常に高いレベルにあることが分かる。紫外線暴露や加齢によって、バリアー機能やドライスキンの増加等、角層のダメージ度合いが高いことはよく知られており、これより、AGEsの存在割合が多いほど、角層のダメージ度合いが大であると判断することができる。 From this, it can be seen that the presence ratio of AGEs in the stratum corneum gradually increases with age at the inner side of the upper arm, which is a non-exposed part. On the other hand, in the outer part of the forearm, which is an ultraviolet-exposed region, the individual difference is very large, and it can be seen that the ratio of AGEs present is very high in those who are exposed to ultraviolet rays due to lifestyle (outdoor activities) or the like. It is well known that the degree of damage to the stratum corneum is high due to exposure to UV rays and aging, such as an increase in barrier function and dry skin. From this, the more AGEs are present, the greater the degree of damage to the stratum corneum. It can be judged that there is.
健常女性2名(30代)の顔面左右頬部を対象に、下記の紫外線防御化粧料A(朝夕2回塗布)の1ヶ月間使用の有無によるAGEsの存在割合への影響を検討した。方法は実施例2と同様にして、使用前と1ヶ月後の染色部総面積の比(%)を算出した。紫外線防御化粧料Aを使用した部位の場合2名の平均で92%に対し、使用しない部位では102%であった。これより、紫外線防御化粧料Aの使用によってAGEs存在割合を抑制する効果があることが分かる。 The effect of the following UV protective cosmetic A (applied twice a day in the morning and evening) on the presence of AGEs was examined on the left and right cheeks of two healthy women (30's). The method was the same as in Example 2, and the ratio (%) of the total area of the stained area before use and after 1 month was calculated. In the case of the part using the UV protective cosmetic A, the average of two persons was 92%, whereas in the part not using it was 102%. From this, it can be seen that the use of the ultraviolet protective cosmetic A has an effect of suppressing the AGEs existing ratio.
紫外線防御化粧料A(W/O型乳液)は、以下に示す処方に従って作成した。即ち、イ、ロの成分を80℃に加熱攪拌し、イにハの成分を加え、ディスパーで分散させて一様な状態とし、これに徐々にロを加えて乳化し、攪拌冷却して紫外線防御化粧料Aを得た。 Ultraviolet protective cosmetic A (W / O type emulsion) was prepared according to the formulation shown below. In other words, the components (a) and (b) are heated and stirred at 80 ° C., the component (c) is added to (b), and dispersed with a disper to obtain a uniform state. A protective cosmetic A was obtained.
イ
ショ糖モノステアリン酸エステル 0.3 質量%
ソルビタンモノラウリン酸エステル 0.4 質量%
ジメチコン(5mPascal・秒) 30 質量%
シクロメチコン 5 質量%
セチルイソオクタネート 5 質量%
ポリエーテル変性シリコーン 2 質量%
ロ
1,3−ブタンジオール 10 質量%
水酸化カリウム 0.4 質量%
クエン酸ナトリウム 1 質量%
硫酸マグネシウム 0.4 質量%
エスクレチン 0.1 質量%
フェノキシエタノール 0.5 質量%
グリチルリチン酸ジカリウム1%水溶液 5 質量%
水 33.4 質量%
ハ
「ベントン−38V」 1.5 質量%
黄色酸化鉄焼結二酸化チタン 0.6 質量%
ステアリン酸アルミニウム被覆微粒子二酸化チタン 1.6 質量%
(平均粒径0.8μm)
ハイドロジェンメチルポリシロキサン被覆酸化亜鉛 2 質量%
(平均粒径0.7μm)
タルク 0.8 質量%
Isho sugar monostearate 0.3% by mass
Sorbitan monolaurate 0.4% by mass
Dimethicone (5mPascal · sec) 30% by mass
Cyclomethicone 5% by mass
Cetyl isooctanoate 5% by mass
Polyether-modified silicone 2% by mass
1,3-butanediol 10% by mass
Potassium hydroxide 0.4 mass%
Sodium citrate 1% by mass
Magnesium sulfate 0.4% by mass
Esculetin 0.1% by mass
Phenoxyethanol 0.5% by mass
1% aqueous solution of dipotassium glycyrrhizinate 5% by mass
Water 33.4% by mass
C “Benton-38V” 1.5% by mass
Yellow iron oxide sintered titanium dioxide 0.6 mass%
Aluminum stearate coated fine particle titanium dioxide 1.6% by mass
(Average particle size 0.8μm)
Hydrogenmethylpolysiloxane-coated zinc oxide 2% by mass
(Average particle size 0.7μm)
Talc 0.8 mass%
本発明によって、角層中におけるAGEsを非侵襲的且つ定量的に測定する方法を提供することができる。その結果、肌状態に適した化粧料の選択、角層のダメージ度合のアドバイスや経時的な角層のモニタリング等、多くの顧客に満足を提供できる。 The present invention can provide a method for noninvasively and quantitatively measuring AGEs in the stratum corneum. As a result, it is possible to provide satisfaction to many customers, such as selection of a cosmetic suitable for the skin condition, advice on the degree of damage to the stratum corneum, and monitoring of the stratum corneum over time.
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