JP6650240B2 - Method of searching for melanin control agent - Google Patents
Method of searching for melanin control agent Download PDFInfo
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- JP6650240B2 JP6650240B2 JP2015201569A JP2015201569A JP6650240B2 JP 6650240 B2 JP6650240 B2 JP 6650240B2 JP 2015201569 A JP2015201569 A JP 2015201569A JP 2015201569 A JP2015201569 A JP 2015201569A JP 6650240 B2 JP6650240 B2 JP 6650240B2
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- melanin
- exosome
- exosomes
- keratinocytes
- cells
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Description
本発明は、メラニン制御剤を探索する方法に関する。 The present invention relates to a method for searching for a melanin controlling agent.
人の皮膚や毛髪の色調は、皮膚及び毛髪に存在する色素メラニンの量によって決定される。メラニンは、皮膚や毛髪の毛球部に存在する色素細胞（メラノサイト）において、酵素チロシナーゼやドーパオキシダーゼによってチロシンから生合成されることから、メラノサイトの活性化又はチロシナーゼの活性化によりメラニン産生が亢進すれば、皮膚は褐色化し、毛髪は黒色化する。 The color tone of human skin and hair is determined by the amount of pigment melanin present in skin and hair. Melanin is biosynthesized from tyrosine by the enzymes tyrosinase and dopa oxidase in pigment cells (melanocytes) present in the hair bulb of the skin and hair. , The skin turns brown and the hair turns black.
皮膚のシミ、ソバカスは、その原因の一つとして、皮膚の紫外線暴露による刺激、ホルモンの異常又は遺伝的要素等によってメラノサイトが活性化され、メラニン産生が亢進することが挙げられる。したがって、従来より、チロシナーゼの活性を阻害してメラニン産生を抑制したり、産生したメラニンを減少させたりする美白剤が開発されてきた。また、表皮角化細胞（ケラチノサイト）に存するメラニン顆粒は、メラノサイトで産生されたメラニン顆粒を、メラノサイトがデンドライトを伸長し、ケラチノサイトと接合して該メラニン顆粒をケラチノサイト内に転送することにより、ケラチノサイトに存するようになる。これは、メラニン色素の皮膚組織への沈着にとって重要な過程であるため、デンドライトの伸長を阻害することは美白剤の開発に有用であるのみならず、皮膚の色素異常症の診断や治療にも役立つと考えられる。 One of the causes of skin spots and freckles is that the melanocytes are activated by the stimulation of the skin by exposure to ultraviolet rays, hormonal abnormalities or genetic factors, and the production of melanin is enhanced. Therefore, conventionally, a whitening agent that inhibits the activity of tyrosinase to suppress melanin production or reduces produced melanin has been developed. In addition, the melanin granules present in the epidermal keratinocytes (keratinocytes), the melanin granules produced in the melanocytes, the melanocytes extend the dendrites, and joined with the keratinocytes to transfer the melanin granules into the keratinocytes, to the keratinocytes Will exist. Since this is an important process for the deposition of melanin on skin tissue, inhibiting dendrite elongation is not only useful for the development of whitening agents, but also for the diagnosis and treatment of skin pigmentation disorders. It is thought to be useful.
一方で、メラニンの産生を亢進して肌を褐色化する需要も多く、日光浴や屋内での紫外線照射により肌を積極的に日焼けさせることや、タンニング剤を利用することが従来から行われている。また、皮膚に於いて、デンドライトの伸長が妨げられてメラニン顆粒の転送に支障を来すと、メラニン産生が妨げられ白斑などを生ずるようになる。したがって、白斑の治療に於いては、メラノサイトのデンドライト伸長を促進することが有効である。 On the other hand, there is also a great demand for increasing the production of melanin and browning the skin, and active suntanning of the skin by sunbathing or ultraviolet irradiation indoors, and use of a tanning agent have been conventionally performed. . In the skin, if the elongation of dendrites is hindered and the transfer of melanin granules is hindered, the production of melanin is hindered and vitiligo and the like occur. Therefore, in the treatment of vitiligo, it is effective to promote dendrite elongation of melanocytes.
また、白髪は、毛母色素細胞の変化によってメラニンが減少する生理的老化現象の一つであるが、その発生機序の多くは未だ解明されておらず、染毛剤による染毛が中心となっているのが現状である。従って、白色化した髪を黒髪へと変化させる方法の開発が求められている。 In addition, gray hair is one of the physiological aging phenomena in which melanin decreases due to changes in hair matrix pigment cells, but the mechanism of its development has not yet been elucidated. That is the current situation. Therefore, development of a method for changing whitened hair to black hair is required.
斯様に、メラノサイトに直接的若しくは間接的に作用してメラニン量やデンドライトの形成を変化させる成分の探索は、美白、肌の褐色化、白髪の防止又は改善等を図るために極めて有用である。 Thus, the search for a component that directly or indirectly acts on melanocytes to change the amount of melanin or the formation of dendrites is extremely useful for achieving whitening, skin browning, preventing or improving white hair, and the like. .
一方、エクソソーム（ｅｘｏｓｏｍｅ）は脂質二重膜で囲まれた膜小胞で、従来は不要な細胞内成分を外に放出するために機能していると考えられてきたが、近年、免疫細胞や腫瘍細胞をはじめ、多くの細胞がエクソソームを放出し、これが分泌細胞（エクソソーム放出細胞）とその標的細胞（エクソソーム受容細胞）の間で蛋白質や脂質を交換する重要なメッセンジャーとなっていることが解明されつつある。近年、ケラチノサイト由来のエクソソームも単離され、線維芽細胞に添加することで、ＭＭＰ−１の発現が上昇すること（非特許文献１）、骨髄由来樹状細胞に添加することで、ＩＬ−６、ＩＬ−１０、ＩＬ−１２の産生が上昇すること（非特許文献２）が報告されている。
しかしながら、ケラチノサイトから放出されるエクソソームがメラノサイトに対して如何なる作用を示すのかは全く知られていない。
On the other hand, exosomes (exosomes) are membrane vesicles surrounded by a lipid bilayer and have conventionally been thought to function to release unnecessary intracellular components to the outside. Many cells, including tumor cells, release exosomes, which are important messengers that exchange proteins and lipids between secretory cells (exosome-releasing cells) and their target cells (exosome-receiving cells). Is being done. In recent years, exosomes derived from keratinocytes have also been isolated and added to fibroblasts to increase the expression of MMP-1 (Non-Patent Document 1), and added to bone marrow-derived dendritic cells to produce IL-6. , IL-10 and IL-12 are reported to increase (Non-Patent Document 2).
However, it is not known at all what effect exosomes released from keratinocytes exert on melanocytes.
本発明は、メラノサイトにおけるメラニン量を制御する物質を簡易に探索する方法を提供することに関する。 The present invention relates to providing a method for easily searching for a substance that controls the amount of melanin in melanocytes.
本発明者は、メラニン制御に関して種々検討したところ、ケラチノサイトから放出されるエクソソームがメラノサイトに受容され、メラノサイトの細胞増殖及びデンドライト形成を顕著に促進し、メラニン産生を促進することを見出した。すなわち、ケラチノサイト由来のエクソソームは、メラノサイトの細胞増殖能及びメラニン産生能をはじめとした細胞活性を正に制御しており、ケラチノサイトから放出されるエクソソームの分泌レベルを指標とすることにより、メラニン制御剤を評価又は選択できることを見出した。 The present inventors have conducted various studies on melanin control, and found that exosomes released from keratinocytes are accepted by melanocytes, remarkably promote cell growth and dendrite formation of melanocytes, and promote melanin production. In other words, exosomes derived from keratinocytes positively control cell activities including melanocyte cell growth ability and melanin production ability, and by using the secretion level of exosomes released from keratinocytes as an index, a melanin regulator Can be evaluated or selected.
すなわち、本発明は、以下の工程ａ）〜ｃ）を含むメラニン制御剤の探索方法に係るものである。
ａ）ケラチノサイトに被験物質を接触させ培養する工程
ｂ）培養細胞からエクソソーム画分を単離し、分泌レベルを測定する工程
ｃ）エクソソームの分泌レベルを変化させる被験物質をメラニン制御剤として選択する工程
That is, the present invention relates to a method for searching for a melanin controlling agent comprising the following steps a) to c).
a) a step of culturing a test substance by contacting it with keratinocytes b) a step of isolating an exosome fraction from cultured cells and measuring a secretion level c) a step of selecting a test substance that changes the secretion level of exosomes as a melanin regulator
本発明によれば、メラノサイトのメラニン量を変化させる素材やデンドライト形成を変化させる素材を探索することができる。すなわち、メラニン産生促進剤、皮膚褐色化剤、白斑予防又は治療剤、白髪予防又は改善剤、デンドライト伸長促進剤、メラニン産生抑制剤、美白剤、デンドライト伸長抑制剤等を評価又は選択することができる。 According to the present invention, it is possible to search for a material that changes the amount of melanin in melanocytes or a material that changes dendrite formation. That is, melanin production promoters, skin browning agents, vitiligo prevention or treatment agents, gray hair prevention or amelioration agents, dendrite growth promoters, melanin production inhibitors, whitening agents, dendrite growth inhibitors, and the like can be evaluated or selected. .
本発明において、「エクソソーム」とは、ケラチノサイトから恒常的に放出されるエクソソームを意味する。ケラチノサイト由来のエクソソームは、直径が３０〜１００ｎｍで、ｃｕｐ形状を有し、Ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ ７０（ＨＳＰ７０）、β−Ａｃｔｉｎ、テトラスパニン（ＣＤ９、ＣＤ６３）等のタンパク質を含むという特徴を有することが報告されているが、本発明においてはこれに限定されるものではない。 In the present invention, “exosome” means an exosome constantly released from keratinocytes. It is reported that exosomes derived from keratinocytes have a diameter of 30 to 100 nm, have a cup shape, and are characterized by containing proteins such as heat shock protein 70 (HSP70), β-Actin, and tetraspanins (CD9, CD63). However, the present invention is not limited to this.
後記実施例に示すように、ケラチノサイト由来のエクソソームは、メラノサイトの細胞活性（ドーパオキシダーゼ活性、細胞呼吸鎖活性）を上昇させ（図３）、メラニン関連タンパク質をコードする遺伝子（Microphthalmia-associated transcription factor（ＭＩＴＦ）、Tyrosinase（ＴＹＲ）、Tyrosinase related protein１（ＴＹＲＰ１）、Dopachrome tautomerase (ＤＣＴ)／Tyrosinase related protein２（ＴＹＲＰ２））の発現を上昇させ（図４）、メラノサイトの細胞増殖及び形態形成を促進させ（細胞数の増加、デンドライトの伸長）（図５）、メラニン関連タンパク質（ＭＩＴＦ、Tyrosinase、ＴＲＰ１、ＴＲＰ２）の発現を上昇させ（図６）、さらにメラニン産生を促進させる（図７）。また、さらに表皮中のエクソソーム含有量とメラノソームに特異的なタンパク質であるＰｍｅｌ１７の発現量は良好に相関する（図８）。したがって、ケラチノサイト由来のエクソソームは、メラノサイトの細胞活性を正に制御し、皮膚色形成に関与すると考えられる。
故に、ケラチノサイトからのエクソソームの分泌レベルを変化させることによって、メラニンの制御が可能であり、当該エクソソームの分泌レベルの増加はメラニン量を増加させ、逆に当該エクソソームの分泌レベルの低減はメラニン量を低下させると云え、実際に、エクソソームの放出促進剤によりメラニン生成が促進され（図９〜１１）、エクソソームの放出抑制剤によりメラニン生成が抑制される（図１２〜１５）。
したがって、ケラチノサイトにおけるエクソソームの分泌レベルを指標とすることにより、メラニン制御剤を評価又は選択することができる。
As shown in Examples below, exosomes derived from keratinocytes increase the cell activity (dopa oxidase activity, cell respiratory chain activity) of melanocytes (FIG. 3), and the gene encoding melanin-related protein (Microphthalmia-associated transcription factor ( Increase expression of MITF), Tyrosinase (TYR), Tyrosinase related protein 1 (TYRP1), Dopachrome tautomerase (DCT) / Tyrosinase related protein 2 (TYRP2) (FIG. 4), and promote cell growth and morphogenesis of melanocytes (cells Increase in number, dendrite elongation) (FIG. 5), increase the expression of melanin-related proteins (MITF, Tyrosinase, TRP1, TRP2) (FIG. 6) and further promote melanin production (FIG. 7). Furthermore, the exosome content in the epidermis correlates well with the expression level of Pmel17, which is a melanosome-specific protein (FIG. 8). Therefore, exosomes derived from keratinocytes positively control the cell activity of melanocytes and are considered to be involved in skin color formation.
Therefore, by changing the secretion level of exosomes from keratinocytes, it is possible to control melanin, an increase in the secretion level of the exosomes increases the amount of melanin, and conversely, a decrease in the secretion level of the exosomes decreases the amount of melanin. In fact, the exosome release enhancer promotes melanin production (FIGS. 9 to 11), and the exosome release inhibitor suppresses melanin production (FIGS. 12 to 15).
Therefore, the melanin regulator can be evaluated or selected by using the secretion level of exosomes in keratinocytes as an index.
本発明において、「メラニン制御」とは、メラノサイトにおけるメラニン量の制御及びデンドライト形成の制御を意味し、具体的にはメラノサイトの細胞増殖活性の上昇又は低下、メラノサイトの細胞数の増加又は減少、メラニン合成の促進又は低下、メラニン合成に関わる因子（メラニン合成のマスター制御因子：Microphthalmia-associated transcription factor (ＭＩＴＦ)、メラニン合成に関わる酵素群：ＴＹＲ，ＴＲＰ１，ＴＲＰ２等）及びその遺伝子の発現量の増加又は減少、デンドライト形成の促進又は低下が包含される。 In the present invention, "melanin control" means control of the amount of melanin and control of dendrite formation in melanocytes, specifically, increase or decrease in cell proliferation activity of melanocytes, increase or decrease in the number of cells of melanocytes, melanin Acceleration or reduction of synthesis, factors related to melanin synthesis (master regulator of melanin synthesis: Microphthalmia-associated transcription factor (MITF), enzymes related to melanin synthesis: TYR, TRP1, TRP2, etc.) and increase in the expression level of their genes Or reduction, promotion or reduction of dendrite formation.
本発明のメラニン制御剤の探索方法は、ａ）ケラチノサイトに被験物質を接触させ培養する工程、ｂ）培養細胞からエクソソーム画分を単離し、分泌レベルを測定する工程、ｃ）エクソソームの分泌レベルを変化させる被験物質をメラニン制御剤として選択する工程、を含む。 The method for searching for a melanin controlling agent of the present invention comprises: a) a step of culturing a test substance in contact with keratinocytes; b) a step of isolating an exosome fraction from cultured cells and measuring a secretion level; Selecting a test substance to be changed as a melanin controlling agent.
本発明において用いられる「ケラチノサイト」としては、自己増殖性を有する正常な表皮角化細胞であれば使用可能であり、ヒト、マウス、ラット、ブタ等の哺乳動物由来の細胞を使用することができるが、ヒトの細胞を用いるのが好ましい。当該ケラチノサイトを用いる場合には、動物よりケラチノサイトを採取し、常法に従って処理して用いることができるが、樹立細胞株として市販されているもの（例えば、ヒト表皮角化細胞株であるHaCaT細胞など）を購入して利用することもできる。ヒトの細胞としては、例えば正常ヒトケラチノサイト（ＮＨＥＫ：Normal Human Epidermal Keratinocytes）が好適に挙げられる。 As "keratinocytes" used in the present invention, any normal keratinocytes having self-proliferation can be used, and cells derived from mammals such as humans, mice, rats, and pigs can be used. However, it is preferable to use human cells. When the keratinocytes are used, keratinocytes can be collected from the animal and treated according to a conventional method, and used. However, those which are commercially available as established cell lines (for example, HaCaT cells which are human epidermal keratinocyte cell lines, etc.) ) Can also be purchased and used. Suitable human cells include, for example, normal human keratinocytes (NHEK: Normal Human Epidermal Keratinocytes).
被験物質の種類は特に限定されず、天然物でも合成物でもよく、また単一物質であっても組成物若しくは混合物であってもよい。接触の形態は、被験物質に依存して、任意の形態であり得る。 The type of the test substance is not particularly limited, and may be a natural substance or a synthetic substance, and may be a single substance, a composition or a mixture. The form of contact can be in any form, depending on the test substance.
被験物質とケラチノサイトとの接触における、被験物質の濃度は、通常０．００００１質量％以上、好ましくは０．０００１質量％以上で、１質量％以下、好ましくは０．１質量％以下であり、また０．００００１〜１質量％、好ましくは０．０００１〜０．１質量％である。当該細胞と被験物質とを接触させる時間は、通常、１２〜１２０時間であり、４８〜７２時間が好ましい。 The concentration of the test substance in the contact between the test substance and keratinocytes is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, and 1% by mass or less, preferably 0.1% by mass or less; It is 0.00001 to 1% by mass, preferably 0.0001 to 0.1% by mass. The time for bringing the cells into contact with the test substance is usually 12 to 120 hours, preferably 48 to 72 hours.
当該細胞と被験物質との接触は、当該細胞が成育可能な条件で培養しながら行われる。例えば、増殖用添加剤（Ｈｕｍｅｄｉａ−ＫＧ）を含むＥｐｉｌｉｆｅ培地（Life Technologies製）、上記、増殖用添加剤のうちｈＥＧＦを含まないＥｐｉｌｉｆｅ培地（Life Technologies製）やCnT-Prime培地（CELLnTEC製）の他、適宜、抗生物質、血清、増殖因子等を添加した培地等の市販の培地中で培養できる。培養は、通常３０℃〜４０℃、１〜１０ｖｏｌ％二酸化炭素存在下で実施すればよく、３７℃、５ｖｏｌ％二酸化炭素存在下で実施するのが好ましい。 The contact between the cell and the test substance is performed while culturing the cell under conditions that allow the cell to grow. For example, an Epilife medium (manufactured by Life Technologies) containing a growth additive (Humedia-KG), an Epilife medium (manufactured by Life Technologies) and a CnT-Prime medium (manufactured by CELLnTEC) containing no hEGF among the growth additives described above are used. Alternatively, the cells can be cultured in a commercially available medium such as a medium to which antibiotics, serum, growth factors and the like are added as appropriate. The cultivation may be carried out usually at 30 ° C. to 40 ° C. in the presence of 1 to 10 vol% carbon dioxide, and preferably at 37 ° C. in the presence of 5 vol% carbon dioxide.
また、被験物質と接触させる際のケラチノサイトの細胞数は、例えば６ｗｅｌｌプレートを用いる場合、通常１０４〜１０６個／ｗｅｌｌであればよく、５×１０４〜５×１０５個／ｗｅｌｌが好ましい。 When a 6- well plate is used, the number of keratinocyte cells to be brought into contact with the test substance may be usually 10 4 to 10 6 cells / well, and preferably 5 × 10 4 to 5 × 10 5 cells / well. .
エクソソームの単離は、培養上清を回収した後、超遠心機を用いて、異なる速度で段階的に遠心処理することにより行うことができる(ref. Thery C et al. (2006) Curr Protoc Cell Biol., 3.22.1-3.22.29)。具体的には、培養上清を回収し、300gで10分間、2,000gで20分間、10,000gで30分間と段階的に遠心分離することにより、余分な細胞(破片)を除去した培養上清に対し、100,000gで70分間の遠心分離を行った後で上清を除去し、Phosphate Buffered Saline (PBS)にて一度洗浄し、さらに、100,000gで70分間遠心分離した後に完全に上清を除去することにより行うことができる。また、超遠心機を用いずに、エクソソーム膜に発現するタンパク質（CD9、CD63、そして、CD81など）に対する抗体でコートされたビーズを用いて、エクソソームを直接単離する免疫吸着法（抗体結合法）を用いることもできる(ref. Wubbolts R et al. (2003) J Biol Chem., 278: 10963-10972)。その他、市販のキット試薬（例えばExosome Precipitation Solution, ExoQuick-TC（System Biosciences社、#EXOTC10A-1 or EXOTC50A-1）やmiRCURY Exosome Isolation Kit (EXIQON社、#300102)、Total Exosome Isolation Reagent (invitrogen社、#4478359)等）を用いることでもよい。 The exosomes can be isolated by collecting the culture supernatant and centrifuging at different speeds using an ultracentrifuge (ref. Thery C et al. (2006) Curr Protoc Cell Biol., 3.22.1-3.22.29). Specifically, the culture supernatant from which the excess cells (debris) have been removed by collecting the culture supernatant and centrifuging stepwise at 300 g for 10 minutes, at 2,000 g for 20 minutes, and at 10,000 g for 30 minutes. After centrifugation at 100,000 g for 70 minutes, the supernatant was removed, washed once with Phosphate Buffered Saline (PBS), and further centrifuged at 100,000 g for 70 minutes to completely remove the supernatant. Removal can be performed. In addition, without using an ultracentrifuge, an immunoadsorption method (antibody binding method) is used to directly isolate exosomes using beads coated with antibodies against proteins (CD9, CD63, and CD81) expressed on exosome membranes. ) Can also be used (ref. Wubbolts R et al. (2003) J Biol Chem., 278: 10963-10972). In addition, commercially available kit reagents (for example, Exosome Precipitation Solution, ExoQuick-TC (System Biosciences, # EXOTC10A-1 or EXOTC50A-1), miRCURY Exosome Isolation Kit (EXIQON, # 300102), Total Exosome Isolation Reagent (invitrogen, # 4478359)).
エクソソームの分泌レベルの変化は、例えば、被験物質を接触させる細胞群と接触させない群（対照細胞）を用意し、エクソソームの分泌レベルを測定し、両者間で比較することが挙げられる。なお、対照細胞としては、被験物質を接触させない代わりに、対照物質を接触させたものを用いてもよい。 The change in the exosome secretion level includes, for example, preparing a cell group to be contacted with the test substance and a group not contacting (control cell), measuring the exosome secretion level, and comparing the two. In addition, instead of not contacting a test substance, a control cell may be used as a control cell.
エクソソームの分泌レベルの測定は、例えば、エクソソーム画分の総タンパク質量の測定、エクソソーム中に含まれるマーカー物質（ＣＤ９、ＣＤ６３、ＣＤ８１、ＨＳＰ７０、Ａｌｉｘ、Ｔｓｇ１０１、Ｈｒｓ、ＧＭ１ガングリオシドなど）の発現量の測定、エクソソームの粒径の分布と濃度の測定、エクソソーム数の測定、エクソソーム中に含まれるＲＮＡ（ｍＲＮＡ、ｓｍａｌｌ ＲＮＡ、ｍｉＲＮＡなど）の発現量の測定等によって行うことができる。好適には、４８〜７２時間の間に培養上清中に放出されたエクソソーム画分のタンパク質定量を行い、それを細胞数で除することにより、単位時間における１細胞あたりのエクソソーム放出量を算出することが挙げられる。
各種測定は、当該分野で通常使用される任意の解析方法を用いればよく、遺伝子発現解析には、例えば、ドットブロット法、ノーザンブロット法、ＲＮａｓｅプロテクションアッセイ法、ルシフェラーゼ等によるレポーターアッセイ、ＲＴ−ＰＣＲ法、ＤＮＡマイクロアレイ等を、タンパク質発現解析には、ウェスタンブロッティング法、免疫染色法、ＥＬＩＳＡ、バインディングアッセイ等を用いることができる。また、市販のキット（例えばCD63 ExoELISA Kit（System Biosciences社、#EXOEL-CD63A-1）等）やナノ粒子解析装置（NanoSight LM10（NanoSight社））を用いることもできる。
The measurement of the secretion level of the exosome includes, for example, the measurement of the total protein amount of the exosome fraction and the expression level of a marker substance (CD9, CD63, CD81, HSP70, Alix, Tsg101, Hrs, GM1 ganglioside, etc.) contained in the exosome. The measurement can be performed by measuring, measuring the distribution and concentration of exosome particle size, measuring the number of exosomes, measuring the expression level of RNA (mRNA, small RNA, miRNA, etc.) contained in exosomes, and the like. Preferably, the protein amount of the exosome fraction released into the culture supernatant during 48 to 72 hours is determined, and the exosome release amount per cell per unit time is calculated by dividing the exosome fraction by the number of cells. It is mentioned.
For various measurements, any analysis method commonly used in the art may be used. For gene expression analysis, for example, dot blot method, Northern blot method, RNase protection assay method, reporter assay using luciferase, etc., RT-PCR For example, Western blotting, immunostaining, ELISA, binding assay and the like can be used for protein expression analysis. Alternatively, a commercially available kit (for example, CD63 ExoELISA Kit (System Biosciences, # EXOEL-CD63A-1) or the like) or a nanoparticle analyzer (NanoSight LM10 (NanoSight)) can be used.
上記のように測定されたケラチノサイトにおけるエクソソームの分泌レベルの変化に基づき、メラニン制御剤を選択することができる。すなわち、被験物質（候補物質）を添加したエクソソームの分泌レベルが被験物質（候補物質）を添加しない対照細胞でのレベルと比較して、低下又は減少していれば、メラニン産生抑制剤、美白剤又はデンドライト伸長抑制剤として評価又は選択でき、増強又は増加していれば、メラニン産生促進剤、皮膚褐色化剤、白斑予防又は治療剤、白髪予防若しくは改善剤又はデンドライト伸長促進剤として評価又は選択することができる。 Based on the change in exosome secretion level in keratinocytes measured as described above, a melanin regulator can be selected. That is, if the secretion level of the exosome to which the test substance (candidate substance) is added is reduced or decreased compared to the level in control cells to which no test substance (candidate substance) is added, a melanin production inhibitor, a whitening agent Or, it can be evaluated or selected as a dendrite elongation inhibitor. be able to.
上述した実施形態に関し、本発明においては更に以下の態様が開示される。
＜１＞以下の工程ａ）〜ｃ）を含むメラニン制御剤の探索方法。
ａ）ケラチノサイトに被験物質を接触させ培養する工程
ｂ）培養細胞からエクソソーム画分を単離し、分泌レベルを測定する工程
ｃ）エクソソームの分泌レベルを変化させる被験物質をメラニン制御剤として選択する工程
＜２＞ｂ）工程における分泌レベルの測定が、エクソソーム画分のタンパク質量を測定し、1細胞あたりのエクソソーム放出量を算出する、＜１＞の方法。
＜３＞エクソソームの分泌レベルを増強又は増加させる被験物質を、メラニン産生促進剤、デンドライト伸長促進剤、皮膚褐色化剤、白斑予防若しくは治療剤、又は白髪予防若しくは改善剤として評価及又は選択する＜１＞又は＜２＞の方法。
＜４＞エクソソームの分泌レベルを低下又は減少させる被験物質を、メラニン産生抑制剤、デンドライト伸長抑制剤又は美白剤としてとして評価又は選択する＜１＞又は＜２＞の方法。
＜５＞前記被験物質の濃度は、通常０．００００１質量％以上、好ましくは０．０００１質量％以上で、１質量％以下、好ましくは０．１質量％以下であり、また０．００００１〜１質量％、好ましくは０．０００１〜０．１質量％である＜１＞〜＜４＞の方法。
＜６＞前記細胞と被験物質とを接触させる時間は、通常、１２〜１２０時間であり、好ましくは４８〜７２時間である＜１＞〜＜５＞の方法。
Regarding the above-described embodiment, the present invention further discloses the following aspects.
<1> A method for searching for a melanin controlling agent comprising the following steps a) to c).
a) a step of contacting and culturing a test substance with keratinocytes; b) a step of isolating an exosome fraction from cultured cells and measuring a secretion level; c) a step of selecting a test substance that changes the secretion level of exosomes as a melanin controlling agent <2> The method according to <1>, wherein the measurement of the secretion level in step b) comprises measuring the amount of protein in the exosome fraction and calculating the amount of exosome released per cell.
<3> Evaluate or select a test substance that enhances or increases the exosome secretion level as a melanin production promoter, a dendrite elongation promoter, a skin browning agent, a vitiligo prevention or treatment agent, or a gray hair prevention or improvement agent < The method according to 1> or <2>.
<4> The method according to <1> or <2>, wherein the test substance that reduces or decreases the secretion level of exosomes is evaluated or selected as a melanin production inhibitor, a dendrite elongation inhibitor or a whitening agent.
<5> The concentration of the test substance is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, 1% by mass or less, preferably 0.1% by mass or less, and 0.00001 to 1% by mass. <1> to <4>, in which the amount is preferably 0.0001 to 0.1% by mass.
<6> The method according to <1> to <5>, wherein the time for contacting the cells with the test substance is generally 12 to 120 hours, preferably 48 to 72 hours.
以下、実施例を示し、本発明をより具体的に説明する。
実施例1：ケラチノサイト由来エクソソームの単離とその特徴付け
（１）細胞培養
正常ヒト新生児包皮由来表皮角化細胞(凍結NHEK (F) Lightly; ケラチノサイト)、表皮角化細胞用増殖培地(Epilife)はLife Technologies社より、その培地用増殖添加剤(Humedia-KG)はクラボウ社より購入した。ケラチノサイトは、37℃ 5Vol%CO2の環境下で培養した。
Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1: Isolation and Characterization of Keratinocyte-Derived Exosome (1) Cell Culture Normal human neonatal foreskin-derived epidermal keratinocytes (frozen NHEK (F) Lightly; keratinocytes) and a growth medium for epidermal keratinocytes (Epilife) The culture medium growth additive (Humedia-KG) was purchased from Life Technologies, Inc. from Kurabo Industries. Keratinocytes were cultured at 37 ° C. in an environment of 5 Vol% CO 2 .
（２）ケラチノサイト由来エクソソームの単離
ケラチノサイトを2.5×106cells/flask (25mL/flask)の細胞密度で175cm2 flaskに播種した。この時の培地としては、ヒト上皮細胞成長因子(hEGF; Human Epidermal Growth Factor)以外のHumedia-KG増殖添加剤（インスリン、ハイドロコーチゾン、BPE、ゲンタマイシン/アンフォテリシンB）を含むEpilife培地を使用した。3日間の培養後、培養上清を回収し、300gで10分間、2,000gで20分間、10,000gで30分間と段階的に遠心分離することにより、余分な細胞(破片)を除去した。この操作を終えた後の培養上清に対し、100,000gで70分間の遠心分離を行った後で上清を除去し、Phosphate Buffered Saline (PBS)にて一度洗浄した。さらに、100,000gで70分間遠心分離した後に完全に上清を除去し、沈殿物をPBSにて懸濁した。100mLの培養上清から遠心分離した場合、最終沈殿物は200μLのPBSにて懸濁し、これを原液として評価に用いた。また、100,000g遠心後の上清はAmicon(R) Ultra Centrifugal Filters Ultra(R)-3K (Millipore)に供し、4,800rpmで45分間遠心処理を行った。遠心処理後にフィルター上部に残留した液を培養上清濃縮物として回収した。また、細胞非培養の培地に対して同様の処理を施した液を培地濃縮物として回収した。
(2) Isolation of exosomes derived from keratinocytes Keratinocytes were seeded at a cell density of 2.5 × 10 6 cells / flask (25 mL / flask) in a 175 cm 2 flask. As a medium at this time, an Epilife medium containing a Humedia-KG growth additive (insulin, hydrocortisone, BPE, gentamicin / amphotericin B) other than human epidermal growth factor (hEGF) was used. After culturing for 3 days, the culture supernatant was recovered, and excess cells (debris) were removed by stepwise centrifugation at 300 g for 10 minutes, 2,000 g for 20 minutes, and 10,000 g for 30 minutes. After the operation, the culture supernatant was centrifuged at 100,000 g for 70 minutes, and then the supernatant was removed and washed once with Phosphate Buffered Saline (PBS). Furthermore, after centrifugation at 100,000 g for 70 minutes, the supernatant was completely removed, and the precipitate was suspended in PBS. When centrifuged from 100 mL of the culture supernatant, the final precipitate was suspended in 200 μL of PBS and used as a stock solution for evaluation. The supernatant after centrifugation at 100,000 g was supplied to Amicon® Ultra Centrifugal Filters Ultra®-3K (Millipore) and centrifuged at 4,800 rpm for 45 minutes. The liquid remaining on the upper part of the filter after the centrifugation was collected as a culture supernatant concentrate. In addition, a liquid obtained by performing the same treatment on a medium in which cells were not cultured was collected as a medium concentrate.
（３）単離したエクソソーム画分の電子顕微鏡観察
単離したエクソソーム画分をPBSもしくはHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) bufferにて適宜希釈した。その溶液を親水化処理したコロジオン膜貼付メッシュ 400メッシュ(日新EM)に5μL滴下し、5分間静置した。膜上の水分を吸い取った後、2% Sodium Dodecatungsto(VI) phosphate n-Hydrate (wako)もしくはEMステイナー(日新EM)を10μL滴下し、1分間もしくは30分間静置することにより、ネガティブ染色を行った。膜上の水分を除いた後、精製水を10μL滴下した。余分な水分を除いた後、H7650透過型電子顕微鏡(日立)を用いて加速電圧100kVで観察した。その結果、既報と同様に、エクソソームに特徴的なサイズ (直径30-100nm)と形態(cup-shaped) を示す小胞を確認した(図1)。
(3) Electron microscopic observation of the isolated exosome fraction The isolated exosome fraction was appropriately diluted with PBS or HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer. 5 μL of the solution was dropped on a hydrocolloid-coated membrane 400 mesh (Nissin EM) and left to stand for 5 minutes. After absorbing the moisture on the membrane, 10% of 2% Sodium Dodecatungsto (VI) phosphate n-Hydrate (wako) or EM Stainer (Nissin EM) is dropped and left for 1 minute or 30 minutes to allow negative staining. went. After removing the water on the membrane, 10 μL of purified water was added dropwise. After removing excess water, observation was performed using an H7650 transmission electron microscope (Hitachi) at an acceleration voltage of 100 kV. As a result, as in the previous report, vesicles showing the characteristic size (30-100 nm in diameter) and morphology (cup-shaped) of exosomes were confirmed (FIG. 1).
（４）単離したエクソソーム画分におけるマーカータンパク質の発現確認
単離したエクソソーム画分をResuspension buffer (ambion)にて溶解し、その溶液についてBovine Serum Albumin (BSA)を標準物質としたBCA（ビシンコニン酸）法によりタンパク質定量を行った後、細胞抽出物、培養上清濃縮物及び培地濃縮物と共に、タンパク質量を揃え、定法に従ってSDS-PAGE及びウェスタンブロットに供した。一次抗体は、anti-CD9 (Santacruz, 1:1000)抗体、anti-CD63 (Santacruz, 1:1000)抗体、anti-Calnexin (Cell Signaling, 1:1000)抗体、anti-HSP70 (Cell Signaling, 1:1000)抗体、あるいはanti-β-actin (Sigma-Aldrich, 1:5000)抗体を用いた。二次抗体は、anti-mouse IgG, HRP-Linked F(ab’)2Fragment Sheep (GE Healthcare Life Science)あるいはanti-rabbit IgG HRP-Linked F(ab’)2 Fragment Donkey (GE Healthcare Life Science) を用いた。その後、ECL plus western blotting detection reagents (GE healthcare bioscience)を用いて発色させ、LAS4000(富士フィルム)を用いて可視化した。その結果、エクソソームに特徴的に発現するマーカーとされるタンパク質群、すなわち、細胞質に存在するHeat shock protein 70、細胞骨格タンパク質であるActin、また、テトラスパニン(CD9、CD63)の発現がエクソソーム画分に確認され、さらにエクソソームには含有されないとして知られるCalnexinの発現が認められないことも確認された(図2)。それと同時に、ケラチノサイトの培養上清濃縮物(図2; lane2)には、Hsp70を除くエクソソームマーカータンパク質の発現が認められなかったことから、エクソソームがその濃縮過程で除去されていることを確認した。なお、Hsp70の発現が培養上清濃縮物で認められたことは既報（Chavez-Munoz C. et al., 2008, J.Cell. Biochem. 104:2165-2173）と同様の結果である。
(4) Confirmation of expression of marker protein in isolated exosome fraction The isolated exosome fraction was dissolved in Resuspension buffer (ambion), and the solution was subjected to BCA (bicinchoninic acid) using Bovine Serum Albumin (BSA) as a standard substance. After the protein quantification was carried out according to the method described above, the protein amount was adjusted together with the cell extract, the culture supernatant concentrate and the medium concentrate, and subjected to SDS-PAGE and Western blot according to a standard method. Primary antibodies are anti-CD9 (Santacruz, 1: 1000) antibody, anti-CD63 (Santacruz, 1: 1000) antibody, anti-Calnexin (Cell Signaling, 1: 1000) antibody, anti-HSP70 (Cell Signaling, 1: 1000). 1000) antibody or anti-β-actin (Sigma-Aldrich, 1: 5000) antibody was used. Secondary antibody, anti-mouse IgG, HRP-Linked F (ab ') 2 Fragment Sheep (GE Healthcare Life Science) or anti-rabbit IgG HRP-Linked F (ab') 2 Fragment Donkey (GE Healthcare Life Science) Using. Thereafter, color was developed using ECL plus western blotting detection reagents (GE healthcare bioscience) and visualized using LAS4000 (Fuji Film). As a result, the expression of exosomes as a group of proteins characteristically expressed in exosomes, that is, Heat shock protein 70 present in the cytoplasm, Actin cytoskeletal protein, and tetraspanin (CD9, CD63) expression in the exosome fraction It was also confirmed that the expression of Calnexin, which is not contained in exosomes, was not observed (FIG. 2). At the same time, the keratinocyte culture supernatant concentrate (Fig. 2; lane2) showed no expression of exosome marker proteins except Hsp70, confirming that exosomes were removed during the enrichment process. . Note that the expression of Hsp70 was observed in the culture supernatant concentrate, which is the same result as reported previously (Chavez-Munoz C. et al., 2008, J. Cell. Biochem. 104: 2165-2173).
以上のことより、ケラチノサイトの培養上清より単離した画分がエクソソーム画分と呼ぶに値するものと判断し、これを用いて、以降の評価を行った。 Based on the above, the fraction isolated from the keratinocyte culture supernatant was judged to be worthy of being called the exosome fraction, and the following evaluation was performed using this.
実施例2：ケラチノサイト由来エクソソームがメラノサイトに及ぼす影響
（１）細胞培養
正常ヒト新生児包皮由来表皮メラニン細胞(凍結NHEM (NB) Darkly; メラノサイト)、表皮メラニン細胞用増殖培地(Medium254)、その培地用増殖添加剤(HMGS)はLife Technologiesより購入した。メラノサイトは、37℃ 5Vol%CO2の環境下で培養した。
Example 2: Effect of keratinocyte-derived exosomes on melanocytes (1) Cell culture Normal human neonatal foreskin-derived epidermal melanocytes (frozen NHEM (NB) Darkly; melanocytes), growth medium for epidermal melanocytes (Medium 254), growth for the medium Additive (HMGS) was purchased from Life Technologies. The melanocytes were cultured at 37 ° C. in an environment of 5 Vol% CO 2 .
（２）メラノサイトに対するケラチノサイト由来エクソソーム画分の処理
メラノサイトを1.0×104 cells/well (100μL/well)、1.0×105 cells/well (500μL/well)及び1.5×105 cells/well (1.5mL/well)の細胞密度で96-well plate、12-well plate及び6-well plateにそれぞれ播種した。この時の試験用培地としては、Medium254 (増殖添加剤（HMGS）のうち、Fetal Bovine Serum (FBS)、Human Fibroblast growth factor-basic (hFGF-B)、ハイドロコーチゾン、インスリン、トランスフェリン、ヘパリン含有、phorbol 12-myristate 13-acetate (PMA)及びbovine pituitary extract (BPE)不含）を用いた。2日後、PBSにて懸濁したエクソソーム画分を添加した後、さらに、96-well plateにおいては5日間、12-well plateにおいては3日間もしくは4日間培養し、それぞれドーパオキシダーゼ活性の計測及び定量的RT-PCR解析に用いた。6-well plateにおいては5日間培養した後に、細胞数の計測及び形態観察、ウェスタンブロッティング解析、さらに、細胞内メラニン定量に用いた。
(2) Treatment of keratinocyte-derived exosome fraction on melanocytes Melanocytes were treated with 1.0 × 10 4 cells / well (100 μL / well), 1.0 × 10 5 cells / well (500 μL / well) and 1.5 × 10 5 cells / well (1.5 mL) / well) at a cell density of 96-well plate, 12-well plate and 6-well plate, respectively. The medium for the test at this time was Medium254 (of Fetal Bovine Serum (FBS), Human Fibroblast growth factor-basic (hFGF-B), hydrocortisone, insulin, transferrin, heparin-containing, phorbol 12-myristate 13-acetate (PMA) and bovine pituitary extract (BPE) were not used). Two days later, after the exosome fraction suspended in PBS was added, the cells were further cultured for 5 days in a 96-well plate, 3 days or 4 days in a 12-well plate, and the measurement and quantification of dopa oxidase activity, respectively. RT-PCR analysis. After culturing for 5 days in a 6-well plate, the cells were used for cell number measurement and morphological observation, Western blotting analysis, and further for intracellular melanin quantification.
（３）ケラチノサイト由来エクソソームがメラノサイトのドーパオキシダーゼ活性に及ぼす影響
96-well plateでの培養終了後、培地を全量置換し(100μL/well)、アラマーブルー(Bio-Rad AbD Serotec Limited)試薬を10μL/wellになるように添加した。37℃、5Vol%CO2の条件下で1時間インキュベートした後、培地の蛍光強度 (Ex544nm/Em590nm)を測定することで細胞呼吸鎖(増殖)活性を評価した。その後、細胞の培養プレートをPBSで3回洗浄して、抽出Buffer (0.1M Tris-HCL (pH:7.2）、1% NP-40、0.01% SDS)を20μL/well、Assay Buffer (4%ジメチルホルムアミド、100mM Sodium phosphate-buffered (pH:7.1))を20μL/well添加した。4℃にて2時間かけて細胞を可溶化し、ドーパオキシダーゼ活性の測定を行ったが、その活性測定は、MBTH法（Winder A. et al., 1991, Eur.J. Biochem. 198:317-326）を基本とした次に示す方法で行った。すなわち、可溶化した細胞溶液の各wellに、上記Assay Bufferを80μL、20.7mM MBTH (3-メチル-2-ベンゾチアゾリノン ヒドラゾン)溶液を60μL、基質として5mM L-DOPA (L-ジヒドロキシフェニルアラニン)溶液を40μL加え、37℃にて30〜60分間反応させた後、その吸光度を505nmの測定波長で測定した。その結果、PBSのみを添加したコントロールと比較して、同画分添加による両活性の有意な上昇を確認した(図3A、B)。
(3) Effect of keratinocyte-derived exosomes on melanocyte dopa oxidase activity
After completion of the culture in the 96-well plate, the medium was completely replaced (100 μL / well), and Alamar Blue (Bio-Rad AbD Serotec Limited) reagent was added to 10 μL / well. 37 ° C., after 1 hour under the conditions of 5 vol% CO 2, cells were assessed respiratory chain (proliferation) activity by measuring the medium fluorescence intensity (Ex 544nm / Em 590nm). After that, the cell culture plate is washed three times with PBS, and the extraction buffer (0.1 M Tris-HCL (pH: 7.2), 1% NP-40, 0.01% SDS) is added at 20 μL / well, and the assay buffer (4% dimethyl Formamide, 100 mM sodium phosphate-buffered (pH: 7.1)) was added at 20 μL / well. The cells were solubilized at 4 ° C. for 2 hours and the dopa oxidase activity was measured. The activity was measured by the MBTH method (Winder A. et al., 1991, Eur. J. Biochem. 198: 317). -326) based on the following method. That is, in each well of the solubilized cell solution, 80 μL of the above-mentioned Assay Buffer, 60 μL of 20.7 mM MBTH (3-methyl-2-benzothiazolinone hydrazone) solution, and 5 mM L-DOPA (L-dihydroxyphenylalanine) as a substrate After adding 40 μL of the solution and reacting at 37 ° C. for 30 to 60 minutes, the absorbance was measured at a measurement wavelength of 505 nm. As a result, a significant increase in both activities was confirmed by the addition of the same fraction as compared to the control to which only PBS was added (FIGS. 3A and 3B).
（４）ケラチノサイト由来エクソソームがメラノサイトのメラニン関連遺伝子の発現量に及ぼす影響
12-well plateでの培養終了後、細胞をPBSで洗浄した後、RNeasy Mini Kit (QIAGEN）を用いて定法に従いtotal RNAを抽出した。その抽出したtotal RNAを鋳型とし、High Capacity RNA to cDNA Kit (Life Technologies)を用いた逆転写反応によりcDNAを合成した。反応には、MH Research社製のPeltier Thermal Cyclerを用いた。続いて合成したcDNA及びTaqMan(R)probeを用いて、定量的PCR法による遺伝子発現解析を行った。各遺伝子に特異的なprobe及びprimerは、Life Technologies社製のTaqMan(R)Gene Expression Assays (P/N 4331182)を使用した。各々の発現量は、内部標準遺伝子であるribosomal protein, large, P0 (RPLP0)の発現量により補正した。反応条件は定法に従い、アプライドバイオシステムズ社製のシークエンスディテクター(ABI PRISM 7500 Real Time PCR System)を用いて行った。その結果、エクソソーム画分を添加して72時間後にMITFの発現上昇を確認し(図4A)、96時間後にはTYR、TYRP1、Dopachrome tautomerase (DCT) /TYRP2の発現上昇を確認した(図4B)。
(4) Effect of keratinocyte-derived exosomes on melanocyte melanin-related gene expression
After completion of the culture in the 12-well plate, the cells were washed with PBS, and then total RNA was extracted using a RNeasy Mini Kit (QIAGEN) according to a standard method. Using the extracted total RNA as a template, cDNA was synthesized by a reverse transcription reaction using a High Capacity RNA to cDNA Kit (Life Technologies). For the reaction, a Peltier Thermal Cycler manufactured by MH Research was used. Subsequently, gene expression analysis was performed by quantitative PCR using the synthesized cDNA and TaqMan (R) probe. Probes and primers specific to each gene used TaqMan (R) Gene Expression Assays (P / N 4331182) manufactured by Life Technologies. Each expression level was corrected by the expression level of the internal standard gene, ribosomal protein, large, P0 (RPLP0). Reaction conditions were performed according to a standard method using a sequence detector (ABI PRISM 7500 Real Time PCR System) manufactured by Applied Biosystems. As a result, an increase in the expression of MITF was confirmed 72 hours after the addition of the exosome fraction (FIG. 4A), and an increase in the expression of TYR, TYRP1, Dopachrome tautomerase (DCT) / TYRP2 was confirmed after 96 hours (FIG. 4B). .
（５）ケラチノサイト由来エクソソームがメラノサイトの細胞増殖能及び形態形成に及ぼす影響
6-well plateでの培養終了後、顕微鏡観察をおこない、画像を記録した。その後、0.25% Trypsin-EDTA 溶液(Life Technologies)処理を施して、細胞接着を剥離した後に回収し、血球計算板を用いて細胞数のカウントを行った。その結果、エクソソーム画分を添加して5日後に、PBSを添加したコントロールに対して、細胞数の有意な増加を確認すると共に(図5A)、デンドライトの顕著な伸長を確認した(図5B)。
(5) Effect of keratinocyte-derived exosomes on cell proliferation and morphogenesis of melanocytes
After completion of the culture in the 6-well plate, microscopic observation was performed and an image was recorded. Thereafter, the cells were treated with a 0.25% Trypsin-EDTA solution (Life Technologies) to remove the cell adhesion, collected, and counted using a hemocytometer. As a result, 5 days after adding the exosome fraction, a significant increase in the number of cells was confirmed with respect to the control to which PBS was added (FIG. 5A), and remarkable elongation of dendrite was confirmed (FIG. 5B). .
（６）ケラチノサイト由来エクソソームがメラノサイトのメラニン関連タンパク質の発現量に及ぼす影響
6-well plateでの培養終了後、細胞をPBSで洗浄し、RIPA buffer (Sigma-Aldrich)を用いて回収してから、超音波処理により細胞を破砕した。その後、15,000rpmで15分間遠心分離し、その上清についてBovine Serum Albumin (BSA)を標準物質としたBCA（ビシンコニン酸）法によりタンパク量を定量した後、各群のタンパク質量を揃え、定法に従ってSDS-PAGE及びウェスタンブロットに供した。一次抗体は、anti-MITF (Santacruz, 1:200)、anti-Tyrosinase related protein (TRP) 1 (Santacruz, 1:1000)、anti-TRP2 (Santacruz, 1:200)、あるいはanti-Tyrosinase (Zymed, 1:1000)を用いた。二次抗体は、anti-mouse IgG, HRP-Linked F(ab’)2 Fragment Sheep (GE Healthcare Life Science)、あるいはanti-goat IgG, HRP-Linked F(ab’)2Fragment Sheep (GE Healthcare Life Science)を用いた。 その後、ECL plus western blotting detection reagents (GE healthcare bioscience)を用いて発色させ、LAS4000 (GE healthcare bioscience)を用いて発現量を可視化した。内部標準としてのβ-actinの発現は、monoclonal antibody specific for β-actin (Sigma-Aldrich, 1:5000)を用いて評価した。上記タンパク質の検出バンドの強度を画像解析により定量した結果、PBSを添加したコントロールに対して、全てのタンパク質が有意に発現上昇していることを確認した(図6)。
(6) Effect of keratinocyte-derived exosomes on melanocyte melanin-related protein expression
After completion of the culture in the 6-well plate, the cells were washed with PBS, collected using a RIPA buffer (Sigma-Aldrich), and then disrupted by sonication. After centrifugation at 15,000 rpm for 15 minutes, the supernatant was quantified by the BCA (Bicinchoninic acid) method using Bovine Serum Albumin (BSA) as a standard substance. The samples were subjected to SDS-PAGE and Western blot. Primary antibodies are anti-MITF (Santacruz, 1: 200), anti-Tyrosinase related protein (TRP) 1 (Santacruz, 1: 1000), anti-TRP2 (Santacruz, 1: 200), or anti-Tyrosinase (Zymed, 1: 200). 1: 1000). Secondary antibody is anti-mouse IgG, HRP-Linked F (ab ') 2 Fragment Sheep (GE Healthcare Life Science) or anti-goat IgG, HRP-Linked F (ab') 2 Fragment Sheep (GE Healthcare Life Science ) Was used. Thereafter, the color was developed using ECL plus western blotting detection reagents (GE healthcare bioscience), and the expression level was visualized using LAS4000 (GE healthcare bioscience). The expression of β-actin as an internal standard was evaluated using a monoclonal antibody specific for β-actin (Sigma-Aldrich, 1: 5000). As a result of quantifying the intensities of the detection bands of the above proteins by image analysis, it was confirmed that the expression of all proteins was significantly increased with respect to the control to which PBS was added (FIG. 6).
（７）ケラチノサイト由来エクソソームがメラノサイトのメラニン産生に及ぼす影響
6-well plateでの培養終了後、細胞をPBSで洗浄し、セルスクレーパーを用いてマイクロチューブに細胞を回収した。各マイクロチューブに2M NaOHを150μL加えた後100℃にて細胞を溶解させ、遠心処理によって得られた上清について405nmの測定波長で吸光度を測定し、メラニン量を算出した。その結果、エクソソーム画分を添加して5日後に、PBSを添加したコントロールに対して、メラニン産生が促進傾向にあることを確認した(図7)。
(7) Effect of keratinocyte-derived exosomes on melanocyte melanin production
After completion of the culture in the 6-well plate, the cells were washed with PBS, and the cells were collected in a microtube using a cell scraper. After 150 μL of 2M NaOH was added to each microtube, the cells were lysed at 100 ° C., and the supernatant obtained by centrifugation was measured for absorbance at a measurement wavelength of 405 nm to calculate the amount of melanin. As a result, 5 days after the addition of the exosome fraction, it was confirmed that melanin production tended to be promoted relative to the control to which PBS was added (FIG. 7).
以上のことより、ケラチノサイトが恒常的に分泌しているエクソソームがメラノサイトの細胞活性及びメラニン産生能を正に制御していることが示された。 From the above, it was shown that exosomes constantly secreted by keratinocytes positively control the cell activity and melanin-producing ability of melanocytes.
実施例３：異なる人種由来のヒト皮膚組織におけるエクソソーム含有量とメラノソームタンパク量との関連性
米国スキンバンクNational Disease Research Interchange (NDRI)から、外科手術由来の正常ヒト皮膚組織を提供頂いた。人種の異なる7名の被験者由来の皮膚組織から外科的に脂肪及び真皮の除去を行った後でタンパク質を抽出し、ウェスタンブロット解析により、メラノソーム特異的なタンパク質であるPmel17の発現を検出し、それらのバンド強度を画像解析により定量した(図8A)。一方、10mLのhomogenization buffer (20mM Sodium phosphate、 pH6.5、0.15M NaCl、10mM ethylenediaminetetraacetic acid (EDTA)、1mM dithiothreitol (DTT)、Halt protease inhibitor cocktail (Thermo Scientific))中でガラス製ホモジナイザーを用いて組織破砕を行った。続いて、300gで10分間、2,000gで20分間、遠心分離することにより、余分な組織(破片)を除去した。この破砕液の上清を回収してタンパク質量を定量した後、約6mg分のタンパク質を含む液に調製し、10,000gで30分間、100,000gで70分間の遠心処理を行った。上清を除去し、PBSにて一度洗浄してから、さらに100,000gで70分間遠心処理を行い、完全に上清を除去して沈殿物をPBSに懸濁することで、エクソソーム画分を単離した。そして、そのタンパク質定量を行うことで、表皮組織中のエクソソーム含有量を算出した(図8B)。双方の結果から、Pmel17の発現量と表皮組織中のエクソソーム含有量の間に統計学的に有意な正の相関が見出された(図8C)。
このことより、実際のヒト皮膚組織において、エクソソームの量に応じてメラニン合成が正に制御され、皮膚色に影響を与えている可能性が示された。
Example 3: Relationship between exosome content and melanosome protein levels in human skin tissues from different races Normal surgical skin-derived human skin tissues were provided by National Skin Research Interchange (NDRI), USA. After surgical removal of fat and dermis from skin tissue from seven subjects of different races, the protein was extracted, and by western blot analysis, the expression of Pmel17, a melanosome-specific protein, was detected. Their band intensities were quantified by image analysis (FIG. 8A). On the other hand, the tissue using a glass homogenizer in 10 mL of homogenization buffer (20 mM Sodium phosphate, pH 6.5, 0.15 M NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), Halt protease inhibitor cocktail (Thermo Scientific)). Crushing was performed. Subsequently, excess tissue (debris) was removed by centrifugation at 300 g for 10 minutes and at 2,000 g for 20 minutes. After collecting the supernatant of the crushed liquid and quantifying the amount of protein, it was prepared into a liquid containing about 6 mg of protein, and centrifuged at 10,000 g for 30 minutes and at 100,000 g for 70 minutes. The supernatant was removed, washed once with PBS, and then centrifuged at 100,000 g for 70 minutes.The supernatant was completely removed, and the precipitate was suspended in PBS to separate the exosome fraction. Released. Then, by quantifying the protein, the exosome content in the epidermal tissue was calculated (FIG. 8B). From both results, a statistically significant positive correlation was found between the expression level of Pmel17 and the exosome content in epidermal tissue (FIG. 8C).
This indicates that in actual human skin tissue, melanin synthesis was positively controlled in accordance with the amount of exosomes, possibly affecting skin color.
実施例4：エクソソーム放出促進剤D609によるメラニン産生の促進
（１）細胞培養
正常ヒト新生児包皮由来表皮角化細胞(凍結NHEK (F) Lightly; ケラチノサイト)、表皮角化細胞用増殖培地(Epilife)はLife Technologies社より、その培地用増殖添加剤(Humedia-KG)はクラボウ社より購入し、また、正常ヒト新生児包皮由来表皮メラニン細胞(凍結NHEM (NB) Darkly; メラノサイト)、表皮メラニン細胞用増殖培地(Medium254)、その培地用増殖添加剤(HMGS)はLife Technologies社より購入した。ケラチノサイト及びメラノサイトは、37℃ 5%CO2の環境下で培養した。
Example 4: Promotion of melanin production by exosome release enhancer D609 (1) Cell culture Normal human neonatal foreskin-derived epidermal keratinocytes (frozen NHEK (F) Lightly; keratinocytes), a growth medium for epidermal keratinocytes (Epilife) From Life Technologies, the culture growth additive (Humedia-KG) was purchased from Kurabo Co., Ltd., and normal human neonatal foreskin-derived epidermal melanocytes (frozen NHEM (NB) Darkly; melanocytes), epidermal melanocyte growth medium (Medium 254) and its culture growth additive (HMGS) were purchased from Life Technologies. Keratinocytes and melanocytes were cultured at 37 ° C. in an environment of 5% CO 2 .
（２）エクソソーム放出促進剤D609がケラチノサイトのエクソソーム放出量に及ぼす影響
ケラチノサイトを1.5×106 cells/flask (25mL/flask)の細胞密度で175cm2 flaskに播種した。この時の培地としては、ヒト上皮細胞成長因子(hEGF; Human Epidermal Growth Factor) 以外のHumedia-KG増殖添加剤（インスリン、ハイドロコーチゾン、BPE、ゲンタマイシン/アンフォテリシンB）を含むEpilife培地を使用した。播種した翌日に、エクソソームの放出促進剤として報告（国際特許公開第2013/054534号）されているD609 (トリシクロデカン-9-イルキサントゲン酸カリウム；Tocris Bioscience社)を終濃度で5μMになるように添加し、37℃、5Vol%CO2の条件下で3日間培養を行った。コントロールとしては、同量のDMSOを添加した。培養終了後、細胞の培養上清から既に記載した遠心分離法によりエクソソーム画分を単離した。培養上清を除去した後に、底面に接着している細胞に対してプロナーゼ溶液(極東製薬工業)処理を施して、細胞接着を剥離した後に回収し、血球計算板を用いて細胞数のカウントを行った。また、エクソソーム画分のタンパク質定量を行い、それを細胞数で除することにより、1細胞あたりのエクソソーム放出量を算出した。その結果、D609の添加により、DMSOを添加したコントロールに対して、その放出量が有意に増加することが確認された(図9)。
(2) Effect of exosome release promoter D609 on exosome release amount of keratinocytes Keratinocytes were seeded at a cell density of 1.5 × 10 6 cells / flask (25 mL / flask) in a 175 cm 2 flask. As a medium at this time, an Epilife medium containing a Humedia-KG growth additive (insulin, hydrocortisone, BPE, gentamicin / amphotericin B) other than human epidermal growth factor (hEGF) was used. The next day after seeding, D609 (potassium tricyclodecane-9-ylxanthogenate; Tocris Bioscience), reported as an exosome release enhancer (International Patent Publication No. 2013/054534), was adjusted to a final concentration of 5 μM. , And cultured at 37 ° C. under 5 Vol% CO 2 for 3 days. As a control, the same amount of DMSO was added. After completion of the culture, the exosome fraction was isolated from the culture supernatant of the cells by the centrifugation method described above. After removing the culture supernatant, the cells adhering to the bottom surface were treated with a pronase solution (Kyokuto Pharmaceutical Co., Ltd.) to remove the cell adhesion, collected, and counted using a hemocytometer. went. In addition, protein quantification of the exosome fraction was performed, and the exosome release amount per cell was calculated by dividing the exosome fraction by the number of cells. As a result, it was confirmed that the addition of D609 significantly increased the release amount as compared to the control to which DMSO was added (FIG. 9).
（３）エクソソーム放出促進剤D609がケラチノサイト及びメラノサイトからなる共培養系においてメラニン産生に及ぼす影響
ケラチノサイトを6-well plateに1×105 cells/well (2mL/well)の細胞密度で播種し、翌日、メラノサイトを2×105 cells/well (1mL/well)の細胞密度で播種した。さらにその翌日に、D609を終濃度で5μMになるように添加し、37℃、5Vol%CO2の条件下で7日間培養を行った。培地には、試験用培地としてヒト上皮細胞成長因子(hEGF; Human Epidermal Growth Factor) 以外のHumedia-KG増殖添加剤（インスリン、ハイドロコーチゾン、BPE、ゲンタマイシン/アンフォテリシンB）を含むEpilife培地を用いた。コントロールとしては、同量のDMSOを添加した。なお、培地交換は3日に1度行った。培養終了後、細胞の培養プレートをPBSで洗浄し、セルスクレーパーを用いてマイクロチューブに細胞を回収した。各マイクロチューブに2M NaOHを150μL加えた後100℃にて細胞を溶解させ、遠心処理によって得られた上清について405nmの測定波長で吸光度を測定し、メラニン量を算出した。その結果、DMSOを添加したコントロールに対して、有意にメラニン産生が促進していることが確認され、また、細胞の黒化は視覚的にも認められた(図10)。
(3) Effect of exosome release enhancer D609 on melanin production in a co-culture system consisting of keratinocytes and melanocytes Keratinocytes were seeded in a 6-well plate at a cell density of 1 × 10 5 cells / well (2 mL / well), and the next day Then, melanocytes were seeded at a cell density of 2 × 10 5 cells / well (1 mL / well). The next day, D609 was added to a final concentration of 5 μM, and the cells were cultured at 37 ° C. and 5 Vol% CO 2 for 7 days. As a medium, an Epilife medium containing a Humedia-KG growth additive (insulin, hydrocortisone, BPE, gentamicin / amphotericin B) other than human epidermal growth factor (hEGF) was used as a test medium. As a control, the same amount of DMSO was added. The medium was changed once every three days. After completion of the culture, the cell culture plate was washed with PBS, and the cells were collected in a microtube using a cell scraper. After 150 μL of 2M NaOH was added to each microtube, the cells were lysed at 100 ° C., and the supernatant obtained by centrifugation was measured for absorbance at a measurement wavelength of 405 nm to calculate the amount of melanin. As a result, it was confirmed that the production of melanin was significantly promoted as compared with the control to which DMSO was added, and the darkening of the cells was visually recognized (FIG. 10).
（４）エクソソーム放出促進剤D609がメラノサイトの単独培養系においてメラニン産生に及ぼす影響
メラノサイトを2×105 cells/well (2mL/well)の細胞密度で播種した。その翌日に、D609を終濃度で1、5、10μMになるように添加し、37℃、5Vol%CO2の条件下で7日間培養を行った。培地には、試験用培地としてMedium254 (増殖添加剤（HMGS）のうち、Fetal Bovine Serum (FBS)、Human Fibroblast growth factor-basic (hFGF-B)、ハイドロコーチゾン、インスリン、トランスフェリン、ヘパリン含有、phorbol 12-myristate 13-acetate (PMA)及びbovine pituitary extract (BPE)不含））を用いた。コントロールとしては、同量のDMSOを添加した。なお、培地交換は3日に1度行った。培養終了後、細胞の培養プレートをPBSで洗浄し、セルスクレーパーを用いてマイクロチューブに細胞を回収した。各マイクロチューブに2M NaOHを150μL加えた後100℃にて細胞を溶解させ、遠心処理によって得られた上清について405nmの測定波長で吸光度を測定し、メラニン量を算出した。その結果、DMSOを添加したコントロールに対して、D609を5μM添加した際にはメラニン産生量の有意な増加は確認されなかった(図11)。
以上のことより、D609はケラチノサイトに作用して、エクソソームの放出を促し、放出されたエクソソームがメラノサイトを活性化させることによって、メラニン産生を促進したものと考えられる。
(4) Effect of exosome release promoter D609 on melanin production in a single culture system of melanocytes Melanocytes were seeded at a cell density of 2 × 10 5 cells / well (2 mL / well). The next day, D609 was added to a final concentration of 1, 5, or 10 μM, and culturing was performed at 37 ° C. and 5 Vol% CO 2 for 7 days. The medium includes Medium254 (containing Fetal Bovine Serum (FBS), Human Fibroblast growth factor-basic (hFGF-B), hydrocortisone, insulin, transferrin, heparin, phorbol 12 -myristate 13-acetate (PMA) and bovine pituitary extract (BPE) not included)). As a control, the same amount of DMSO was added. The medium was changed once every three days. After completion of the culture, the cell culture plate was washed with PBS, and the cells were collected in a microtube using a cell scraper. After 150 μL of 2M NaOH was added to each microtube, the cells were lysed at 100 ° C., and the supernatant obtained by centrifugation was measured for absorbance at a measurement wavelength of 405 nm to calculate the amount of melanin. As a result, no significant increase in the amount of melanin production was confirmed when 5 μM of D609 was added to the control to which DMSO was added (FIG. 11).
From the above, it is considered that D609 acts on keratinocytes to promote the release of exosomes, and the released exosomes activate melanocytes, thereby promoting melanin production.
実施例5：エクソソーム放出抑制剤GW4869によるメラニン産生の抑制
（１）細胞培養
正常ヒト新生児包皮由来表皮角化細胞(凍結NHEK (F) Lightly; ケラチノサイト)、表皮角化細胞用増殖培地(Epilife)はLife Technologies社より、その培地用増殖添加剤(Humedia-KG)はクラボウ社より購入し、また、正常ヒト新生児包皮由来表皮メラニン細胞(凍結NHEM (NB) Darkly; メラノサイト)、表皮メラニン細胞用増殖培地(Medium254)、その培地用増殖添加剤(HMGS)はLife Technologies社より購入した。ケラチノサイト及びメラノサイトは、37℃ 5%CO2の環境下で培養した。
Example 5: Inhibition of melanin production by exosome release inhibitor GW4869 (1) Cell culture Normal human neonatal foreskin-derived epidermal keratinocytes (frozen NHEK (F) Lightly; keratinocytes), a growth medium for epidermal keratinocytes (Epilife) From Life Technologies, the culture growth additive (Humedia-KG) was purchased from Kurabo Co., Ltd., and normal human neonatal foreskin-derived epidermal melanocytes (frozen NHEM (NB) Darkly; melanocytes), epidermal melanocyte growth medium (Medium 254) and its culture growth additive (HMGS) were purchased from Life Technologies. Keratinocytes and melanocytes were cultured at 37 ° C. in an environment of 5% CO 2 .
（２）エクソソーム放出抑制剤GW4869がケラチノサイトのエクソソーム放出量に及ぼす影響
ケラチノサイトを1.5×106 cells/flask (25mL/flask)の細胞密度で175cm2 flaskに播種した。この時の培地としては、ヒト上皮細胞成長因子(hEGF; Human Epidermal Growth Factor) 以外のHumedia-KG増殖添加剤（インスリン、ハイドロコーチゾン、BPE、ゲンタマイシン/アンフォテリシンB）を含むEpilife培地を使用した。播種した翌日に、エクソソームの放出抑制剤として報告（国際特許公開第2013/054534号）されているNeutral Sphingomyelinaseの阻害剤GW4869 (Ｎ，Ｎ’−ビス［４−（４，５−ジヒドロ−１Ｈ−イミダゾール−２−イル）フェニル］−３，３’−ｐ−フェニレン−ビス−アクリルアミド ２塩酸塩、Sigma Aldrich)を終濃度で20μM（溶媒はDMSO）になるように添加し、37℃、5Vol%CO2の条件下で3日間培養を行った。コントロールとしては、同量のDMSOを添加した。培養終了後、細胞の培養上清から既に記載した遠心分離法によりエクソソーム画分を単離した。培養上清を除去した後に、底面に接着している細胞に対してプロナーゼ溶液(極東製薬工業)処理を施して、細胞接着を剥離した後に回収し、血球計算板を用いて細胞数のカウントを行った。また、エクソソーム画分についてBovine Serum Albumin (BSA)を標準物質としたBCA（ビシンコニン酸）法によりタンパク質定量を行い、それを細胞数で除することにより、1細胞あたりのエクソソーム放出量を算出した。その結果、GW4869の添加により、DMSOを添加したコントロールに対して、その放出量が有意に減少することが確認された(図12)。
(2) Effect of Exosome Release Inhibitor GW4869 on Exosome Release of Keratinocytes Keratinocytes were seeded at a cell density of 1.5 × 10 6 cells / flask (25 mL / flask) in a 175 cm 2 flask. As a medium at this time, an Epilife medium containing a Humedia-KG growth additive (insulin, hydrocortisone, BPE, gentamicin / amphotericin B) other than human epidermal growth factor (hEGF) was used. The day after seeding, Neutral Sphingomyelinase inhibitor GW4869 (N, N'-bis [4- (4,5-dihydro-1H-), which is reported as an exosome release inhibitor (International Patent Publication No. 2013/054534). Imidazol-2-yl) phenyl] -3,3′-p-phenylene-bis-acrylamide dihydrochloride (Sigma Aldrich) to a final concentration of 20 μM (solvent: DMSO), 37 ° C., 5 Vol% Culture was performed for 3 days under the condition of CO 2 . As a control, the same amount of DMSO was added. After completion of the culture, the exosome fraction was isolated from the culture supernatant of the cells by the centrifugation method described above. After removing the culture supernatant, the cells adhering to the bottom surface were treated with a pronase solution (Kyokuto Pharmaceutical Co., Ltd.) to remove the cell adhesion, collected, and counted using a hemocytometer. went. In addition, the exosome fraction was subjected to protein quantification by the BCA (bicinchoninic acid) method using Bovine Serum Albumin (BSA) as a standard substance, and the exosome fraction was divided by the number of cells to calculate the amount of exosome released per cell. As a result, it was confirmed that the addition of GW4869 significantly reduced the release amount as compared to the control to which DMSO was added (FIG. 12).
（３）エクソソーム放出抑制剤GW4869がケラチノサイト及びメラノサイトからなる共培養系においてメラニン産生に及ぼす影響
ケラチノサイトを6-well plateに1×105cells/well (2mL/well)の細胞密度で播種し、翌日、メラノサイトを2×105cells/well (1mL/well)の細胞密度で播種した。さらにその翌日に、GW4869を終濃度で5、10、20μMになるように添加し、37℃、5Vol%CO2の条件下で7日間培養を行った。培地には、試験用培地としてhEGF以外のHumedia-KG増殖添加剤（インスリン、ハイドロコーチゾン、BPE、ゲンタマイシン/アンフォテリシンB）を含むEpilife培地を用いた。コントロールとしては、同量のDMSOを添加した。なお、培地交換は3日に1度行った。培養終了後、細胞をPBSで洗浄し、RIPA buffer (Sigma-Aldrich)を用いて回収してから、超音波処理により細胞を破砕した。その後、15,000rpmで15分間遠心分離し、その上清についてBovine Serum Albumin (BSA)を標準物質としたBCA（ビシンコニン酸）法によりタンパク量を定量した後、各群のタンパク質量を揃え、定法に従ってSDS-PAGE及びウェスタンブロットに供した。一次抗体はanti-Pmel17 (Dako, 1:250)を用いた。二次抗体は、anti-mouse IgG, HRP-Linked F(ab’)2 Fragment Sheep (GE Healthcare Life Science)を用いた。 その後、ECL plus western blotting detection reagents (GE healthcare bioscience)を用いて発色させ、LAS4000 (GE healthcare bioscience)を用いて発現量を可視化した。内部標準としてのβ-actinの発現は、monoclonal antibody specific for β-actin (Sigma-Aldrich, 1:5000)を用いて評価した。Pmel17の検出バンドの強度を画像解析により定量した結果、DMSOを添加したコントロールに対して、GW4869を20μM処理した際に、それが有意に発現低下していることを確認した(図13)。Pmel17はメラノソームに特異的なタンパク質であることから、GW4869処理によりメラニン量が減少したものと考えられる。
(3) Effect of exosome release inhibitor GW4869 on melanin production in a co-culture system consisting of keratinocytes and melanocytes Keratinocytes were seeded in a 6-well plate at a cell density of 1 × 10 5 cells / well (2 mL / well), and the next day Then, melanocytes were seeded at a cell density of 2 × 10 5 cells / well (1 mL / well). The next day, GW4869 was added to a final concentration of 5, 10, or 20 μM, and culturing was performed at 37 ° C. and 5 Vol% CO 2 for 7 days. As a medium, Epilife medium containing a Humedia-KG growth additive (insulin, hydrocortisone, BPE, gentamicin / amphotericin B) other than hEGF was used as a test medium. As a control, the same amount of DMSO was added. The medium was changed once every three days. After completion of the culture, the cells were washed with PBS, collected using a RIPA buffer (Sigma-Aldrich), and then disrupted by sonication. After centrifugation at 15,000 rpm for 15 minutes, the supernatant was quantified by the BCA (Bicinchoninic acid) method using Bovine Serum Albumin (BSA) as a standard substance. The samples were subjected to SDS-PAGE and Western blot. The primary antibody used was anti-Pmel17 (Dako, 1: 250). As the secondary antibody, anti-mouse IgG, HRP-Linked F (ab ') 2 Fragment Sheep (GE Healthcare Life Science) was used. Thereafter, the color was developed using ECL plus western blotting detection reagents (GE healthcare bioscience), and the expression level was visualized using LAS4000 (GE healthcare bioscience). The expression of β-actin as an internal standard was evaluated using a monoclonal antibody specific for β-actin (Sigma-Aldrich, 1: 5000). As a result of quantifying the intensity of the detection band of Pmel17 by image analysis, it was confirmed that the expression of GW4869 was significantly reduced when treated with 20 μM of DMSO-added control (FIG. 13). Since Pmel17 is a protein specific to melanosomes, it is considered that the treatment with GW4869 reduced the amount of melanin.
（４）エクソソーム放出抑制剤GW4869がメラノサイトの単独培養系においてメラニン産生に及ぼす影響
メラノサイトを2×105cells/well (2mL/well)の細胞密度で播種した。その翌日に、GW4869を終濃度で5、10、20μMになるように添加し、37℃、5Vol%CO2の条件下で7日間培養を行った。培地には、試験用培地としてMedium254 (増殖添加剤（HMGS）のうち、Fetal Bovine Serum (FBS)、Human Fibroblast growth factor-basic (hFGF-B)、ハイドロコーチゾン、インスリン、トランスフェリン、ヘパリン含有、phorbol 12-myristate 13-acetate (PMA)及びbovine pituitary extract (BPE)不含）を用いた。コントロールとしては、同量のDMSOを添加した。なお、培地交換は3日に1度行った。培養終了後、細胞の培養プレートをPBSで洗浄し、セルスクレーパーを用いてマイクロチューブに細胞を回収した。各マイクロチューブに2M NaOHを150μL加えた後100℃にて細胞を溶解させ、遠心処理によって得られた上清について405nmの測定波長で吸光度を測定し、メラニン量を算出した。その結果、DMSOを添加したコントロールに対して、GW4869を添加した際にメラニン産生量に対する影響は確認されなかった(図14)。
(4) Effect of exosome release inhibitor GW4869 on melanin production in a single culture system of melanocytes Melanocytes were seeded at a cell density of 2 × 10 5 cells / well (2 mL / well). The next day, GW4869 was added to a final concentration of 5, 10, or 20 μM, and the cells were cultured at 37 ° C. and 5 Vol% CO 2 for 7 days. The medium includes Medium254 (containing Fetal Bovine Serum (FBS), Human Fibroblast growth factor-basic (hFGF-B), hydrocortisone, insulin, transferrin, heparin, phorbol 12 -myristate 13-acetate (PMA) and bovine pituitary extract (BPE) were not used). As a control, the same amount of DMSO was added. The medium was changed once every three days. After completion of the culture, the cell culture plate was washed with PBS, and the cells were collected in a microtube using a cell scraper. After 150 μL of 2M NaOH was added to each microtube, the cells were lysed at 100 ° C., and the supernatant obtained by centrifugation was measured for absorbance at a measurement wavelength of 405 nm to calculate the amount of melanin. As a result, no effect on melanin production was observed when GW4869 was added to the control to which DMSO was added (FIG. 14).
（５）エクソソーム放出抑制剤GW4869が表皮メラニン量に及ぼす影響
米国スキンバンクNational Disease Research Interchange (NDRI)から、外科手術由来の正常ヒト皮膚組織を提供頂いた。ヒト皮膚組織は、皮下脂肪をトリミングした後に、ナイフで約1 cm×1 cmの大きさに小片化し、6-well plateを使用して37℃ 5Vol%CO2の環境下にて培養した。培養には、Fetal Bovine Serum (FBS)を10% (v/v)含むAdvanced DMEM培地 (いずれもLife Technologies)を使用した。ヒト皮膚組織を6-well plateに静置し、GW4869を終濃度で20μMになるように培地中に添加した上で組織培養を実施した。2〜3日毎に培地を交換しながら、培養開始から7日間経過後に皮膚組織を回収、定法に従って凍結組織切片を作成後にフォンタナマッソン染色を実施し(図15A)、その染色像に関して、Image Jソフトウェアを用いて二値化を行うことにより、画像解析を行った。表皮と真皮の境界における単位長あたりの表皮メラニン量を算出した結果、DMSOを添加したコントロールに対して、GW4869を処理した皮膚では、有意にメラニン量が低下していることが確認された(図15B)。
(5) Effect of Exosome Release Inhibitor GW4869 on Epidermal Melanin Amount Normal human skin tissue derived from surgery was provided by the National Skin Research Interchange (NDRI) of the United States Skin Bank. After trimming the subcutaneous fat, the human skin tissue was cut into small pieces having a size of about 1 cm × 1 cm with a knife, and cultured in a 6-well plate at 37 ° C. in an environment of 5 Vol% CO 2 . For the culture, an Advanced DMEM medium containing 10% (v / v) of Fetal Bovine Serum (FBS) (both Life Technologies) was used. The human skin tissue was allowed to stand in a 6-well plate, and GW4869 was added to the medium at a final concentration of 20 μM, followed by tissue culture. While changing the medium every 2-3 days, the skin tissue was collected after elapse of 7 days from the start of culture, and a frozen tissue section was prepared according to a standard method, followed by Fontana Masson staining (FIG. 15A). Image analysis was performed by performing binarization using. As a result of calculating the amount of epidermal melanin per unit length at the boundary between the epidermis and the dermis, it was confirmed that the amount of melanin was significantly reduced in the skin treated with GW4869 as compared to the control to which DMSO was added (FIG. 15B).
以上のことより、GW4869はケラチノサイトに作用して、エクソソームの放出を抑制し、エクソソームによるメラノサイトの活性化を抑制させることで、メラニン産生を抑制したものと考えられる。 From the above, it is considered that GW4869 acts on keratinocytes, suppresses the release of exosomes, and suppresses the activation of melanocytes by exosomes, thereby suppressing melanin production.
実施例４及び５の結果から、ケラチノサイトのエクソソーム放出量を指標に、メラニン制御剤のスクリーニングを行うことが可能である。 From the results of Examples 4 and 5, it is possible to screen for a melanin regulator using the exosome release amount of keratinocytes as an index.
Claims (3)
ａ）ケラチノサイトに被験物質を接触させ培養する工程
ｂ）培養細胞からエクソソーム画分を単離し、分泌レベルを測定する工程
ｃ）エクソソームの分泌レベルを変化させる被験物質をメラニン制御剤として選択する
工程 A method for searching for a melanin controlling agent comprising the following steps a) to c), wherein a test substance that enhances or increases the secretion level of exosomes is used as a melanin production promoter, dendrite growth promoter, skin browning agent, vitiligo prevention Or a method of evaluating or selecting as a therapeutic agent or a gray hair preventing or improving agent .
a) a step of culturing a test substance by contacting it with keratinocytes b) a step of isolating an exosome fraction from cultured cells and measuring a secretion level c) a step of selecting a test substance that changes the secretion level of exosomes as a melanin regulator
ａ）ケラチノサイトに被験物質を接触させ培養する工程a) a step of culturing a test substance in contact with keratinocytes
ｂ）培養細胞からエクソソーム画分を単離し、分泌レベルを測定する工程b) a step of isolating the exosome fraction from the cultured cells and measuring the secretion level
ｃ）エクソソームの分泌レベルを変化させる被験物質をメラニン制御剤として選択する工程c) a step of selecting a test substance that changes the secretion level of exosomes as a melanin controlling agent
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