内皮素-1对毛囊外根鞘无色素黑素细胞黏附和移行及其细胞骨架形态的影响

Adhesion and migration of amelanotic melanocytes in hair out root sheath induced by endothelin-1 and effects on its cytoskeleton in vitro

目的:体外研究内皮素-1(ET-1)对毛囊外根鞘无色素黑素细胞(AMMC)黏附和移行的作用,并观察其对细胞骨架蛋白微丝、微管的形态影响。方法:3种浓度的ET-1作用体外纯化培养的人AMMC,观察其在细胞外基质蛋白如纤维连接蛋白、层黏连蛋白、Ⅳ型胶原包被培养板上的黏附,以及通过Boyden小室微孔滤膜的情况。采用免疫荧光双重染色法对AMMC分别进行罗丹明(红色)和异硫氰酸(绿色)标记纤维型-肌动蛋白(F-actin)、β微管蛋白,激光共聚焦显微镜观察3种浓度ET-1处理前后AMMC纤维型-肌动蛋白、β微管蛋白形态的变化。结果:与空白对照组相比,ET-1促进了AMMC在纤维连接蛋白上的黏附,20ng/mLET-1作用最明显(P<0.01),而200ng/mL时对细胞的黏附不继续增加。但对层黏连蛋白和Ⅳ型胶原上的黏附无明显影响(P>0.05)。另外,ET-1呈浓度依赖性地促进AMMC通过微孔滤膜。激光共聚焦显微镜观察显示,>20ng/mLET-1可明显促进AMMC胞质中束状应力纤维形成,并集中分布于细胞膜内侧,但对微管结构无明显影响。结论:ET-1在体外可以促进AMMC的黏附和移行,其作用可能与诱导束状应力纤维形成和促进其向细胞膜内侧分布有关。

Objective： To study the effects of endothelin-1 on the adhesion and migration of amelanotic melanocytes in hair out root sheath and observe its role on cytoskeleton. Methods： Amelanotic melanocytes were treated with three different concentrations of endothelin-1 to observe adhesion on culture dishes which were respectively coated with fibronectin, laminin, collagen Ⅳ. And then, migration on fibronectin coated micropore filters was investigated by Boyden Chemotaxis Chamber. Cellular microfilament and microtubule were immunostained with rhodamine labeled F-actin and FITC labeled β tubulin. The effects of cytoskeleton to endothelin-1 were observed with laser scanning confocal microscopy. Results： It showed that amelanotic melanocytes treated with endothelin-1 increased the adhesion and migration on fibronectin （P〈 0.01）, 〉20 ng/mL endothelin-1 induced F-actin to form stress fibre fasciculus which was distributed inside the cell membrane. Conclusions： endothelin-1 may directly stimulate the adhesion and migration of amelanotic melanocytes on fibronectin, which maybe induced by the formation of stress fibre fasciculus inside the cell membrance.