原子力显微镜对角质形成细胞与黑素细胞共培养模型的观察研究

目的：用原子力显微镜观察人表皮黑素细胞（MC）、角质形成细胞（KC）单独培养和共培养的表面形态以及α-促黑素（α-MSH）对细胞表面形态的影响。方法：分别纯化培养来自人包皮的表皮MC和KC，MC以自配的添加MC生长物质的MCDB153培养基培养，KC以KC无血清培养基（K-SFM）常规培养。传第2代后以1：10的比例将两细胞接种到3Cm×3Cm的小培养皿中，以K-SFM培养基继续培养，单独或混合培养的细胞经添加含或不含100nMα-MSH的培养基干预3天后，0．5％戊二醛固定10min，原子力显微镜常温常压下，触摸式扫描。结果：正常人表皮MC有3个树突，每个树突有明显的二级分枝，除主干和分支见到膨出的颗粒物质，我们在树突的侧缘底侧和顶端还发现有丝状伪足结构，经α-MSH刺激后树突明显变长、变细，主干和分支表面膨出颗粒物质更为密集，许多已脱离枝干，丝状伪足则未有明显变化。表皮KC表面可见许多片状或钩状突起。共培养后，KC与MC接触部位可见明显的丝状伪足样结构，未连接部位则未见到丝状伪足样结构，添加α-MSH后，两细胞连接处的丝状伪足样结构明显增多。结论：通过胞吐和丝状伪足输送可能是黑素小体从MC向KC传递的两种主要方式，α-MSH可能通过促进这两种结构的发生而发挥促黑素传递的作用。

Morphologic comparision of co-cultured human epidermal melanocytes and keratinocytes observed by atomic force microscopy

Objective To image the surface structure of mono-culture and co-culture of human epidermal melanocytes and keratinocytes by atomic force microscopy （AFM） and observe the morphological influence ofα-MSH on them. Methods Human epidermal metanocytes and keratinocytes were isolated and co-cultured with 1：10 ratio in a defined K-SFM medium, the cells with or without 100 nMα-MSH treatment were fixed by 0.5% glutaraldehyde and AFM images of scanning observation were captured by contacting and tapping modes under normal atmospheric pressure and temperature. Results Human epidermal melanocytes had three grades of branches. Except for globular structures, there were also some fitopodias protruded on the tops and lateral sides of the branches. After administration withα- MSH, the branches became thinner and longer and globular structures became obvious and intensive. Many detached globular structures were observed. Meanwhile the filopodias was less changed. Many hook-like or lamellar enations were observed on the surface of keratinocyte. In co-culture model, many filopodias were observed on the junction of the keratinocytes and melanocytes. Following exposure to α-MSH, the filopodias became more obviously. Conclusions The exocytosis and filopodia formation may be two main ways of melanosome transfer between melanocytes and keratinocytes, α-MSH may play a important role in melanosome transfer by promoting exocytosis and filopodia formation.