表皮真皮分离方法的探索

Method for separating the epidermal-dermal junction

目的 探索一种适合细胞培养的分离真表皮的酶消化法。方法 将分离酶配成 0 1%、0 2 %、0 5 % 3个浓度 ,在 4℃或 3 7℃条件下消化头皮或包皮 ,摸索消化的时间 ,并与 0 2 5 %胰蛋白酶消化法进行对比。结果 0 5 %分离酶在 4℃条件下消化 16～ 18h ,或 3 7℃条件下消化 1h ,能够很好地分离表皮与真皮 ,再用胰蛋白酶将表皮游离成角质形成细胞 ,活细胞率高 ,培养后细胞融合时间短。结论 分离酶能够选择性消化基底膜成分而起到很好的分离表皮与真皮的作用 ,分离酶和胰蛋白酶联合消化法是一种非常理想的分离角质形成细胞的方法 ,同时又能够避免成纤维细胞的污染。

Objective To find a method for separating the epidermal-dermal junction for cell culture. Methods Dispase was dispensed to 0.1%, 0.2%, and 0.5% for the digestion of skin pieces at 4 ℃ or 37 ℃ for different time. The digestion time was compared with that of 0.25% trypsin. Results Skin pieces could be digested by 0.5% dispase at 4 ℃ for 16-18 h or at 37 ℃ for 1 h and could separate the epidermis from the dermis completely. The separated epidermis, digested with 0.25% trypsin and isolated into keratinocytes, could result in high survival rate of cells and shorted time for confluence. Conclusion Dispase can selectively digest the basement membrane to separate the epidermis from the dermis. The combination of dispase and trypsin digestion is a very good method for isolating keratinocytes without contamination of fibroblasts.