获取SD仔鼠角朊细胞和真皮成纤维细胞6种方法比较

目的 :探讨如何以简便快捷的方法获得大量最佳生长状态的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞 .方法 :用 2 .5 g·L -1 胰酶、35 g·L -1 热溶素和 9.0× 1 0 3 U·L-1 Dispase分别在不同条件下处理皮肤标本 ,观察表皮与真皮的分离情况 ,并分别培养角朊细胞和真皮成纤维细胞 ,观察比较细胞的贴壁率和克隆形成率 ,通过MTT和BrdU检测观察细胞的生长状态 ,并分别用不同方法获得的细胞构建组织工程皮肤 .结果 :在各种条件下 ,Dispase处理组获得的细胞数量、细胞贴壁率和克隆形成率均优于其他消化液处理组 ,细胞纯度高 ,生长状态良好 ;用其他方法获得的细胞生长状态相似 ,都可成功构建组织工程皮肤 .结论 :使用9.0× 1 0 3 U·L-1 Dispase可以简便快捷的获得大量生长状态良好的角朊细胞和真皮成纤维细胞 ,以作为皮肤组织工程的种子细胞 ,合适的消化方法、时间和温度可以最大限度保持原代细胞的活力

Isolation of keratinocytes and dermal fibroblasts from new-born SD rat skin for skin replacement

AIM: To develop a rapid and reproducible method for the isolation of the two populations of cells (fibroblasts and keratinocytes) from SD rat skin biopsy. METHODS: The keratinocytes and dermal fibroblasts were isolated and cultured by six different methods. The biological conditions of the cells were examined by detection of their attaching efficiency and colony forming efficiency, MTT detection and BrdU analysis. RESULTS: All the six methods were successful in separating fibroblasts and keratinocytes from SD rat skin biopsy, but histological cross sections showed that the Dispase was the best in separating the dermis and the epidermis. By Dispase, the greatest number of high quality keratinocytes and dermal fibroblasts could be attained and the attaching efficiency and colony forming efficiency were better than the other methods. CONCLUSION: The greatest number of keratinocytes and dermal fibroblasts in good conditions can be attained rapidly by Dispase.