山羊表皮干细胞的分离培养及EGFP基因转染研究

Study on the isolation and culture of goat epidermalstem cells and EGFP gene transfection

采用组织块法和酶消化法对山羊表皮干细胞进行分离，对分离的表皮干细胞进行了免疫组化染色鉴定，并对其进行了增强型绿色荧光蛋白（EGFP）基因转染研究。结果表明，2种方法均能得到山羊表皮干细胞，其中组织块培养法简单易行，组织块可重复利用，但原代细胞脱出时间较长，平均为8d，且培养季节对细胞脱出时间、原代传代时间、传代数有显著影响；酶消化法周期短，8d左右即可传代，不同培养季节对细胞克隆形成时间、细胞传代时间和传代数无显著影响，但处理过程繁琐。EGFP基因在转染表皮干细胞24h后大量表达，转染率达20％～30％，72h时转染强度最大，荧光可持续5周；转染使细胞增殖能力明显下降。

Goat epidermal stem ceils [EpiSCs] was respectively obtained by enzymic digestion and tissue piece culture,then the cultured cells were identified by immunohistochemistry stain. EpiSCs was transfected by pEGFP-C1 plasid. Results show that the both two ways can obtain EpiSCs,the former is quicker than the latter. Tissue piece culture method is simple and the tissue piece can be used repeatedly. But the migrating time of primary cells ,about 8 days ,is too long. With this method,seasons have remarkable effect on migrating time,passaging time and passaging numbers while DispaselI enzyme digestion method has a short period. It can be passaged in 8 days. With this method,seasons have no remarkable effect on migrating time,passaging time and passaging numbers. But its disposal is complex. GFP gene considerably expressed after 24 hours,reached 20-30 percent. The highest intensity appeared at the 72th hour. It lasted 5 weeks. The amplification ability of transfected cells was greatly decreased.