碱性成纤维细胞生长因子基因转染人表皮细胞及其表达

目的 观察碱性成纤维细胞生长因子基因真核表达载体(pEGFP-C3-bFGF)能否转染体外培养的人表皮细胞并表达.方法 采用脂质体(LipofectamineTM 2000)转染法,将pEGFP-C3-bFGF转染至人表皮细胞系(HEKa细胞)中,在荧光显微镜下观察其瞬时表达及细胞形态.以同期培养的表皮细胞作为阴性对照,免疫细胞化学染色和免疫荧光标记法检测实验组及对照组中β1整合素、CKl9、CK14及CK10在表皮细胞中表达的差异.结果 荧光显微镜下观察基因转染率为31.6%,转染的细胞体积小,呈圆形或圆梭形,核质比大.免疫细胞化学染色结果显示,实验组细胞中CK19、CK14表达增强,CK10表达减弱.免疫荧光染色结果显示,转染阳性细胞β1整合素弱表达分布在胞膜上,CK19、CK14在胞膜和胞质中表达,CK10表达为阴性.结论 pEGFP-C3-bFGF能够转染至人表皮细胞并表达,为探讨bFGF调控表皮细胞去分化的分子机制提供实验参考.

Expression of pEGFP-bFGF by human epidermal cells after transfected with pEGFP-bFGF

Objective To investigate whether human epidermal ceils could be transfected with the eukaryotic expressive vector of pEGFP-C3-bFGF and express the exogenous gene.Methods pEGFP-C3-bFGF was transferred into the human epidermal cells by using liposome-mediatedmethod.Subsequent-ly, the phenotypic changes of epidermal cells were detected by using immunohistochemical staining and im-munofluorescent analysis 36 h later.For controls, epidermal cells were cultured simultaneously in the ab-sence of exogenous gene transfection.Results Specific green fluorescence could be observed by the fluo-rescence microscopy in the gene positive groups.Immunohistochemical staining revealed that the expression levels of CK19 and CK14 were up-regulated, while those of CK10 significantly down-regulated.Meanwhile,immunofluorescent analysis indicated that β1 integrin was expressed on the membrane of positive cells in lower level,CK19 and CK14 were expressed on the membrane and in the cytoplasm of those cells, and CK10 was negative.Conclusion The pEGFP-C3-bFGF is successfully transferred into human epidermal ceils, which can induce epidermal cells to reverse their differentiated process and thus express some undif-ferentiated proteins of epidermal stem cells.