摘要
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（LipofectamineTM2000）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、CK19、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。
Objective To investigate whether human epidermal cells 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-mediated method. Subsequently, 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 absence of exogenous gene transfection. Results Specific green fluorescence could be observed by the fluorescence microscopy in the gene positive groups. Immnnohistochemical staining revealed that the expression levels of CKI9 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 CKIO was negative. Conclusion The pEGFP-C3-bFGF is successfully transferred into human epidermal cells, which can induce epidermal ceils to reverse their differentiated process and thus express some undifferentiated proteins of epidermal stem cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第3期286-288,409,共4页
Chinese Journal of Experimental Surgery
基金
基金项目：国家重点基础研究发展计划资助项目（2005CB522603）
