脂质体介导外源基因体外转染牦牛乳腺上皮细胞条件的优化(英文)

Optimization of Parameters of Exogenous Gene Mediated by Liposome to Transfect Yak Mammary Epithelial Cells in Vitro

[目的]优化脂质体介导外源基因体外转染牦牛乳腺上皮细胞的条件。[方法]通过胶原酶消化法和组织块贴壁法,体外分离培养得到牦牛乳腺上皮细胞。采用免疫细胞化学技术检测细胞角蛋白在细胞内的表达。以绿色荧光蛋白为报告基因,通过脂质体介导外源基因转染牦牛乳腺上皮细胞,统计在不同细胞融合度、脂质体浓度、脂质体与质粒的比例、转染时间下的转染效率,寻求最佳转染条件。[结果]采用胶原酶消化法,24h内细胞团块贴壁生长,纤维样细胞沿着细胞团块生长,后上皮样细胞爬出。上皮细胞与纤维细胞界限明显,细胞呈椭圆形,排列紧密,岛屿状生长。纯化至5代左右可得到较纯的上皮样细胞。采用组织块贴壁法,6d左右组织块周围出现细胞晕,有少量纤维样细胞生长;8d左右,上皮样细胞长出,并往外不断铺展。在荧光显微镜下观察,试验组细胞角蛋白检测呈阳性,细胞质呈绿色,细胞核复染呈红色。牦牛乳腺上皮细胞在细胞融合度为81%～90%,脂质体的量为1.8μl,脂质体与质粒比例为2∶1,转染时间为4h时,转染效率最高。[结论]该研究可为乳腺生物反应器构建提供试验依据。

[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...