摘要
[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]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 was to optimize conditions of exogenous gene transfection of yak mammary epithelial cells in vitro mediated by liposome. [Method] Yak mammary epithelial cells were obtained by using the methods of collagenase digestion and tissue adhesion in vitro. Use immunocytochemistry technique to authenticate the expression of cytokeratin in yak mammary epithelial cell. With green fluorescence protein as report gene,through exogenous gene transfection of yak mammary epithelial cells in vitro mediated by liposome,the transfection efficiency under different cell fusion degree,liposome concentration,liposome and plasmid ratio,transfection time was obtained to find the optimum transfection conditions. [Result] The results showed that the highest transfection efficiency would be obtained when cell confluence was 81%-90%,the quantity of liposome was 1.8 μl,liposome and plasmid ratio was 2∶1 and the transfecting time was 4 h. [Conclusion] The study will provide test basis for constructing mammary gland bioreactor.
出处
《安徽农业科学》
CAS
北大核心
2010年第9期4604-4606,4613,共4页
Journal of Anhui Agricultural Sciences
基金
国家自然科学基金资助项目(30771550)
关键词
牦牛乳腺上皮细胞
免疫细胞化学
转染
Yak mammary epithelial cell
Immunocytochemistry
Transfection
