稳定表达TMEM16A蛋白TE-1细胞株的构建与鉴定

医学研究与教育 ›› 2013, Vol. 30 ›› Issue (1): 15-19.

稳定表达TMEM16A蛋白TE-1细胞株的构建与鉴定

耿仙¹,冯承保²,吴建民³,杨会茹⁴,宋新梅²

1.河北大学基础医学院药理教研室,河北保定 071002
2.保定市第二医院肿瘤科,河北保定 071002
3.新乐市医院外科,河北新乐 050700
4.新乐市医院儿科,河北新乐 050700

收稿日期:2016-10-09 修回日期:2016-10-09 出版日期:2013-02-25 发布日期:2013-02-25

Transfection of TMEM16A gene into TE-1 cells stably by liposome and its identification

Received:2016-10-09 Revised:2016-10-09 Online:2013-02-25 Published:2013-02-25

摘要/Abstract

摘要： 目的建立稳定表达TMEM16A蛋白的TE-1肿瘤细胞株.方法用Lipofectamine?2000转染试剂将TMEM16A基因转染到TE-1细胞,经G418筛选,使用激光共聚焦显微镜和Western blot技术检测TMEM16A基因是否在TE-1细胞中已经稳定表达.结果通过使用G418进行筛选,阳性克隆于4周后培养形成.激光共聚焦显微镜观察稳定表达TMEM16A蛋白的TE-1细胞可见细胞发出绿色荧光.Western blot结果表明,未稳转细胞和稳转细胞系统均表达有TMEM16A蛋白,而稳转细胞系统的蛋白水平明显高于未转染细胞系统的蛋白水平.结论利用脂质体转染方法成功构建了稳定表达TMEM16A基因的TE-1细胞系.

关键词: TMEM16A, TE-1, 脂质体2000, 激光共聚焦显微镜, 免疫印迹

Abstract: Objective To get stably expressing TMEM16A proteins by transfecting TE-1 cells. Methods TMEM16A gene was transfected into TE-1 cells by Lipofectamine?2000, then we picked up cell clones in the presence of G418. TMEM16A proteins were identified by confocal microscope and western blot. Results After 4 weeks, the cell clones of colony-like formed after G418 screening. The stable TMEM16A-expression TE-1 cell lines were observed by confocal microscope showing signal of GFP co-transfected. Western blot results showed that TMEM16A protein expressed in control and stable TMEM16A-expression TE-1 cells, and the expression was significantly increased in TMEM16A stable cell lines. Conclusion Stable TMEM16A-expression TE-1 cell lines are created with liposome method successfully.

Key words: TMEM16A, TE-1, Lipofectamine?2000, confocal microscope, Western blot

中图分类号:

R735.1

耿仙,冯承保,吴建民,杨会茹,宋新梅. 稳定表达TMEM16A蛋白TE-1细胞株的构建与鉴定[J]. 医学研究与教育, 2013, 30(1): 15-19.

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