肝癌细胞中Connexin32蛋白可调控表达亚克隆的建立

Establishment of subclones with controllable expression of connexin32 in hepatocellular carcinoma cells

目的:利用Tet-off基因表达体系建立人间隙连接蛋白Connexin32基因可调控表达的肝癌细胞系,为探讨Connexin32蛋白表达与肝癌细胞的增殖、运动和侵袭能力的相关性奠定基础。方法:利用基因重组方法建立人Connexin32cDNA的逆病毒表达载体pRevTRE-Cx32;利用逆病毒感染法将Connexin32表达载体和调控质粒pRevTet-off转入人肝癌细胞系HuH7中,筛选出稳定整合了两种载体的细胞克隆;western-blot法检测Connexin32蛋白表达的诱导率。结果:建立了可以通过向细胞培养液中添加或去除Doxycycline调控Connexin32蛋白表达的肝癌细胞HuH7亚克隆,western-blot结果显示当从细胞培养液中去除Doxycycline后,Connexin32表达明显升高,呈现4倍左右的诱导率。结论:成功建立了Connexin32蛋白可诱导表达的肝癌细胞HuH7 Tet-offCx32亚克隆。

Objective：To establish stable subelones with Tot- off gene expression system in HuH7 hepatoeellular carcinoma cell line,in which expression of gap junction protein connexin32 can be controlled by doxyeycline supplement/depletion in hepatocellular carcinoma. Methods ： Retroviral expression vetor of eonnexin32, pRevTRE - Cx32, was contrueted by gene recombination and thereafter transfected along with Tet - responsive element pRevTet - off to human HuH7 hepatoeellular carcinoma cells by means of retroviral infection. Stable clones with high inducibility were selected after eonnexin32 screening by western - blot. Results ： HuH7 Tet - off Cx32 subclone was selected, which showed approximately 4 - fold inducibility when doxyeycline was removed from culture medium, comparing to doxyeyeline- supplemented medium. Conclusion： HuH7 subclones in which connexin32 can be controlled by doxycycline were successfully established.