鼻咽癌CD44抑制表达细胞株的建立

目的:构建CD44新剪接变异体siRNA质粒表达载体,建立CD44新变异体抑制表达的鼻咽癌细胞株.方法:合成CD44新变异体特异性干扰DNA片段,干扰DNA片段亚克隆于带绿色荧光蛋白的pGenesil -1.3质粒表达载体中,双酶切和测序鉴定重组表达质粒载体；采用脂质体将重组表达质粒载体转染入鼻咽癌5 -8F细胞系,进行G418筛选；Western blot分析CD44表达.结果:重组CD44干扰DNA片段质粒表达载体的碱基序列和插入方向正确；细胞转染效率达70％；G418筛选获得GFP表达的单克隆鼻咽癌5 -8F细胞株；Western blot分析表明CD44表达受抑制.结论:建立了CD44新变异体抑制表达的鼻咽癌细胞株,为CD44新变异体在鼻咽癌中的生物学功能提供了基础.

Establishment of CD44 Silencing Nasopharyngeal Carcinoma Cell Line

Objective: To construct CD44 new variant of siRNA plasmid expression vector,and establish nasopharyngeal carcinoma cell line of CD44 new variant inhibited expression.Method:Synthesizing specific interference DNA fragment of new CD44 variant,interference DNA fragment cloned in green fluorescent protein pGenesil-1.3 plasmid expression vector,double enzyme digestion and DNA sequencing to confirm recombinant expression plasmid;recombinant expression plasmid vector transfected 5-8F cell line by using liposome,nasopharyngeal carcinoma cell lines of GFP expression were screened by G418,CD44 expression was analyzed by Western blot.Result:Obtained recombinant CD44 interference expression plasmid with right DNA fragment base sequence and direction,The transfected efficiency was about 70%;obtained monoclonal 5-8F nasopharyngeal carcinoma cell line with GFP expression were screened by G418;CD44 expression was inhibited by western blot identification.Conclusion:The established nasopharyngeal carcinoma cell line is CD44 new variant knock-down cell line,this provides the basis for exploring the biological functions of CD44 new.