HO-1 siRNA表达载体的构建及其稳定转染SGC7901细胞系的建立

Screening of HO-1 gene silencing stable transfection cell lines of gastric cancer cell line SGC7901

目的:构建携带绿色荧光蛋白的HO-1基因siRNA表达载体,通过将载体转染入胃癌细胞株SGC7901筛选稳定转染细胞株。方法:参照前期工作,构建可用于筛选阳性克隆并带有绿色荧光的HO-1基因siRNA表达载体,采用脂质体转染法将载体转入SGC7901细胞中,并用G418进行阳性克隆筛选,获得稳定转染细胞系。结果:经酶切和测序验证,证实表达载体构建成功,将HO-1 siRNA表达载体转入SGC7901后,通过G418筛选获得稳定转染细胞系,经检测HO-1的表达水平明显低于为未转染的细胞。结论:本实验成功地构建了HO-1基因siRNA表达载体,筛选出HO-1基因沉默的稳定转染胃癌细胞系,为今后研究HO-1基因的作用机制提供了实验基础。

Objective： To construct siRNA expression vector of HO-1 which carry green fluorescent protein gene, and to transfect the vector into gastric cancer cell line SGC7901 to screen positive clones. Methods： According to our previous work, we constructed a recombinant plasmid. The new vector carrying green fluorescent gene could be used for screening positive clones. Then we transfected siRNA vector into SGC7901 cells, screened HO- 1 gene silencing stable transfected cell lines using C,418 for positive screening. Results： By restriction endonuclease and sequencing, eukaryotic expression plasmid were proved to be successfully constructed. Through G418 screening, HO- 1 gene silencing stable transfection cell lines was got, the HO- 1 level of the cell lines was less than that of the untransfected cell lines. Conclusion： The expression vector have been successfully constructed. HO-1 gene silencing stable transfection cell lines of gastric cancer is got successfully. Our results may provide some ideas for the gene therapy in gastric cancer in future.