ERK2-siRNA质粒的构建及其诱导宫颈癌HeLa细胞凋亡的实验研究

目的:合成、克隆、筛选ERK2-siRNA干扰片段,构建含有ERK2-siRNA的骨架质粒。明确其抑制基因ERK2在HeLa细胞中表达及诱导HeLa细胞凋亡。方法:利用RNA干扰方法构建真核表达载体pGenesil1.1-ERK2-siRNA。体外培养人宫颈癌HeLa细胞,利用脂质体法将该质粒转染HeLa细胞。利用western blot法检测该质粒转染后ERK2基因的表达,筛选最佳干扰质粒。通过TUNEL法HeLa细胞凋亡染色及HeLa细胞Caspase-3表达的免疫组化染色检测其诱导HeLa细胞的凋亡。结果:成功构建及筛选出ERK2-siRNA质粒。该质粒在体外能够有效诱导HeLa细胞凋亡。结论:干扰ERK2基因的表达能够诱导HeLa细胞的凋亡,应用该方法可以为宫颈癌的治疗提供新思路。

Construction of ERK-siRNA plasmids and its apoptosis effect on HeLa cells

Objective：Synthesizing,cloning and screening ERK2-siRNA interference fragment,to construct the ERK2-siRNA plasmids,and to identify ERK2 gene inhibition in HeLa cells and its apoptosis effect on HeLa cells.Methods：To construct the eukaryotic expression vector pGenesil1.1-ERK2-siRNA.HeLa cells were cultured in vitro and transfected with ERK2-siRNA plasmids by LipofectAMINE2000.Western blot was applied to detect the expression of ERK2 gene and screen the best RNA interference plasmid.HeLa cells apoptosis were assessed by TUNEL staining and caspase-3 protein immunohistochemistry staining.Results：To obtain the efficient interference fragment ERK2-siRNA.The plasmid can induce cell apoptosis in vitro.Conclusion：ERk2gene inhibition can induce HeLa cell apoptosis,and using this method may be a promising therapeutic strategy for uterine cervix cancer.