论文部分内容阅读
目的构建针对HPV16 E6基因的逆转录病毒载体,感染HPV16阳性宫颈癌细胞,筛选稳定表达HPV16 E6 siRNA的细胞克隆。方法采用DNA重组技术构建表达靶向HPV16 E6基因的pSUPER.retroRNAi逆转录病毒载体,脂质体法将逆转录病毒载体转染入包装细胞PA317,G418筛选稳定产生逆转录病毒的细胞克隆,收集病毒上清,感染靶细胞SiHa,G418筛选出稳定表达HPV16 E6 siRNA细胞克隆,RT-PCR检测细胞中E6 mRNA表达,细胞增殖实验检测细胞增殖力。结果获及稳定产生逆转录病毒的细胞克隆,病毒感染靶细胞SiHa后,筛选出稳定表达HPV16 E6 siRNA的细胞克隆,RT-PCR示HPV16 E6 mRNA表达受到抑制。细胞增殖实验示克隆细胞增殖率明显下降。结论成功建立稳定表达HPV16 E6 siRNA细胞克隆,特异性的siRNA能抑制细胞生长、HPV16 E6 mRNA表达,HPV16 E6 siRNA可能成为治疗宫颈癌的一种新方法。
Objective To construct a retroviral vector targeting HPV16 E6 gene, HPV16-positive cervical cancer cells and screen for cell clones stably expressing HPV16 E6 siRNA. Methods Recombinant plasmid pSUPER.retroRNAi was constructed by DNA recombination technique. The retroviral vector was transfected into the packaging cell line PA317 by lipofectamine 2000, and the cell clone stably producing retrovirus was screened by G418. The supernatant was transfected into SiHa and G418 cells. The stable clone of HPV16 E6 siRNA was screened out. The expression of E6 mRNA was detected by RT-PCR and the cell proliferation was measured by cell proliferation assay. Results The stable clones producing retrovirus were obtained. After virus infected target cells SiHa, the cell clones stably expressing HPV16 E6 siRNA were screened out. The expression of HPV16 E6 mRNA was inhibited by RT-PCR. Cell proliferation experiments showed that the proliferation of clonal cells decreased significantly. Conclusion The successful cloning of HPV16 E6 siRNA cell line has been successfully established. The specific siRNA can inhibit cell growth and express HPV16 E6 mRNA. HPV16 E6 siRNA may become a new method for the treatment of cervical cancer.