稳定表达HPV16 E6 siRNA细胞克隆的建立

ESTABLISHMENT OF CELL CLONE WITH STABLE EXPRESSION OF HPV16 E6 siRNA

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 recombinant defective vector targeting HPV16 E6 gene,transfect into the HPV po sitive cervical carcimona cells and select clone cells with stable expression of HPV16 E6 siRNA. Methods The pSUPER, retro RNAi retrovlrus vector expressing HPV16 E6 siRNA was constructed by recombinant DNA technology. The packaging cell PA317 was transfected with this recombinant plasmid using liposome-based transfection method and the stable integrant was selected using G-418, the virus supernatant was collected and then infected SiHa ceils, the cell clone that silenced of HPV16 E6 gene was obtained. The cell proliferative activity, HPV16 E6 messenger RNA （mRNA） expression were measured before and after the transfection by proliferation assay and reverse transcriptase-polymerase chain reaction, respectively. Results The SiHa cells that stably expressed E6 siRNA were obtained. Compared with non-infected groups, the expression of E6 mRNA level was reduced remarkably. The proliferation was suppressed significantly in clone cells. Conclusion The cell clone with stable expression of HPV16 E6 siRNA was constructed. The specific siRNA demonstrates inhibitory effect on cell growth and mRNA expression. This study indicates that siRNA HPV16 E6 can be used as a novel therapeutic approach for cervical cancer.