中文摘要：

目的建立反转录病毒介导的MDC1基因RNA干扰表达体系并观察其在宫颈癌细胞（HeLa）中对MDC1表达的影响。方法将人MDC1基因的RNA干扰双链DNA片段重组到反转录病毒质粒pSilencer5.1-H1Retro中,构建携带人MDC1基因的RNA干扰反转录病毒载体psiRNA-MDC1。经PT67细胞包装后,产生的重组反转录病毒感染宫颈癌细胞株HeLa细胞,并用嘌呤霉素筛选稳定的细胞克隆。采用RT-PCR和Western blotting检测细胞中MDC1mRNA和蛋白表达的变化。结果重组psiRNA-MDC1质粒经测序鉴定正确;重组反转录病毒感染HeLa细胞后用嘌呤霉素筛选出稳定的细胞克隆;RT-PCR和Western blotting检测人MDC1mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。结论携带人MDC1基因RNA干扰双链DNA片段的反转录病毒感染HeLa细胞后能明显抑制MDC1mRNA和蛋白表达,为进一步研究MDC1在宫颈癌中的作用奠定了基础。

英文摘要：

Objective To construct a retroviral-mediated expression system containing double strands DNA for RNA interference on human mediator of DNA damage checkpoint protein 1 （MDC1）, and investigate the effects of the system on the expression of MDC1 in HeLa cell line. Methods A recombinant retroviral vector pSit~NA MDC1 was constructed by cloning a double strands DNA for RNA interference on human MDC1 gene into a retroviral vector pSilencer5. 1-H1 Retro. The pSiRNA-MDC1 plasmid was transfected into PT67 packaging cell line, the retroviral supernant was collected 3 days after the transfection from the PT67 cells transfected with pSiRNA-MDC1 plasmid using 0. 45 micrometer filter. HeLa cells were infected with retroviral supernant supplemented with polybrene. HeLa cell clones in fected with retroviral supemant were screened 72 hours later with puromycin, the concentration of which was 0. 6μg/ml. After 12 clays screening, stably infected Hela cell clones were generated. The expression of MDC1 in stably infected HeLa cell clones was examined by RT PCR and Western blotting for mRNA and protein levels. Results The pSiRNA-MDC1 recombinant retroviral vector had been con structed correctly and verified by sequencing. Stably infected HeLa cell clones were generated successfully after screening with puromycin. The expression of MDC1 in stably infected HeLa cells was decreased significantly at rnRNA and protein levels compared with that of nega tive control group and normal group. Conclusions The retrovirus containing pSiRNA MDC1 retroviral vector shows effective inhibition on the expression of MDC1 at mRNA and protein levels. The successfully constructed stably infected HeLa cell clones provide a favorable foundation for further study on the function of MDC1 in cervical cancer.