目的:研究RNAi 技术抑制宫颈癌细胞CaSKi中HPV16 E6 癌基因的表达以及对细胞生物学特性的影响.方法:构建真核细胞表达特异性shRNA的pGENESIL-1/E6 (PS6)重组质粒,脂质体转染细胞后用半定量RT-PCR技术检测CaSKi细胞中E6mRNA 水平的变化,然后用western blotting检测RNA干扰前后p53和p21蛋白的表达来验证RNAi效应.用透射电子显微镜观察CaSKi/PS6细胞形态变化;应用流式细胞仪检测细胞的凋亡和细胞周期的变化,最后利用细胞计数和裸鼠成瘤实验测定CaSKi/PS6 细胞增殖能力.结果:成功构建了真核细胞表达特异性siRNA的PS6 重组质粒;在RNA干扰24、48和72小时后CaSKi/PS6细胞中E6mRNA分别下降了21.50%、52.60%和65.60%;CaSKi/PS6 细胞出现凋亡的特征性改变;RNA干扰24h、48h和72h后CaSKi/PS6细胞的凋亡率分别为27.94%、31.95%和56.63%,细胞生长周期停滞于S期.结论:应用RNA干扰技术能有效地、特异地抑制宫颈癌细胞中HPV16 E6基因的表达,为RNA干扰应用于研究宫颈癌发病分子机制、临床治疗和干预性预防提供了新的方法.%Objective :To specifically inhibit the expression of oncogene E6 of HPV16 in cervical carcinoma cell CaSKi by RNA interference ( RNAi )and investigate the RNAi effects on cellular biological features. Methods : The recombinant plasmid pGENESIL - 1/E6 ( PS6 ) which expressed small hairpins RNA ( shRNA ) in vivo targeting E6 gene was constructed. E6mRNA was detected by semi - quantitative RT - PCR and p53 and p2lproteins were detected by western blotting. The morphologic variations in CaSKi/PS6 were observed by transmission electron microscope. Cell growth curve and tumor formation assay in nude mice were performed to detect the ability of proliferation of CaSKi/PS6 cells. Flow cytometry ( FCM ) was carried out to detect apoptosis and cell cycles. Results: The plasmids pGENESIL - 1/Negative ( PSN ) and pGENESIL - 1/E6 ( PS6 ) were constructed successfully. The amount of E6 mRNA in CaSKi/PS6 was decreased 21. 50％ ,52. 60％ and 65. 60％ respectively at 24h,48h and 72h after RNAi. The expression of p53 and p2lproteins was increased in CaSKi/PS6. Transmission electron microscope assay showed the apoptosis of CaSKi/PS6. At 24h , 48h and 72h after RNAi , the apoptosis rates of CaSKi/PS6 were 27. 94％ , 31. 95％ and 56. 63％ respectively and the cell cycle stage was blocked at S phase. Conclusion: Specific small hairpin RNA ( shRNA ) targeting E6 in HPV16 successfully inhibited the expression of E6 in CaSKi containing integrated HPV16. These findings suggested that RNA interference was effective and might provide new strategy for prevention and treatment of cervical carcinoma infected by HPV16.
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