【摘要】 目的构建并筛选出最有效的HPV16E6基因特异的小干扰RNA(smallinterferingRNA,siRNA)表达载体,观察其对宫颈癌细胞中HPV16E6基因表达的长期影响,探讨E6基因在宫颈癌发生过程中的分子作用机制,为临床HPV感染及宫颈癌治疗探索新方法。方法构建HairpinsiRNA质粒,稳定转染宫颈癌SiHa细胞,鉴定转染细胞中的质粒DNA,通过Real-TimeRT-PCR检测细胞中HPV16E6mRNA表达,采用Western-blot检测p53、p21等蛋白的变化。MTT法(四甲基偶氮唑盐微量酶反应比色法)检测SiHa细胞转染siRNA后细胞增殖曲线。结果HPV16E6AhairpinsiRNA表达载体转染人宫颈癌SiHa细胞,可以在细胞内长期表达siRNA,有效抑制细胞内HPV16E6基因的表达。E6AsiRNA能抑制细胞生长,作用持续达4个月以上。结论利用siRNA表达载体抑制整合在细胞中的外源HPVE6病毒癌基因可能是治疗HPV感染和宫颈癌的一种新的理想方法。更多还原

【Abstract】 Objective To Find a most effective short hairpin RNA(shRNA) for human papillomavirus(HPV) 16 E6 mRNA and investigate the effects of the HPV16E6 shRNA on cervical carcinoma cell.Methods HPV16 E6-specific hairpin siRNA expression vectors were constructed,sequenced and transfected into the cervical carcinoma SiHa cells,stable expression cell populations were selected with G418,pooled and passaged.HPV16 E6 mRNA levels in siRNA transfected cells were determined by Real-time RT-PCR and the most effective siRNA was screened.p53,p21 proteins were examined by western blot.Results HPV16 E6A siRNA was selected as the most effective siRNA,which could provide a 90% reduction of HPV16 E6 mRNA in the transfected cells.HPV16 E6A hairpin siRNA expression vector could effectively inhibit the expression of HPV16 E6 mRNA at 4 months after transfection.p53,p21 proteins levels increased greatly in transfected cells.The E6A siRNA transfected cells showed a proliferation inhibition.Conclusions HPV16 hairpin siRNA expression vector can stable express siRNA and effectively inhibit HPV viral genes expression in SiHa cells for more than 4 months after transfection.The E6A siRNA can inhibit cell proliferation.更多还原