靶向HPV16-E6的siRNA对宫颈癌CaSki细胞的抑制作用

以RNA干扰技术为手段，HPV编码的癌蛋白E6为靶标．探讨靶向HPV16-E6的siRNA对宫颈癌细胞生物学行为的影响，并试图阐明该实验的临床意义．构建靶向HPV16-E6的siRNA表达载体，应用体外转染试剂转染HPV16-E6阳性的宫颈癌CaSki细胞．以RT—PCR检测CaSki细胞中E6蛋白的mRNA的表达．借助细胞色素c测定来分析细胞凋亡相关分子的表达和活性．从而研究靶向HPV16-E6的siRNA诱导细胞凋亡的分子机制．RT．PCR检测结果表明，将靶向HPV16-E6的siRNA的表达载体瞬时转染到HPV16-E6阳性的CaSki细胞后，其所舍E6蛋白质和mRNA的表达下调；Westernblotting栓出抑凋亡蛋白Bcl．2的表达亦告下调；细胞色素C释放实验结果显示，HPV16-E6siRNA能够诱导细胞色素c从线粒体释放到细胞浆中。从而诱导细胞凋亡．靶向HPV16-E6的siRNA能够有效抑制细胞增殖并诱导细胞凋亡．靶向HPV16-E6的siRNA为研究重要致瘤蛋白HPV16-E的功能开辟了新途径，给HPV16-E6阳性肿瘤的靶向基因治疗提供新的实验依据．并探索了HPV感染及宫颈癌的新基因疗法．

Depressant Effects of the HPV16-E6-Targeted siRNA on the CaSki Cell of Cervical Cancer

To study the effects of the HPV16-E6-targeted siRNA on the biological behavior of cervical cancer cells by using RNA interference techniques and the HPV-encoded oncoprotein HPV16-E6 as a target. Moreover, we attempt to elucidate the clinical significance of its experiment. The expression vectors of HPV16-E6-targeted siRNA were constructed and transfected into the HPV16-E6-positive CaSki cells by in vitro transfection reagents. The expression levels of the HPV16-E6 mRNA and the mRNA encoded oncoproteins in cervical cancer CaSki cells were detected by RT-PCR and Western blotting, respectively. The expression and activities of the apoptosis-associated molecules were analyzed through cytochrome c assay in order to investigate the molecular mechanism of cell apoptosis induced by HPV16-E6-targeted siRNA. RT-PCR assay demonstrated that after the expression vectors of HPV16-E6-targeted siRNA were transiently transfected into the HPV 16-E6-positive CaSki cells, a variety of considerable changes occurred: the expression of E6 protein and mRNA in CaSki cells were down-regulated. Western blotting showed that the expression of anti-apoptotic protein Bcl-2 was also down-regulated; the cytochrome c liberation test displayed its release from mitochondria into cytoplasm, thus inducing the cell apoptosis. HPV16-E6-targeted siRNA can significantly inhibit the proliferation of transplanted tumor cells and induce cell apoptosis. It finds out a new approach for the functional study of this important oncoprotein HPV16-E and provides new experimental evidences for the gene-targeted treatment of HPV16-E6-positive tumors, especially exploring a new gene therapy for HPV infection and cervical cancer.