【摘要】 目的:研究短发夹状RNA(short hairpin RNA,shRNA)构建的人乳头瘤病毒(human papillomavirus,HPV)16 E7 siRNA表达载体转染宫颈癌细胞株CaSki细胞后,在体内外对CaSki细胞E7基因的抑制作用。方法:利用脂质体将构建有HPV16 E7特异性小干扰RNA(small interfering RNA,siRNA)的表达载体P1、P2、P3转染CaSki细胞,以实时荧光定量RT- PCR及流式细胞仪检测不同时间点E7 mRNA和蛋白的变化,并将载体P1转染后的细胞接种到裸鼠皮下,4周后观察裸鼠体内皮下移植瘤体积和质量的差异,并用免疫组化检测瘤体组织中E7蛋白的表达变化。结果:表达载体P1、P2、P3均能抑制CaSki细胞E7 mRNA和蛋白的表达。其中载体P1抑制作用最强,在抗性克隆形成后1周,对E7 mRNA和蛋白的抑制率分别为92．86%、84．21%;在抗性克隆形成后4周,抑制率仍分别为68．95%、62．50%。在接种后4周载体P1组裸鼠体内皮下移植瘤的体积和重量明显小于空载体组和对照组,同时E7蛋白的表达也被有效抑制,抑制率为74．75%。结论:构建有shRNA的E7 siRNA表达载体在体内外可明显抑制E7基因的表达。更多还原

【Abstract】 Objective:To investigate the inhibitory effect of human papillomavirus(HPV)16E7-specific shRNA expression vector on HPV16 E 7 gene expression in cervical cancer CaSki cells in vitro and in vivo.Methods:The siRNA vectors,P1,P2 and P3,were transfected into CaSki cells by liposome mediation,respectively.The HPV16E7 mRNA and protein expression were detected by real-time reversed transcriptase polymerase chain reaction(RT-PCR)and flow eytometry analysis(FCM)at different time points after transfection.CaSki cells transfected with vector P1 were inoculated Subcutaneously into the nude mice. The volume and weight of tumor in nude mice at the 4th week after inoculation were recorded.The expressions of E7 protein in tumor tissues was detected by immunohistochemical method.Results:The expression of HPV16 E7 mRNA and protein were in- hibited by vector Pl,P2,and P3.Among them the inhibitory effect of vector P1 was stronger than P2 and P3.At the 1st week after the formation of CaSki resistant clone the E7 mRNA and protein inhibition rate was 92.86% and 84.21%,respectively; and at the 4th week it decreased to 68.95% and 62.50%,respectively.The tumor volume was significantly smaller and tumor weight was significantly lighter in vector P1 group than that of empty vector group and CaSki group at the 4th week after inocula- tion.E7 protein expression in tumor tissues was reduced by 74.75% simultaneously.Conclusion:The expression of E7 gene in CaSki cells is inhibited effectively by HPV16E7-specific shRNA expression vector.更多还原