不可逆性电穿孔介导HPV16E6shRNA对宫颈癌.doc

不可逆性电穿孔转染HPV 16 E6 shRNA 对宫颈癌SiHa细胞增殖的影响 王智亮1，余腾骅2，秦琴1，吴雨桐1，张文倩1，华媛媛1，熊正爱1，周玮3 （400016 重庆， 重庆医科大学：附属第二医院妇产科1，400010，附属第一医院内分泌乳腺外科2；400013 重庆，重庆市妇幼保健院产科3 ) [摘要] 目的 探讨利用亚致死剂量脉冲电场产生不可逆电穿孔（Irreversible electroporation;IRE）转染HPV 16 E6 shRNA的可行性，以及二者共同作用对宫颈癌SiHa细胞增殖的影响。方法 构建HPV 16 E6 shRNA真核表达载体，通过特定电场参数的IRE，作用于SiHa细胞与HPV 16 E6 shRNA干扰质粒的混悬液。在荧光显微镜下观察SiHa细胞绿色荧光蛋白的表达，判断转染效率；RT-PCR检测HPV 16 E6 mRNA的变化，Western Blot检测HPV 16 E6蛋白、P53及PCNA的表达，CCK-8法检测细胞增殖能力的变化。结果 成功构建HPV 16 E6 shRNA真核表达载体，IRE处理后24小时细胞即可见到绿色荧光；RT-PCR提示IRE转染干扰质粒后，与IRE组比较，E6 mRNA表达下降(63.23±8.17)%（P＜0.05），Western blot提示E6蛋白降低（51.06±6.32)%，P53蛋白表达增高（253.53±12.33)%、PCNA表达下降(63.26±7.17)%；CCK-8法提示转染干扰质粒后，细胞增殖受到抑制；IRE转染组较单纯转染组抑制细胞增殖作用更明显(P＜0.05)。结论 IRE可促进外源基因进入细胞；且与shRNA或有协同作用，可显著抑制宫颈癌SiHa细胞增殖。 [关键词] 不可逆性电穿孔；干扰质粒；宫颈癌；增殖 Effect of irreversible electroporation transfect HPV 16 E6 shRNA on the proliferation of cervical cancer SiHa cell Wang Zhiliang1，Yu Tenghua2，Qin Qin1，Wu Yutong1，Zhang Wenqian1，Hua Yuanyuan1，Xiong Zhengai1，Zhou Wei3（1Department of gynecology and obstetrics，Second Affiliated Hospital，Chongqing Medical University，Chongqing，400010； 2Department of Endocrine [基金项目]国家自然科基金81301928）；重庆市卫生局医学科研项目（2011-2-155） [通信作者]周玮，电话：（023E-mail: dr.zhouwei@163.com and Breast Surgery，First Affiliated Hospital，Chongqing Medical University，Chongqing，400016；3Department of obstetrics，Chongqing Health Center for Women and Children,Chongqing 400013） [Abstract] Objective To verify the feasibility of using irreversible electroporation(IRE) mediating HPV 16 E6 shRNA into cervical cancer cell line SiHa，and the co-effect on the proliferation of SiHa. Methods A HPV 16 E6 gene targeted shRNA vector was designed to transfect SiHa by IRE according fixed parameters.The expression of GFP and transfection efficiency was confirmed by inverted fluorescence microscope 24h after IRE transfected the vector.The expression of E6 at mRNA and protein levels was detected by RT-PCR and Western blotting, which was also applied to de