靶向HPV18 E6基因不同锌指结构编码序列的siRNA对宫颈癌HeLa细胞作用的比较_医学论文.docVIP

靶向HPV18 E6基因不同锌指结构编码序列的siRNA对宫颈癌HeLa细胞作用的比较_医学论文 靶向HPV18 E6基因不同锌指结构编码序列的siRNA对宫颈癌HeLa细胞作用的比较_医学论文 作者：殷广洁 罗兵 王言奎 【摘要】 　　目的 探讨靶向HPV18 E6基因不同锌指区编码序列的siRNA对HeLa细胞的影响。方法 分别设计靶向E6蛋白不同锌指区编码序列的两对siRNA，脂质体法转染HeLa细胞。半定量RT检测E6 mRNA转录表达水平；四甲基偶氮唑盐比色法（MTT法）检测细胞增殖活性；流式细胞仪Annexin V双染检测细胞凋亡情况。结果 与siRNASCR转染组比较，RT结果显示，siRNA1和siRNA2转染后E6表达水平明显降低（F=8.709，0.05）， 且siRNA2组较siRNA1组下降更为显著（t=3.61，0.05）。MTT结果提示，2对siRNA转染后48 h和72 h的细胞增殖活性均明显下降（F=60.583、102.421，0.05），siRNA2组较siRNA1组下降更为明显（t=4.717、7.932，0.01）；2对siRNA转染后均未检测到明显的细胞凋亡。结论 靶向锌指2区编码序列的siRNA抑制E6基因表达和HeLa细胞增殖的效果更好，锌指2区可能是使HeLa细胞发生恶性转化并使其增殖失调控的关键区域。 【关键词】 RNA干扰 锌指 乳头状瘤病毒 人 E6基因 宫颈肿瘤 HeLa细胞 　　［ABSTRACT］ Objective To investigate the biological changes of HeLa cells after HPV18 E6 was knocked down at the comically synthetic siRNA targeting to HPV18 E6 was transfected into HeLa cells by Lipofectamine 2000, then the mRNA levels of HPV18 E6 were tested by RTPCR MTT and flow cytometry used to detect proliferation and cell apoptosis,respectively. Results Compared with siRNASCR, the expression of E6 showed with RTPCR was significantly decreased after siRNA1 and siRNA2 transfection (F=8.709,0.05), with the decreasing more obvious in siRNA2 group (t=3.61,0.05). MTT results at 48 h and 72 h showed that both siRNA1 and siRNA2 could inhibit the proliferation of HeLa cells (F=60.583,102.4210.05) with siRNA2 showing a more apparente effect (t=4.717,7.9320.01). Flow cytometry revealed no significant apoptosis. Conclusion The siRNA targeting to the zinc finger Ⅱ more effectively inhibit the expression of E6 and the proliferation of HeLa cells. Maybe zinc finger Ⅱ is the key structure leading to the malignant transformation and outofcontrol proliferation of HeLa cells. 　　［KEY WORDS］ RNA interference Zinc fingers Human papillomavirus 6 Uterine cervical neoplasms HeLa cells 　　HPV (human papillomavirus)感染是公认的宫颈癌致病因子，其E6基因及其产物在诱导宫颈上皮细胞永生化过程中常常导致细胞基因失活、变异和异常表达，是其最主要癌蛋白编码基因。E6蛋白主要结构特征是含有2个锌指结构，其中锌指1区（zinc fingers，简称ZF）位于第30～66氨基酸，锌指2区（zinc fingers，简