【摘要】 目的构建Survivin基因特异性小干扰RNA(siRNA),检测siRNA-Survivin对Survivin基因表达的抑制,在人肝癌细胞株HepG2中研究survivin和survivin siRNA对细胞凋亡的影响。方法设计survivin siRNA序列,构建靶向Survivin siRNA真核载体。利用脂质体转染人肝癌HepG2细胞,通过相差显微镜下观察、四甲基偶氮唑盐微量酶反应比色法(MTT法)及反转录-聚合酶链反应(RT-PCR)观察转染后survivin的表达,以及HepG2细胞的凋亡。结果成功构建了survivin siRNA表达载体,并且si-survivin对survivin有明显抑制效应,可显著抑制HepG2细胞的发生凋亡。转染Survivin-siRNA细胞活性受到显著抑制(P<0.05),光镜下出现明显的凋亡形态,DNA电泳出现典型的凋亡"梯状"带。RT-PCR结果显示细胞转染24 h、48 h和72 h后HepG2 Survivin mRNA分别减少了56%、78%和50%,而siR-NA阴性对照与未转染细胞相比差异不显著。结论转染的Survivin-siRNA可特异性抑制肝癌细胞株HepG2凋亡抑制基因的表达,从而为肿瘤的基因治疗提供新的实验基础。更多还原

【Abstract】 Objective To study siRNA gene transfection on expression of endogenous survivin in hepatoma carcinoma cell line HepG2 cells.Methods The siRNA gene transfection on expression of endogenous survivin in hepatoma carcinoma cell line HepG2 cells was studied and analyzed.The reverse repeated sequence of survivin siRNA gene was designed and synthesized.Survivin gene sequence specific siRNA vectors were constructed.The hepatoma carcinoma cell line HepG2 cells were transfected with survivin siRNA expression vectors via Lipofectamine TM 2000.MTT assay had been used to detect the rate of cellular inhibition.The expression level of survivin mRNA had been assayed by RT-PCR,morphological observation and flow cytometry analysis.Results There were obvious morphological changes in HepG2 cells after transfection of siRNA under optical microscopy.The cell growth and viability in survivin-siRNA transfected group were significantly inhibited(P<0.05).DNA electrophoresis showed a typical DNA-ladder of apoptosis.The expression of survivin mRNA in cells treated by survivin-siRNA at 24,48 and 72 h later was significantly reduced by about 56%,78% and 50% respectively,as compared with those of negative and blank control groups.The latter two groups had similar expression levels.Conclusion Survivin-siRNA designed and transfected in this current study can remarkably inhibit the viability of HepG2 cells and induce their apoptosis,thus it may shed a new experience in gene-therapy for carcinoma.更多还原