【作者】 曾柯；

【导师】 吴小候；

【作者基本信息】 重庆医科大学 ， 外科学， 2007， 硕士

【摘要】 目的初步探讨用RNAi沉默survivin基因对膀胱癌T24细胞增殖和调亡的影响。方法扩增并鉴定重组质粒;实验分为空白对照组,阴性对照组和实验组,将重组质粒(pTZU6+1-shRNA-survivin)、阴性质粒分别转染至膀胱癌T24细胞,用RT-PCR检测T24细胞survivin基因表达的变化;用MTT法观察T24细胞增殖抑制率;用AO/EB荧光染色检测和膜联蛋白V-PI(annexinV/PI)染色流式细胞技术检测T24细胞调亡的情况;用透射电镜观察T24细胞形态的改变。结果酶切及测序鉴定结果表明:重组质粒目的片段插入正确; RT-PCR实验显示:转染pTZU6+1-shRNA-survivin 24h后,实验组T24细胞survivin基因的表达受到显著抑制,抑制率为41.77%,与阴性对照组相比有差异显著性,(P<0.05);MTT实验显示:转染48h后,实验组T24细胞增殖抑制率达到最高,为52.53%,与空白对照组及阴性对照组相比有差异显著性,(P<0.05);annexinV/PI染色法流式细胞术显示:转染24h后,实验组T24细胞凋亡率为(16.76±1.31)%,与空白对照组及阴性对照组相比有差异显著性,(P<0.05);转染24h后,实验组透射电镜观察可见各期凋亡细胞及凋亡小体,阴性对照组无明显改变。结论转染pTZU6+1-shRNA-survivin可以显著下调survivin基因的表达,并可以显著抑制T24细胞增殖并诱导其自发凋亡。RNAi技术有可能应用于膀胱癌的治疗。更多还原

【Abstract】 Objective To investigate the proliferation supperring and apoptosis inducing effects of RNAi targeted to Survivin on T-24 cells, a bladder cancer cell line.Methods An siRNA eukaryotic expression vector targeted to Survivin, pTZU6+1-shRNA-survivin, was emplified in system and identitified by sequencing method. It was transfected into T24 cells following lipofectamineTM2000 protocols. The changes of transcriptional level of survivin gene were detected by semi-quantitive RT-PCR.The effects of proliferation suppressing on T24 cell were detected by MTT assay. The effects of apoptosis inducing on T-24 cells were detected by flow cytometry assay.Rusult The recombined vector was successfully emplified, and was proved to have no mutation sites by sequencing. 24 hours after trancfection the transcriptional level of Survivin gene in T24 cells was significently suppressed at the rate of 41.77%, detected by semi-quantative RT-PCR. The proliferation suppressing effects of RNAi targeted to survivin were observed by MTT assy, 48 hours after transfecion. The proliferation inhibitory rate reached to 52.53%. Meanwhile, 24h after transfection, it was observed that silence survivin by RNAi could significently induce the spontaneous apoptosis of T-24 cells at the rate 16.76%±1.31 , detected by flow cytometry assay and comfirmed by electron microscope.Conclusion Silencing exogenous Suvivin by RNAi can significantly suppress the proliferation of T-24 cells and induce spontaneous apoptosis. RNAi targeted to survivin has a potential role in gene therapy of bladder cancer.更多还原