【摘要】 目的观察生存素(survivin)基因干扰对SW480细胞增殖的影响,探讨其干扰对hTERT基因表达的影响及机制。方法构建携带survivin小发夹干扰RNA的重组表达载体pGenesil-1.1-survivin,转染结肠癌SW480细胞,MTT检测细胞增殖,流式细胞仪检测细胞增殖周期;荧光定量PCR和Western blot检测survivin、hTERT和Sp1的mRNA和蛋白表达,TRAP-ELISE法检测端粒酶活性。构建靶向Sp1基因的RNA干扰载体转染SW480细胞,检测hTERT基因mRNA和蛋白水平及端粒酶活性。结果 Survivin基因干扰后,SW480细胞增殖受到明显抑制,增殖抑制率为34.44%(P<0.05),G0/G1期细胞增加,S期和G2/M期细胞减少(P<0.01)。hTERT和Sp1基因的mRNA和蛋白表达水平显著降低,端粒酶活性降低(P<0.01)。Sp1基因干扰后,hTERT基因的mRNA和蛋白表达水平及端粒酶活性下降(P<0.01)。结论靶向沉默survivin基因对SW480细胞增殖有明显的抑制作用;同时下调hTERT基因表达,其机制可能与Sp1基因表达下调有关。更多还原

【Abstract】 Objective Observation of survivin gene interference effect on the proliferation of SW480 cells,to investigate the effects of the gene expression of hTERT and its mechanism of interference. Methods Small hairpin RNA targeting survivin was synthesized and recombinant plasmid pGenesil-1. 1-survivin was constructed. The siRNA recombinant was transfected into SW480 cell lines. Cell proliferation activity was detected by MTT and the cell cycles were analyzed by flow cytometry. The expression of hTERT,Sp1 mRNA and protein were analyzed by The real-time fluorescence quantitative PCR and western blot. Telomerase activity was examined by TRAP-ELISA analysis. To construct the interference vector targeting Sp1 and transfect into SW480 cell,hTERT mRNA and protein,telomerase activity were examined. Results The cell proliferation rate of SW480 cells transfected with siRNA-survivin recombinant vector was significantly decreased,the inhibitory rate of cell proliferation was 34. 44%( P < 0. 05),an increase in the percentage of cells in the G0/G1 phase and a decrease in the percentage of cells in the S and G2/M phase ( P <0. 01). that expression of hTERT and Sp1 mRNA and protein,telomerase activity decreased in shRNA-survivin and shRNA-Sp1 transfected cells( P < 0. 01). Conclusions Silencing survivin gene significantly inhibited the proliferation of SW480 cells and down-regulatied hTERT gene expression,its mechanism may be related with down-regulaed Sp1 gene expression.更多还原