【摘要】 目的利用RNA干扰技术下调凋亡抑制因子survivin在人成神经细胞瘤细胞系SH-SY5Y中的表达,并评价其对肿瘤细胞凋亡的影响。方法将设计合成的寡核苷酸片段退火后连入pBSHH1构建survivin的短发夹RNA表达质粒pBSHH1-survivin;通过RT-PCR和Western印迹评价其对SH-SY5Y细胞内源性survivin表达的抑制作用;运用Hoechst33258核染色、DNA凝胶电泳以及AnnexinFITC染色检测survivin表达下调对SH-SY5Y细胞凋亡的影响。结果酶切和DNA测序证实survivin的短发夹RNA表达质粒构建成功;RT-PCR和Western印迹表明质粒转染SH-SY5Y细胞后能明显下调survivin的表达;Hoechst33258核染色、DNA凝胶电泳以及AnnexinⅤFITC染色显示下调survivin的表达能诱导SH-SY5Y细胞凋亡。结论成功构建了有效针对survivin的短发夹RNA表达质粒;下调survivin的表达能诱导SH-SY5Y细胞凋亡。更多还原

【Abstract】 Objective To investigate the effects of RNA interference of survivin on cell apoptosis of SH-SY5Y cells. Methods The short hairpin RNA(shRNA) expression vector of survivin gene RNAi (pBSHH1-survivin) was constructed by ligating the designed oligonucleotides into pBSHH1; RT-PCR and western blot were used to estimate the effects of pBSHH1-survivin on down-regulating the expression of survivin gene in SH-SY5Y cells; Nuclear DNA staining by Hoechst 33258,Gel electrophoresis of DNA and Annexin FITC staining were used to ascertain whether cell apoptosis could be induced by down-regulating the expression of survivin gene in SH-SY5Y cells. Results Enzyme digestion analysis and DNA sequencing proved that shRNA expressing vector of survivin was constructed successfully; RT-PCR and Western Blot showed that the expression of survivin was repressed significantly by pBSHH1-survivin; Nuclear DNA staining by Hoechst 33258,Gel electrophoresis of DNA and Annexin FITC staining showed that Knockdown of survivin expression by RNAi induced apoptosis in SH-SY5Y cell. Conclu-sion The effective shRNA expressing vector of survivin has been successfully constructed; Down-regulating the expression of survivin by RNAi may induce apoptosis in human neuroblastoma cell line SH-SY5Y.更多还原