【摘要】 目的构建表达靶向抑制survivin基因的表达短发夹结构(shRNA)的RNA干扰载体,导入人胶质瘤细胞U251中,研究靶向抑制survivin基因对U251细胞的凋亡诱导以及细胞周期阻滞作用.方法在survivin全长序列中选取设计3条含19个核苷酸(19nt)靶序列两条反向重复序列,中间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,分步酶切连接,构建出含有3条shRNA模板且能独立编码shRNA的重组干扰载体pGenesil-1/survivin;采用Metafectene转染试剂将干扰质粒导入到胶质瘤细胞U251;采用荧光定量PCR以及Westernbloting分别从mRNA和蛋白水平检测干扰效果;采用Annexin-V/PI双色标记的流式细胞仪法检测siRNA诱导的细胞凋亡,PI染色检测细胞周期阻滞.结果实时荧光定量PCR以及Westernblotting检测显示,survivin基因的mRNA转录水平以及蛋白水平的表达均得到显著抑制;流式细胞仪检测分析显示,survivin基因表达被抑制后,U251细胞凋亡率明显增高,细胞周期出现明显的G2/M阻滞.结论靶向survivin基因的重组siRNA干扰载体pGenesi-l/survivin介导的RNAi显著靶向抑制了survivin基因在人胶质瘤细胞U251中的表达,并明显地诱导了U251细胞发生凋亡和G2/M期细胞周期阻滞.更多还原

【Abstract】 AIM: To construct a recombinant short hairpin RNA (shRNA) expression vector targeting survivin and investigate apoptosis and cell cycle arrest in glioma cell line U251 induced by survivin-targeting small interfering RNA (siRNA). METHODS: Three siRNA sequences of 19 nucleotides were derived from the full-length coding region of survivin gene and then 2 complementary oligonucleotides with 9 bases for loop sequence were designed for shRNA DNA templates. The shRNA templates were cloned into siRNA expression vector pGenesil-1 to generate survivin-targeting siRNA expression vector pGenesil-1/survivin encoding 3 shRNAs targeting survivin by digestion and ligation step by step. pGenesi-l/survivin was transfected into U251 by Metafectene. Real -time quantitive PCR and Western blotting were performed to validate the interfering efficacy of survivin at mRNA level and protein level respectively. Annexin-V/PI double staining by flow cytometry analysis was used to evaluate apoptosis. PI staining was conducted to evaluate cell cycle arrest. RESULTS: The expression of survivin at both RNA level and protein level were significantly down-regulated as validated by real-time quantitive RT-PCR and Western bloting. Flow cytometry analysis revealed glioma cell line U251 suffered a notable apoptosis and G2/M arrest after RNA interference mediated by siRNA targeting survivin. CONCLUSION: RNA interference (RNAi) mediated by the siRNA expression vector pGenesi-l/survivin could significantly down-regulate the expression of survivin and induce remarkable apoptosis and G2/M cell cycle arrest in glioma cell line U251.更多还原