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目的:探讨转染新构建的UpIb启动子调控携Smac基因的真核表达质粒pcDNA3-UpIbpromoter-Smac对膀胱癌细胞的促凋亡效用。方法:用脂质体将质粒转染到膀胱癌BIU-87细胞系中,24h后用RT-PCR检测Smac的表达,以低剂量丝裂霉素C诱导凋亡,利用倒置光学显微镜、Wrighs-Gimesa染色、DNA凝胶电泳技术、TUNEL-荧光标记技术、流式细胞术检测凋亡。结果:丝裂霉素C处理的各组在倒置光学显微镜和Wrighs-Gimesa染色油镜下可见到典型的凋亡形态学改变,DNA凝胶电泳呈特征性的梯形条带。TUNEL-荧光标记技术及流式细胞术检测转染pcDNA3-UpIbpromoter-Smac组凋亡率显著高于单用丝裂霉素C组。结论:转染pcDNA3-UpIbpromoter-Smac能够通过提高Smac的表达而有效促进丝裂霉素C诱导的膀胱癌细胞凋亡,提示该质粒配合凋亡诱导药物可能成为膀胱癌新的靶向基因治疗手段。
AIM: To investigate the apoptosis-promoting effect of pcDNA3-UpIbpromoter-Smac transfected with a newly constructed UpIb promoter on bladder cancer cells by regulating the expression of Smac gene. Methods: The plasmid was transfected into bladder cancer cell line BIU-87 by lipofectamine. The expression of Smac was detected by RT-PCR 24 h later. The apoptosis was induced by low dose of mitomycin C. The expression of Wrighs- Gimesa staining, DNA gel electrophoresis, TUNEL-fluorescent labeling and flow cytometry. RESULTS: Mitomycin C treatment showed typical apoptosis morphological changes under inverted light microscope and Wrighs-Gimesa staining. The DNA ladder electrophoresis showed a characteristic trapezoidal band. The apoptosis rate of pcDNA3-UpIbpromoter-Smac transfected with TUNEL-fluorescence and flow cytometry was significantly higher than that of mitomycin C alone. CONCLUSION: Transfection of pcDNA3-UpIbpromoter-Smac can effectively promote the apoptosis of bladder cancer cells induced by mitomycin C by increasing the expression of Smac, suggesting that this plasmid may be a new targeted gene therapy for bladder cancer .