重组pcDNA3-UpIb promoter-Smac质粒转染靶向膀胱癌的促凋亡效应

Apoptosis-Promoting Effectiveness of Plasmid Carrying UpIb Promoter and Smac Gene in Targeted Gene Therapy of Bladder Cancer

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。

Abstract Objectives： To transfect a bladder cancer cell line with pcDNA3-Uplb-promoter- Smac, a eukaryotic expression vector carrying the Uplb promoter and the Smac gene and to study the effectiveness of its apoptosis-promoting capabilities. Methods： The bladder cancer cell line BIU-87 was transfected with plasmid by liposome. After 24 hours, the expression of Smac was detected by RT-PCR. Low-dose mitomycin C induced apoptosis which was detected by inverted microscopy with Wright- Giemsa staining, DNA agarose gel electrophoresis, TUNEL fluorescence-labeled technique and flow cytometry. Results： In all groups exposed to mitomycin C, typical morphological features of apoptosis could be detected by inverted microscopy with Wright-Giemsa staining, and DNA ladder could be detected by DNA agarose gel electrophoresis. The apoptosis rate of BIU-87 cells transfected with pcD- NA3-Uplb- promoter-Smac was significantly higher than that of the control cells that were not transfected as detected by TUNEL fluorescence-labeled technique and flow cytometry. Conclusions： The transfection of BIU-87 cells with pcDNA3-Uplb-promoter-Smac increased the expression of Smac and effectively promoted the apoptosis of BIU-87 cells induced by mitomycin C. Cooperative application of this vector and apoptosis-inducing drugs provides a new option for targeted gene therapy of bladder cancer.