改良型TAT-凋亡素体外抗人膀胱肿瘤活性的研究  

Anti-cancer Effect of Improved TAT-VP3 Gene on Human Bladder Cancer in Vitro

作　　者：王春晖[1] 王剑松[1] 詹辉[1] 李鸿钧[2] 丁明霞[1] 颜汝平[1] 柯昌兴[1] 徐鸿毅[1] 

机构地区：[1]昆明医学院第二附属医院泌尿外科,云南省泌尿外科研究所,昆明市650101 [2]中国医学科学院,北京协和医学院医学生物学研究所

出　　处：《中国肿瘤临床》2011年第5期246-249,254,共5页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金资助(编号:30760251)~~

摘　　要：目的:探讨改良型TAT-凋亡素(TAT-VP3)融合基因在不同人膀胱癌细胞中的表达及诱导凋亡的效应方法:利用改良型TAT-VP3原核表达载体pGEX-6p-1-TAT-VP3构建真核表达载体PcDNA3-TAT-VP3,经BamH I/Xho I双酶切鉴定和基因测序无误后采用脂质体介导的基因转染法转染人膀胱癌BIU-87及EJ细胞,利用RT-PCR技术检测改良型TAT-VP3基因在细胞中的表达状况。通过透射电镜扫描观察转染后不同时段肿瘤细胞的超微结构变化,MTT法检测转染重组质粒后对人膀胱癌细胞的增殖活性的影响,流式细胞术TUNAL法检测转染重组质粒24、48、72h后BIU-87及EJ细胞的凋亡率,对实验结果进行统计学分析。结果:成功构建重组质粒PcDNA3-TAT-VP3并经测序无误,转染人膀胱癌细胞后的RT-PCR结果证实改良型TAT-VP3基因在人膀胱癌细胞中获得表达。透射电镜观察到BIU-87及EJ细胞凋亡早期染色质聚集,线粒体固缩,凋亡中晚期核固缩及凋亡小体等超微形态学改变。MTT法检测结果显示转染重组质粒PcDNA3-TAT-VP3后的BIU-87及EJ细胞增殖活性明显下降(P<0.01)。流式细胞术TUNAL法检测显示BIU-87及EJ细胞发生早期凋亡,其凋亡率随时间的推移呈逐渐上升趋势,转染72 h后BIU-87和EJ细胞的凋亡率分别为(30.44±1.47)%、(42.27±2.66)%,与对照组比较差异有统计学意义(P<0.01) 结论:改良型TAT-VP3基因的表达能早期、高效地诱导不同人膀胱癌细胞发生凋亡,为纯化改良型TAT-VP3融合蛋白以及将其应用于膀胱灌注治疗膀胱转移肿瘤的研究奠定实验基础。Objective： To explore the expression and apoptosis-inducing effects of improved TAT-VP3 fusion gene on different human bladder cancer cell lines. Methods： The recombinant plasmid PcDNA3-TAT-VP3 was constructed using the prokaryotic expression vector pGEX-6p-1-TAT-VP3. After double enzymatic digestion with BamH I and Xho I and verification by sequencing, lipo- some-induced gene transfection was used to transfect BIU-87 and EJ human bladder carcinoma cells. RT-PCR was used to detect the expression of improved TAT-VP3 in the cells. Transmission electron microscopy was used to observe changes in the ultrastructure of the tumor cells at different periods of time after the transfection. MTT assay was conducted to detect the effect of the recombinant plas- mid on proliferative activity of the bladder carcinoma cells, and flow cytometry TUNAL was conducted to detect the apoptotic rate at 24, 48 and 72 hours after transfection. Statistical analysis of the experimental results was conducted. Results： The eukaryotic expression vector PcDNA3-TAT-VP3 was successfully constructed. The results of RT-PCR after the transfection confirmed the expression of improved TAT-VP3 mRNA in bladder cancer cells at 24 hours after transfection. The typical morphological changes in early apoptosis such as cell shrinkage, chromatin condensation, nuclear fragmentation and apoptotic bodies were observed using transmission electron microscopy. MTT assay showed that the proliferative activity of bladder cancer cells distinctively decreased （ P 〈 0.01 ） after transfection. The flow cytometry TUNAL detection showed that early apoptosis occurred in BIU-87 and EJ cells with a gradual increase in the apoptotic rate over time. Seventy two hours after the transfection, the apoptotic rate was （ 30.44±1 .47 ） % in BIU-87 cells and （ 42.27 ± 2.66 ） % in EJ cells. There was statistical significance between the experimental group and the control group （ P 〈 0.01 ）. Conclusion： Expression of improved TAT-VP3 fusion gene can effecti

关 键 词：细胞凋亡 VP3蛋白 膀胱肿瘤 

分 类 号：R737.14[医药卫生—肿瘤]