凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应  

Construction of recombinant adeno-associated virus expressing apoptin and anti-tumor effect on human bladder cancer in vitro

作　　者：王春晖[1] 王剑松[1] 王文举[2] 余力锐[1] 詹辉[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,650101 [2]中国科学院昆明动物研究所动物模型与人类疾病机理重点实验室

出　　处：《中华实验外科杂志》2012年第6期1009-1012,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金资助项目（30760251）;教育部博士点基金资助项目（20070678002）;云南省社会发展科技计划项目（2009CA005）;云南省创新团队高层次人才培引工程基金资助项目（20080C015）;昆明医学院博士创新基金资助项目（2011D04）

摘　　要：目的探讨携带凋亡素基因的重组腺相关病毒构建方法，观察其体外抑制膀胱癌的效应。方法构建重组质粒pAAV-VP3，经EcoRI/SalI双酶切鉴定和基因测序无误后，采用三质粒共转染法包装重组腺相关病毒rAAV—VP3。收获病毒后聚合酶链反应（PCR）扩增病毒液中的目的基因鉴定重组病毒，对其进行纯化后透射电镜观察病毒颗粒，并检测病毒滴度。按每个细胞5×10^5个载体基因组的剂量感染EJ细胞后，逆转录（RT）-PCR检测VP3基因在EJ细胞中的转录，Westernblot法检测凋亡素蛋白的表达。透射电镜扫描观察感染重组病毒后肿瘤细胞的超微结构变化，流式细胞术（FCM）检测重组病毒对EJ细胞的影响。结果转染72h后重组腺相关病毒rAAV-VP3包装成功，滴度为5．1×10^11个载体基因组／ml，电镜下可见病毒颗粒。感染EJ细胞后，可检测到VP3基因的转录和凋亡素蛋白表达，电镜观察到凋亡形态学改变，FCM检测S期细胞比例降低和G2／M期细胞比例增高，与对照组比较差异有统计学意义（P〈0．01）。结论重组腺相关病毒rAAV—VP3在体外能够发挥生物学活性，其介导的凋亡素表达抑制膀胱癌的体外效应为体内实验奠定了基础。Objective To construct the recombinant adeno-associated virus （AAV） expressing apoptin and investigate the anti-tumor capabilities of AAV-mediated expression of VP3 to human bladder cancer cells in vitro. Methods In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3 gene. The VP3 gene was cloned into the expression vector pAAV- MCS and the recombinant plasmid pAAV-VP3 was confirmed by double enzymatic digestion using EcoR I and Sal I. The recombinant expression plasmid was co-transfected into the AAV-293 cells with pHelper and pAAV-RC to produce recombinant AAV viral particles. The morphological features of the virus particles were examined under the electron microscopy. The physical titer of recombinant AAV was measured through digoxigenin-labeled CMV probe dot blot method, and the recombinant virus was verified by polymerase chain reaction （PCR） of the exogenous interest genes of apoptin. EJ cells were infected by recombinant virus with 5 × 10^5 vector genomes per cell. Reverse transcription （ RT）-PCR and Western blotting were performed to detect the transcription and expression of apoptin. Concerning on the anti-tumor activity in vitro, transmission electron microscopy was used to determine the apoptotic morphological changes of EJ cells after rAAV-VP3 infection. The cell cycle and apoptosis of EJ cells were quantified by using flow cytometry at 4th day after infection of rAAV-VP3. Results The expression vector pAAV-VP3 was successfully constructed and the sequence of the VP3 was confirmed by DNA sequencing. The rAAV-VP3 was obtained by three plasmids cotransfection into AAV-293 cells after 72 h. The recombinant adeno-associated virus had a high titer of 5. 1 × 10^11 v. g./ml, and virus particles could be observed under the electron microscopy. After infection of EJ cells with rAAV-VP3, the transcription and expression of VP3 gene were detected by RT-PCR and Western blotting. The rAAV-VP3-induced morphological changes of apoptosis suc

关 键 词：VP3蛋白 膀胱肿瘤 基因治疗 

分 类 号：R737[医药卫生—肿瘤]