中国科学院机构知识库网格系统: 凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应

凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应

文献类型：期刊论文


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
刊名	中华实验外科杂志
出版日期	2012
卷号	29 期号:6 页码:1009-1012
关键词	VP3蛋白膀胱肿瘤基因治疗
其他题名	Construction of recombinant adeno-associated virus expressing apoptin and anti-tumor effect on human bladder cancer in vitro
通讯作者	jiangsongwang@yahoo.com
合作状况	其它
中文摘要	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。
英文摘要	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 such as fragmentation and apoptotic body were observed under the transmission electron microcopy.Moreover,the proliferation of EJ cells was slowed down,and the proportion of cells at S phase was decreased and that at G_2/M phase was blocked after rAAV-VP3 infection as compared with control group (P<0.01).Conclusion AAV-mediated VP3 gene transfer may be a new therapeutic technique for the treatment of bladder neoplasms,which may lay a foundation for further studies on anti-tumor effect of bladder cancer in vivo.
资助信息	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）
语种	中文
公开日期	2013-04-09
源URL	[http://159.226.149.42:8088/handle/152453/7322]
专题	昆明动物研究所_分子免疫生物学昆明动物研究所_动物模型与人类重大疾病机理重点实验室
作者单位	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,650223
推荐引用方式 GB/T 7714	王春晖,王剑松[*],王文举,等. 凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应[J]. 中华实验外科杂志,2012,29(6):1009-1012.
APA	王春晖,王剑松[*],王文举,余力锐,詹辉,&徐鸿毅.(2012).凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应.中华实验外科杂志,29(6),1009-1012.
MLA	王春晖,et al."凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应".中华实验外科杂志 29.6(2012):1009-1012.

入库方式： OAI收割

来源：昆明动物研究所

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凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应

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