5/35嵌合型溶瘤腺病毒SG635对肝癌细胞的抑制作用  

Inhibitory effect of 5/35 chimeric oncolytic adenovirus SG635 on hepatocarcinoma cells

作　　者：武玉强[1] 张琪[1] 陈伟[1] 刘炜[1] 台艳[1] 陈规划[1] 

机构地区：[1]中山大学附属第三医院肝移植中心暨广东省肝脏疾病研究重点实验室,广东广州510630

出　　处：《中国肿瘤生物治疗杂志》2011年第4期373-377,共5页Chinese Journal of Cancer Biotherapy

基　　金："十一五"国家科技重大专项课题资助项目(No.2008ZX10002-025;No.2008ZX10002-026);国家重点基础研究发展计划(973计划)资助项目(No.2009CB522404);教育部新世纪优秀人才基金资助项目(No.NCET-08-0583);全国优秀博士论文专项基金资助项目(No.FANEDD 200774)~~

摘　　要：目的:研究5/35嵌合型溶瘤腺病毒SG635在体外对肝癌HepG2和SMMC-7721细胞的特异性杀伤作用。方法:将SG600载体中5型腺病毒(Ad5)纤毛蛋白的knob和shaft结构域替换为35型腺病毒(Ad35)纤毛蛋白的相应结构域,构建成5/35嵌合型溶瘤腺病毒SG635。流式细胞术检测5/35嵌合型腺病毒Ad5/35-EGFP对HepG2和SMMC-7721细胞的感染效率,体外病毒增殖实验观察溶瘤腺病毒SG635的增殖能力,Western blotting检测SG635感染后肝癌细胞中E1A蛋白的表达,CCK-8实验检测SG635对肝癌HepG2和SMMC-7721细胞的杀伤作用。结果:在肝癌HepG2和SMMC-7721细胞中,Ad5/35-EGFP的感染效率明显强于5型腺病毒Ad5-EGFP;5/35嵌合型溶瘤腺病毒SG635在HepG2和SMMC-7721细胞中72 h的增殖倍数高于5型溶瘤腺病毒SG600(15 848.93vs6 309.57,6 309.57vs5 011.87,均P<0.01),而在人正常成纤维细胞BJ中几乎不增殖。SG635感染后,HepG2和SMMC-7721细胞中E1A蛋白表达高于SG600感染,在BJ中则无E1A表达。在一定MOI范围内,SG635对于HepG2细胞和SMMC-7721细胞的杀伤作用逐渐增强,且杀伤率明显强于SG600(MOI为1时,90%vs60%;MOI为10时,90%vs50%),对BJ无杀伤作用。结论:5/35嵌合型溶瘤腺病毒SG635能够高效感染并特异性杀伤肝癌细胞,具有较好的靶向性和安全性。Objective：To investigate the specific cytotoxicity effect of 5/35 chimeric oncolytic adenovirus SG635 on hepatocellular carcinoma HepG2 and SMMC-7721 cells.Methods： The knob and shaft domains of type 5 adenovirus（Ad5） in SG600 plasmid were replaced by the domains of type 35 adenovirus（Ad35）,and chimeric oncolytic adenovirus Ad5/35 was established.Flow cytometry was used to examine the infection efficiency of chimeric adenovirus Ad5/35（Ad5/35-EGFP） in HepG2 and SMMC-7721 cells;replication assay was used to evaluate the replication of oncolytic adenovirus SG635;Western blotting analysis was used to examine the expression of E1A in cells after SG635 infection;and Kit-8 assay was used to assess the cytotoxicity of SG635 and SG600 on HepG2 and SMMC-7721 cells.Results： The infection efficiency of Ad5/35-EGFP in HepG2 and SMMC-7721 cells was obviously enhanced compared with Ad5-EGFP.The replication activity of SG635 in HepG2 and SMMC-7721 cells was higher than that of SG600 72 h after infection（15 848.93,6 309.57 vs 6 309.57,5 011.87,P〈0.01）,but SG635 did not replicate in normal BJ cells.Moreover,SG635 induced a higher expression of E1A protein in HepG2 and SMMC-7721 cells than SG600,but did not induce E1A expression in normal BJ cells.At a certain MOI,SG635 showed increasing cytotoxicity on HepG2（MOI=1,90% vs 60%） and SMMC-7721（MOI=10,90% vs 50%） cells,and the cytotoxicity was stronger than SG600,without causing significant cytotoxicity on normal BJ cells.Conclusion： The 5/35 chimeric oncolytic adenovirus SG635 can effectively infect and specifically kill hepatocarcinoma cells with satisfactory safety and specificity.

关 键 词：溶瘤腺病毒 肝癌 病毒治疗 E1A蛋白 

分 类 号：R730.5[医药卫生—肿瘤] R735.7[医药卫生—临床医学]