新型增殖型腺病毒CNHK500-hγ对肝癌细胞的杀伤作用  

Cytotoxicity of a novel replicative adenovirus CNHK500-hγ against hepatocellular carcinoma cells

作　　者：孙立臣[1] 张柏和[2] 张琪[2] 苏长青[2] 李根丛[2] 吴红平[2] 吴孟超[2] 钱其军[2] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院肝胆外科,山东烟台264000 [2]第二军医大学东方肝胆外科医院病毒基因实验室,上海200438

出　　处：《中国肿瘤生物治疗杂志》2011年第6期635-640,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金国际合作重大项目资助(No.30120160823);国家高技术研究发展"863"计划(No.2001AA217031)~~

摘　　要：目的:探讨一种新型增殖型腺病毒CNHK500-hγ[腺病毒E1A、E1B基因分别由人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子和缺氧反应元件(hypoxia response element,HRE)启动子双重调控的、并携带hIFN-γ的重组腺病毒]对肝癌细胞的体外杀伤作用。方法:TCID_(50)法和MTF检测增殖型腺病毒CNHK500-hγ在两种端粒酶阳性肝癌细胞株HepG2和Hep3B以及一株端粒酶阴性的成纤维细胞株BJ中的扩增能力和对细胞的杀伤作用。用携带绿色荧光蛋白的CNHK500-GFP感染BJ、Hep3B和HepG2细胞,观察其扩增情况。Western blotting和ELISA法检测CNHK500-hγ感染后细胞和细胞上清中hIFN-γ的表达。结果:CNHK500-hγ感染HepG2、Hep3B和BJ细胞48 h后,CNHK500-hγ在HepG2和Hep3B细胞中的扩增是BJ细胞中的16003和2116倍,对BJ细胞杀伤的ED_(50)值分别是杀伤HepG2和Hep3B细胞的500和10 000倍(P<0.01),且明显优于阳性对照增殖型腺病毒ONYX-015。CNHK500-hγ感染后HepG2及Hep3B细胞中hIFN-γ的表达显著高于非增殖型腺病毒Ad-hγ感染后细胞中hIFN-γ的表达(P<0.01)。结论:增殖型腺病毒CNHK500-hγ可在肝癌细胞内特异性扩殖,高效表达hIFN-γ基因,是一种具备治疗肝癌潜力的新型腺病毒。[ Abstract] Objective： To investigate the eytotoxicity of the novel replicative adenovirus CNHK500-h3,, a recombinant adenovirus with the adenovirus E IA and E1 B genes driven by human telomerase reverse transcriptase （hTERT） and hypoxia response element （HRE） promoters respectively and carrying the hIFN-γ, against hepatocellular carcinoma （HCC） cells in vitro. Methods： The amplification and cytotoxicity of replicating adenovirus CNHK500-hγn two telomerase positive HCC cell lines （HepG2 and Hep3B） and one telomerase negative normal cell line （BJ） were analyzed by TCID50 and MTT assays. B J, Hep3B and HepG2 cells were infected with CNHK500-GFP carrying green fluorescent protein and the amplification of CNHK500-GFP was observed. The expressions of hIFN-7 in cells and cell supematants after CNHK500- hγnfection were detected by Western blotting and ELISA assays. Results： Forty-eight hours after infection, the amplifi- cation of CNHK500-GFP in HepG2 and Hep3B cells were 16 003 and 2 116 times of that in BJ cells, and the eytotoxicity ED50 of CNHK500-GFP against BJ cells was respectively 500 and 10 000 times of that against HepG2 and Hep3B cells, and superior to the positive control of replieative adenovirus ONYX-015. Furthermore, hIFN-γ expression in HeoG2 and Hep3B cells after CNHK500-hγnfection was significantly higher than that after non-replicative adenovirus Ad-hγ infection （P 〈 0.01 ）. Conclusion： Replieative adenovirus CNHK500-hγan specifically amplify in HCC ceils and effectively express hIFN-γgene, which holds potential for the treatment of HCC.

关 键 词：肝癌 CNHK500-hγ 病毒治疗 基因治疗 端粒酶 人干扰素-γ 

分 类 号：R735.7[医药卫生—肿瘤] R730.54[医药卫生—临床医学]