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MTT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16 003和2 116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。

Objective : To investigate the cytotoxicity of the novel replicative adenovirus CNHK500-hγ, a recombinant adenovirus with the adenovirus E1A and E1B 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-hγ on two telomerase positive HCC cell lines (HepG2 and Hep3B) and one telomerase negative normal cell line (BJ) were analyzed by TCID50 and MTT assays. BJ, 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-γ in cells and cell supernatants after CNHK500-hγ infection were detected by Western blotting and ELISA assays. Results: Forty-eight hours after infection, the amplification of CNHK500-GFP in HepG2 and Hep3B cells were 16 003 and 2 116 times of that in BJ cells, and the cytotoxicity ED50 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 replicative adenovirus ONYX-015. Furthermore, hIFN-γ expression in HepG2 and Hep3B cells after CNHK500-hγ infection was significantly higher than that after non-replicative adenovirus Ad-hγ infection (P<0.01). Conclusion: Replicative adenovirus CNHK500-hγ can specifically amplify in HCC cells and effectively express hIFN-γ gene, which holds potential for the treatment of HCC.

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）