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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。
OBJECTIVE: To investigate a new type of proliferating adenovirus CNHK500-hγ [adenovirus E1A and E1B genes respectively composed of human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE ) Promoter double regulatory and carrying hIFN-γ recombinant adenovirus] in vitro killing of liver cancer cells. Methods: TCID_ (50) and MTF were used to detect the amplification ability of proliferating adenovirus CNHK500-hγ in two telomerase positive hepatoma cell lines HepG2 and Hep3B and one telomerase negative fibroblast cell line BJ Cell killing effect. BJ, Hep3B and HepG2 cells were infected with CNHK500-GFP carrying green fluorescent protein and the amplification was observed. Western blotting and ELISA were used to detect the expression of hIFN-γ in cells and cell supernatant after CNHK500-hγ infection. Results: After HepG2, Hep3B and BJ cells were infected with CNHK500-hγ for 48 h, the proliferation of CNHK500-hγ in HepG2 and Hep3B cells was 16003 and 2116 times that of BJ cells, respectively. The ED 50 values of BJ cells were HepG2 and Hep3B cells were killed 500 and 10,000 times (P <0.01), and were significantly superior to the positive control proliferating adenovirus ONYX-015. The expression of hIFN-γ in HepG2 and Hep3B cells after CNHK500-hγ infection was significantly higher than that in non-proliferating adenovirus Ad-hγ-infected cells (P <0.01). CONCLUSION: Proliferating adenovirus CNHK500-hγ can specifically proliferate in hepatocellular carcinoma cells and express hIFN-γ gene efficiently. It is a novel adenovirus with the potential of treating hepatocellular carcinoma.