表达凋亡素基因的重组禽痘病毒对人喉癌细胞Hep-2的抑制效应研究

Anti-tumor effects on human laryngeal carcinoma Hep-2 of recombinant fowlpox virus expressing chicken anemia virus Apoptin gene

目的:探讨含鸡贫血病病毒(CAV)凋亡素(Apoptin)基因的重组禽痘病毒vFVApoptin诱导人喉癌细胞Hep-2凋亡的作用及其作用方式。方法:用vFVApoptin感染体外培养的人喉癌细胞Hep-2,运用MTT染色法检测重组质粒对肿瘤细胞的抑制作用;并运用流式细胞仪检测肿瘤细胞线粒体跨膜电位(ΔΨm)和细胞内活性氧(ROS)水平变化情况;通过免疫印迹检测细胞色素c(Cytoc)释放;应用底物显色法检测Caspase-3/9活性。结果:vFVApoptin感染可明显抑制Hep-2肿瘤细胞,并具有下调肿瘤细胞ΔΨm,上调其ROS水平,促进Cytoc释放和激活Caspase-3/9的功能。结论:vFVApoptin通过上调Hep-2肿瘤细胞内线粒体ROS产生,造成Cytoc的释放,激活Caspase-9,进而激活Caspase-3等下游凋亡因子,最终通过线粒体途径诱导细胞凋亡抑制Hep-2肿瘤细胞。

Objective：To investigate the anti tumor effects and the mechanism of the recombinant fowlpox virus expressing Apoptin gene on human laryngeal carcinoma Hep-2. Method： Hep-2 cells cultured in vitro were in- fected with vFVApoptin. The anti tumor effects on Hep 2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential （△ψm） and reactive oxygen species （ROS） were analyzed by flow cytome- try. Western blot was used to detect the release of cytochrome c （Cyto c）. Caspase-3/9 activities were measured by colorimetric assay. Result：vFVApoptin could restrain Hep-2 cells significantly and, had the function of down- regulating △ψm, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9. Conclusion： Cyto c were released from mitochondria by the function of up-regulating ROS of vFVApoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase 9, downstream apoptotic factors, such as caspase-3, were activated. Eventu- ally, Hep-2 cells were suppressed by mitochondrial pathway apoptosis induced by vFVApoptin.