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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 effect of vFVApoptin, a recombinant fowlpox virus containing Apoptin gene of chicken anemia virus (CAV) on Hep-2 cell apoptosis in human laryngeal squamous cell carcinoma and its possible mechanism. Methods: Human laryngeal carcinoma cell line Hep-2 was infected with vFVApoptin. The inhibitory effect of the recombinant plasmid on tumor cells was detected by MTT assay. The mitochondrial transmembrane potential (ΔΨm) and intracellular activity of tumor cells were detected by flow cytometry (ROS) levels were measured. Cytoc release was detected by Western blotting. Caspase-3/9 activity was assayed by substrate chromogenic assay. Results: vFVApoptin infection significantly inhibited Hep-2 tumor cells, down-regulated the ΔΨm of tumor cells, up-regulated the ROS level, promoted the release of Cytoc and activated the function of Caspase-3/9. Conclusion: vFVApoptin can up-regulate the production of mitochondrial ROS in Hep-2 tumor cells, resulting in the release of Cytoc, activation of Caspase-9, activation of downstream apoptotic factors such as Caspase-3, and ultimately apoptosis of Hep-2 cells through mitochondrial pathway .