双特异性重组腺病毒Ad-HT对肝癌细胞的体内、外抑制效应

Antitumor effects of the dual specific recombinant adenovirus Ad-HT on hepatoma carcinoma cell in vivo and in vitro

目的:探讨双特异性抗肿瘤重组腺病毒Ad-HT对肝癌细胞的体内、外抑制作用和作用方式。方法:运用噻唑兰法检测重组腺病毒Ad-HT对BEL-7402细胞的抑制作用;应用AO/EB染色法、DAPI染色法、Annexin V检测、Caspase检测、线粒体膜电位检测和活性氧检测等多种方法分析Ad-HT对BEL-7402细胞抑制作用的方式和途径;构建C57BL/6小鼠荷H22肿瘤模型,瘤内注射重组腺病毒,通过非放射性乳酸脱氢酶细胞毒性检测方法,检测NK活性、CTL活性,利用酶联免疫吸附方法检测IL-2、IL-4、IL-10和IFN-γ等细胞因子水平,探讨Ad-HT对体内实体肿瘤的抑制作用和对免疫系统的影响。结果:Ad-HT能够抑制BEL-7402细胞生长,且其作用具有一定时效和剂效关系趋势;Ad-HT感染导致BEL-7402细胞磷脂膜外翻、细胞核皱缩、细胞膜通透性增加、Caspase酶活性增强、线粒体膜电位下降和活性氧水平升高;Ad-HT实验组模型动物平均期在30天以上,显著高于对照组;另外,Ad-HT具有增强NK和CTL活性,上调IL-2和IFN-γ等细胞因子水平的功能。结论:Ad-HT能够通过诱导细胞凋亡,抑制BEL-7402肿瘤细胞增殖,而这种抑制作用可能通过线粒体途径实现。另外,Ad-HT能够有效延长模型动物平均生存期,活化免疫功能细胞,并使免疫趋向Th1优势。

Objective：To investigate the antitumor effects and mechanism of the dual cancer-specific recombinant adenovirus Ad-HT in vivo and in vitro. Methods： MTT assay was used to evaluate the antitumor effects of Ad-HT on BEL-7402 cells. AO/EB staining, DAPI staining, Annexin V assay and Caspase assay were used to identify the antitumor pathway of Ad-HT on BEL-7402 cells. H22 solid tumor model was used to evaluate the in vivo anti-tumor effects of Ad-HT. C57BL/6 mice bearing H22 solid tumor model was es- tablished and the model mice were injected introtumor with the recombinant adenoviruses. Non-radioactive cytotoxicity assay was then used to detect the activity of NK and CTL, and the enzyme linked immunosorbent assay was used to examine the contents of IL-2, IL-4, IL-10 and IFN-γ. Results： Ad-HT suppressed BEL-7402 cells successfully. The in vitro antitumor effects of Ad-HT depended on the treat time and the treat dose. The infection of Ad-HT resulted in the exposure of phosphatidylserine,the shrink of nucleus, the perme- ation of membrane, the activation of caspase, the downregulation of mitochondrial membrane potentials, and the upregulation of reactive oxygen species. Ad-HT treatment significant increased the mean survival （above 30 d） the animal model in comparison with the other recombinant adenoviruses. Furthermore,in the animal experiment, the administration of Ad-HT resulted in the activation of NK/CTL and upregulation of the cytokines such as IL-2 and IFN-3,. Conclusion： Ad-HT could effectively suppress BEL-7402 cells by inducing apoptosis through mitochondrial pathway, and prolong the mean survival of the animal model. Ad-HT also activated NK, CTL and Thl type cytokines secretion.