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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 inhibitory effect and mechanism of bispecific antitumor recombinant adenovirus Ad-HT on hepatoma cells in vitro and in vivo. Methods: The inhibitory effect of recombinant adenovirus Ad-HT on BEL-7402 cells was detected by thiazolyl method. The expression of Caspase, mitochondrial membrane potential and reactive oxygen species were detected by AO / EB staining, DAPI staining, Annexin V assay, H22 tumor model was established in C57BL / 6 mice and the recombinant adenovirus was injected intratumorally. The cytotoxicity of non-radioactive lactate dehydrogenase IL-2, IL-4, IL-10 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA). The inhibitory effect of Ad-HT on the solid tumor in vivo and its effect on the immune system were also investigated. Results: Ad-HT could inhibit the growth of BEL-7402 cells, and its effect had some time-effect and dose-effect relationship. Ad-HT infection led to the eversion of phospholipid membrane, nuclear shrinkage and cell membrane permeability of BEL-7402 cells, Enzyme activity increased, mitochondrial membrane potential decreased and reactive oxygen species level increased; Ad-HT experimental group model animals average more than 30 days, significantly higher than the control group; In addition, Ad-HT has enhanced NK and CTL activity, upregulated IL- 2 and IFN-γ and other cytokine levels. Conclusion: Ad-HT can inhibit the proliferation of BEL-7402 tumor cells by inducing apoptosis, and this inhibitory effect may be achieved by the mitochondrial pathway. In addition, Ad-HT can effectively prolong the average survival of model animals, activate immune function cells and make the immune tendency to Th1 predominance.