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目的比较HCV不同基因型核心蛋白(core)在肝母细胞瘤细胞凋亡中的作用。方法构建6a基因型HCV core的表达载体pcDNA3.1/myc-His(-)-core(6a)[pCore(6a)];分别将1b、3a和6a基因型HCV core质粒以及空质粒pcDNA3.1/myc-His(-)(pNC)瞬时转染HepG2细胞,48小时后利用流式细胞术检测细胞凋亡发生的情况;利用Real-time PCR检测胱冬肽酶-3的mRNA水平变化;利用化学发光法检测胱冬肽酶-3的活性变化,比较HCV不同基因型core对细胞凋亡作用的差异。结果成功构建了6a基因型HCV core的表达载体pcDNA3.1/myc-His(-)-core(6a)[pCore(6a)];与pNC组相比,表达1b、3a和6a基因型HCV core的HepG2细胞中,Annexin V+细胞数均显著减少,其中6a基因型core较1b和3a基因型core作用显著(P=0.000、0.001);同时实验组中凋亡效应分子胱冬肽酶-3的mRNA水平及其活性较对照组均降低;其中与1b和3a基因型core相比较,6a基因型core显著降低胱冬肽酶-3的mRNA水平,而在抑制胱冬肽酶-3活性方面无显著差别。结论 HCV不同基因型core对细胞凋亡的作用不同;基因6a型core对细胞凋亡的抑制作用更为显著;HCV基因6型核心蛋白可能更易导致肝癌的发生,有待进一步研究。
Objective To compare the role of core of different genotypes of HCV in hepatoblastoma cell apoptosis. Methods The HCV core plasmids of 1b, 3a and 6a genotypes and empty plasmid pcDNA3.1 (pcDNA3.1 / myc-His (-) - core HepG2 cells were transiently transfected with myc-His (-) (pNC) and the apoptosis was detected by flow cytometry 48 hours later. The mRNA level of caspase-3 was detected by Real-time PCR. The change of activity of caspase-3 was detected by chemiluminescence method, and the difference of apoptosis effect between different genotypes of HCV was compared. Results The HCV core expression vector pcDNA3.1 / myc-His (-) - core (6a) [pCore (6a)] was successfully constructed. Compared with the pNC group, Of HepG2 cells, the number of Annexin V + cells was significantly reduced, of which the 6a genotype core than the 1b and 3a genotype significant effect (P = 0.000,0.001); at the same time experimental group apoptosis effector caspase-3 mRNA and activity of caspase-3 were significantly lower than those of control group. Compared with core of 1b and 3a genotypes, core 6a significantly decreased caspase-3 mRNA level, while no effect on caspase-3 activity Significant difference. CONCLUSION: The effect of core of different genotypes of HCV on apoptosis is different. The inhibition of apoptosis on gene type 6a core is more significant. The HCV core protein 6 may be more likely to cause hepatocellular carcinoma, which needs further study.