丙型肝炎病毒不同基因型核心蛋白对肝母细胞瘤细胞凋亡影响的比较

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。

Effects of HCV core of different genotypes on cell apoptosis of HepG2 cell

Objective To compare the effects of HCV core of different genotypes on cell apoptosis of HepG2 cell. Methods The plasmid pcDNA3.1/myc-His （-）-core of genotype 6a pCore （6a）] was constructed. And then pCore （1b）, pCore （3a）, pCore （6a） and negative control pcDNA3.1/myc-His （-） （pNC） were transiently transfected into HepG2 cells respectively. Flow cytometry was used to observe cell apoptosis post 48 hours while the mRNA levels and activities of caspase-3 were detected by Real-time PCR and caspase-glo 3/7 luminometer respectively. Then, compare the different effects of HCV core of different genotypes on cell apoptosis were compared. Results The plasmid pcDNA3.1/myc-His （-）-core of genotype 6a pCore（6a）] was successfully constructed. Compared with control group, the number of Annexin V-positive cells were all decreased in core （1b）, core （3a） or core （6a） expressed HepG2 cells, among which the effect of core （6a） was more remarkable. Meanwhile, the mRNA levels and activities of caspase-3 were all reduced in core （1b）, core （3a） or core （6a） expressed HepG2 cells. Moreover, compared with core （1b） and core （3a）, the effect of core （6a） is more signiifcant in terms of caspase-3 mRNA level, but not caspase-3 activity. Conclusions Different genotypes of HCV core have different effects on cell apoptosis. Inhibitory effect of genotype 6a core on apoptosis is more signiifcant. HCV core protein of genotype 6a may be easier to cause liver cancer, which needs further study.