摘要
目的初步探讨乙型肝炎病毒野生型核壳蛋白和L60V、I97L变异核壳蛋白影响HepG2细胞凋亡的分子机制。方法将构建的野生型核壳蛋白融合表达载体(pEGFP-WT),L60V变异核壳蛋白(pEGFP-V60)和I97L变异核壳蛋白(pEGFP-L97)表达载体HepG2阳性细胞株复苏培养;Western blot检测各核壳蛋白表达;TNF-α、Act-D诱导各种HepG2细胞株凋亡,48h采用流式细胞术检测细胞凋亡,Western blot检测NF-κB信号传导通路中IKK-α、IκB-α、NF-κBP65蛋白表达与caspase-8、caspase-3蛋白表达情况。结果Western blot检测pEGFP-WT组、pEGFP-V60组和pEGFP-L97组核壳蛋白表达基本相同;流式细胞术检测显示,与pEGFP-C1细胞株相比,48h时pEGFP-WT、pEGFP-V60和pEGFP-L97表达细胞株的细胞凋亡率均明显偏低(P<0.01);4组细胞株IKK-α、IκB-α、NF-κBP65蛋白表达水平无明显差异,而pEGFP-WT组caspase-8、caspase-3蛋白表达水平明显低于pEGFP-V60和pEGFP-L97组,且三组表达均明显低于pEGFP-C1组。结论乙型肝炎病毒野生型和L60V、I97L变异核壳蛋白影响HepG2细胞凋亡可能与核壳蛋白影响细胞内caspase-3,8表达有关。
Objective To investigate the mechanisms of HBV wild type and L60V, I97L mutant core proteins on the apoptosis of HepG2 cells. Methods HepG2 cells transfected with pEGFP-WT, pEGFP-V60 and pEGFP-L97 vectors were cultured and analyzed for the expression of wild-type and mutant （L60V and I97L） core proteins by Western blot. Apoptosis of the HepG2 cells was induced by TNF-α and Act-D. Apoptosis was measured by flow cytometry. Signal transduction proteins, such as IKK-α, IKB-α, NF-KB P65, caspase-8 and caspase-3, were detected by Western blot. Results The expression levels of wild-type and mutant core proteins in the transfected HepG2 cells were similar. Compared with the pEGFP-C1 control group, the degree of apoptosis in pEGFP-WT, pEGFP-V60 and pEGFP-L97 group was markedly decreased. Significant difference in the levels of IKK-α, IkB-α and NF-kB P65 proteins was not observed. In contrast, the expression of caspase-8 and caspase-3 was obviously decreased in pEGFP-WT group. Conclusion Overexpression of wild-type and mutant core protein was found to decrease TNF-α-induced apoptosis of HepG2 cells. The effect may be partly due to the down-regulation of the intracellular caspase-8 and caspase-3 expression.
出处
《热带医学杂志》
CAS
2009年第7期735-737,741,共4页
Journal of Tropical Medicine
