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观察乙型肝炎病毒X蛋白(HBx)对肿瘤坏死因子相关的凋亡诱导配体(TNF—related apoptosis—inducing ligand,TRAIL)诱导肝细胞凋亡的影响并初步探讨其分子机制.构建包含HBx 基因的真核表达载体pcDNA-HBx,转染BEL7402肝癌细胞,建立可稳定表达HBx的肝癌细胞系BEL7402-HBx,同时设立空载体pcDNA3转染对照组细胞BEL7402-cDNA3.台盼蓝染色计数, Caspase3活性检测和TUNEL法检测TRAIL诱导BEL7402,BEL7402-cDNA3,BEL7402-HBx细胞凋亡的情况,并通过流式细胞术分析3组细胞表面TRAIL受体的表达水平.此外,利用硫代反义寡核苷酸封闭HBV全基因转染肝癌细胞系HepG2.2.15中HBx蛋白的表达,观察阻断前后对TRAIL诱导凋亡敏感性的改变,进一步反向验证HBx对TRAIL诱导凋亡的调节作用.台盼蓝染色计数提示TRAIL对BEL7402,BEL7402-cDNA3,BEL7402-HBx均有剂量依赖性的细胞毒作用,但在相同浓度TRAIL作用下,BEL7402-HBx细胞较BEL7402,BEL7402-cDNA3细胞有更高的敏感性.Caspase3活性检测结果分析发现,TRAIL作用后BEL7402-HBx细胞在较短时间内有更高的Caspase3活化水平.TUNEL结果显示,10μg/LTRAIL作用下,BEL7402-HBx细胞凋亡率可达(41.4±7.2)%,显著高于对照组细胞.反义封闭HepG2.2.15细胞中HBx基因的表达可部分阻断TRAIL诱导的凋亡.两?
Observation of hepatitis B virus X protein (HBx) on tumor necrosis factor-related apoptosis inducing ligand (TNF-related apoptosis-inducing ligand, TRAIL) induced apoptosis of hepatocytes and to explore the molecular mechanism. HBx gene construct comprising the eukaryotic expression vector pcDNA-HBx, transfected BEL7402 hepatoma cells, establishing stable expressing HBx hepatoma cell line BEL7402-HBx, while setting up the empty vector pcDNA3 transfected control cells BEL7402-cDNA3. trypan blue staining, of Caspase3 activity TUNEL assay and detection TRAIL induced BEL7402, BEL7402-cDNA3, BEL7402-HBx apoptosis of cells, and the level of cell surface expression of TRAIL receptors three groups analyzed by flow cytometry. in addition, the use of phosphorothioate antisense oligonucleotides The expression of HBx in HepG2.2.15 hepatocellular carcinoma cell line transfected with acid-blocked HBV gene was observed, and the change of sensitivity to TRAIL-induced apoptosis was observed before and after blocking, further verifying the regulative effect of HBx on the TRAIL-induced apoptosis. Tip staining for TRAIL on BEL7402, BEL7402-cDNA3, BEL7402-HBx were dose-dependent cell cytotoxicity, but in the same concentration effect of TRAIL, BEL7402-HBx cells than BEL7402, BEL7402-cDNA3 fine .Caspase3 is more sensitive activity assay analysis results found that the effect of TRAIL BEL7402-HBx cells have a relatively short time a higher activation level .TUNEL Caspase3 results show that, under 10μg / LTRAIL action, apoptosis BEL7402-HBx (41.4 ± 7.2)%, which was significantly higher than that of the control cells.The expression of HBx gene in antisense HepG2.2.15 cells partially blocked the TRAIL-induced apoptosis.However,