TRAIL诱导HBV转染肝癌细胞BEL-7402凋亡的作用及机制研究

The apoptosis of TRAIL induce hepatoma BEL-7402 transfected with HBV and its mechanism

目的 研究肝癌细胞BEL- 74 0 2感染HBV前后,TRAIL诱导其凋亡的敏感度变化及作用机制。方法 构建含1.1倍HBV全基因组的真核表达载体pcDNA3 1.1HBV ,转染人肝癌细胞BEL -74 0 2 ,G4 18稳定筛选,建立HBVadr亚型体外转染的细胞模型。原位末端标记(TUNEL)检测TRAIL诱导的凋亡反应,并通过设立TRAIL联合其可溶性受体sDR5 (sDR5与TRAIL结合后,可特异性阻断TRAIL诱导凋亡) ,以证实该凋亡是由TRAIL特异性诱导的。琼脂糖凝胶电泳(DNAladder)检测染色体DNA的断裂情况。流式细胞术、半定量逆转录聚合酶链反应检测HBV感染前后细胞表面TRAIL各受体的表达。双荧光素酶报告基因系统检测抑制凋亡的核转录因子NF κB的活性。结果 成功构建了包含HBV全基因组的真核表达载体pcDNA3 1.1HBV ,转染BEL 74 0 2、经G4 18稳定筛选后得到HBVadr亚型转染的细胞模型BEL 74 0 2 1.1HBV。TRAIL诱导BEL 74 0 2、BEL 74 0 2 pcDNA3及BEL 74 0 2 1.1HBV的凋亡率分别为30 .5 %、2 9.8%和76 % (P <0 .0 1) ;经sDR5特异性阻断后,凋亡率分别为0 .9%、0 .8%和0 .9% ,证明凋亡是由TRAIL特异性诱导的。TRAIL作用2 4h后,琼脂糖凝胶电泳可见BEL- 74 0 2 1.1H-BV较BEL- 74 0 2、BEL 74 0 2 pcDNA3染色体DNA断裂的现象更为明显。无论RNA水平还?

Objective To investigate whether hepatitis B virus t ransfection influence TNF-related apoptotosis inducing ligand TRAIL induced apoptosis of hepatoma BEL-7402. Methods HBV gene eukar yotic expression vector containing 1.1-fold-overlength genome of HBV was const ructed and 1.1 HBV transfected hepatoma cell model was established. The two huma n hepatocellular carcinoma cell lines, BEL-7402 and its 1.1HBV genome transgeni c cell line named BEL-7402/1.1HBV were examined. The apoptosis were examined by DNA agarose gel electrophoresis and TUNEL. The specificity of TRAIL induced apo ptosis was proved by the apoptosis of TRAIL plus sDR5 group. The expression of T RAIL receptors was assessed by flowcytometry and RT-PCR. NF-κB activity was e valuated by dual luciferase reporter assay. Results Euka ryotic expression vector pcDNA3/1.1HBV containing 1.1-fold-overlength geno me of HBV was successfully constructed. HBV transfeceted cell model BEL-7402/1. 1HBV was established. In contrast to BEL-7402 and BEL-7402/pcDNA3 cell lines, the apoptosis rate of BEL-7402/ 1.1HBV to TRAIL was significantly higher (76 %, 30.5%, 29.8%, P<0. 01) after addition of sDR5(specific block TRAIL induc ed apoptosis), the apoptosis rate was reduced to 0.9%, 0.8% and 0.9%. In DNA aga rose gel electrophoresis, there is much more spliced DNA in BEL-7402/1.1HBV tha n BEL-7402 and BEL-7402/ pcDNA3. There is no difference among BEL-7402, BE L-7402/pcDNA3 and BEL-7402/1.1HBV, in terms of expression TRAIL receptors, but the NF-κB activity was drastically lower on BEL-7402/1.1HBV cells(P< 0. 05). Conclusion Our results indicated that HBV could ma ke cells sensitive to TRAIL-induced apoptosis, this may be explained by the dow n-regulation of NF-κB activity. It looks like that HBV infection could lead t o immoderate hepatocyte apoptosis, may facilitate the exploring pathogenesis of HBV related disease.