抗人DR5单链抗体诱导HepG2细胞凋亡的实验研究

Apoptosis of hepG2 cells induced by anti-human DR5 scFv(scFv) ZF1

目的探究抗人DR5单链抗体(scFv)ZF1对肝癌细胞株HepG-2的凋亡作用及机制。方法 MTT法检测ZF1对HepG-2的细胞毒效应;流式细胞术检测ZF1诱导HepG2的凋亡率;荧光显微镜观察经ANNEXIN-V/PI双染的HepG-2细胞;DNA Ladder检测HepG-2细胞的DNA特点;Western blotting检测Bcl-2、PARP蛋白的表达。结果 MTT结果显示ZF1抑制HepG-2细胞生长呈剂量依赖性,ZF1终浓度分别为0.225、0.45、0.9mg/ml、1.2mg/ml200μl时,HepG-2细胞的生长抑制率分别为28.8%、52.3%、65.3%、89.8%;流式细胞仪检测结果显示,终浓度分别为0.225、0.45、1.2mg/ml2ml作用HepG-2细胞4h,凋亡率分别为32.9%、56%、83.2%;荧光显微镜下可见早期凋亡细胞,细胞膜呈绿色荧光,细胞内有凋亡小体的形成,伴有核结构的变化;DNALadder出现明显的彗星尾状条带;Western blotting检测到ZF1诱导凋亡的细胞内Bcl-2、PARP蛋白表达上调。结论 ZF1可诱导HepG2细胞株凋亡,这种作用与HepG2细胞株表达Bcl-2、PARP蛋白蛋白表达上调相关。

We aimed to explore the apoptotic effects of anti-human DR5 scFv ZF1 on the hepatoma cell line HepG-2 and its mechanism.We employed MTT assay to detect the ZF1 cytotoxicity against HepG-2 cell,and found that ZF1 inhibited the growth of HepG-2 cells in a dose-dependent manner（ZF1 final concentrations of 0.225,0.45,0.9,1.2 mg/ml correspond to HepG-2 cell growth inhibition rates of 28.8%,52.3%,65.3%,89.8%）;HepG2 cells were respectively treated with 0.225,0.45,1.2 mg/ml ZF1 for 4 hours,and flow cytometry indicated the corresponding apoptosis rates were 32.9%,56%,83.2%.Then the ZF1-treated HepG2 cells were stained with ANNEXIN-V/PI for fluorescence microscope observation,through which we found early apoptotic cells,cell membrane showing as green fluorescence,and apoptotic body formation accompanied by changes in nuclear structure.DNA ladder of the treated cells appeared comet tail-shaped band;western blotting showed intracellular Bcl-2,PARP protein expression was increased in ZF1treated HepG2 cells.Thus,we concluded that ZF1 can induce apoptosis in HepG2 cell line,which was associated with up-regulation of Bcl-2 and PARP protein expression in HepG2 cell lines.