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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。
Objective To investigate the apoptosis of HepG-2 cells induced by anti-human DR5 scFv and its mechanism. Methods The cytotoxic effect of ZF1 on HepG-2 was detected by MTT assay. The apoptosis rate of HepG2 induced by ZF1 was detected by flow cytometry. HepG2 cells stained with ANNEXIN-V / PI were observed by fluorescence microscopy. Cell DNA characteristics; Western blotting detection of Bcl-2, PARP protein expression. Results MTT results showed that the growth of HepG-2 cells was inhibited by ZF1 in a dose-dependent manner. The final growth inhibitory rates of ZF1 were 0.225,0.45,0.9mg / ml and 1.2mg / ml respectively. The growth inhibition rates of HepG-2 cells were 28.8%, 52.3 %, 65.3%, 89.8% respectively. The results of flow cytometry showed that the apoptotic rates of HepG-2 cells treated with 2ml 0.225,0.45,1.2mg / ml 4h were 32.9%, 56%, 83.2% Early apoptotic cells were observed under the microscope, the green fluorescence of the cell membrane, the formation of apoptotic bodies in the cells with changes in nuclear structure; obvious comet tail bands in DNALadder; the apoptosis of ZF1-induced cells was detected by Western blotting Bcl-2, PARP protein expression upregulation. Conclusion ZF1 can induce apoptosis in HepG2 cells. This effect is related to the up-regulation of Bcl-2 and PARP protein expression in HepG2 cells.