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背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)及其死亡受体的功能性单克隆抗体具有杀伤肿瘤细胞的活性。我们研制了抗人死亡受体5(death receptor,DR5)功能性单克隆抗体(mDRA-6),本研究对其诱导Jurkat细胞凋亡的Caspase分子机制进行初步探讨。方法:mDRA-6作用后,琼脂糖凝胶电泳检测Jurkat细胞的DNA ladder;MTT法检测细胞存活情况;Annexin V-FITC/PI双染流式细胞术定量分析细胞凋亡情况;观察Caspase-10、-9、-8、-3等的抑制剂对mDRA-6诱导细胞凋亡的抑制作用;免疫印迹技术检测凋亡信号蛋白Caspase-10、-9、-8、-3,多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)、BH3相关结构域死亡激动剂(BH3interactingdomain death agonist,Bid)、短缩的Bid(truncated Bid,tBid)、细胞色素C(cytochrome c,Cyto C)等活化裂解的情况。结果:琼脂糖凝胶电泳显示DNA呈现明显梯状带型;mDRA-6对Jurkat细胞具有明显的诱导凋亡作用且呈量-效关系。2.0μg/mL mDRA-6作用Jurkat细胞0.25h、0.5h、1h、2h,其凋亡率分别为16.2%、28.3%、69.2%、78.2%;Caspase-8抑制剂能明显抑制mDRA-6诱导的细胞凋亡,抑制率为77.9%,Caspase-3和Caspase-9抑制剂作用的抑制率分别为54.2%、8.7%,而Caspase-10的抑制剂无抑制作用;免疫印迹技术检测显示Caspase-8、-3、-9均呈现随时间延长酶原逐渐减少、活化片段增加的现象,PARP的降解片段亦增加,Bid激活降解为tbid,Cyto C大量释放,而Caspase-10酶原无明显改变、无活化片段出现。结论:mDRA-6诱导Jurkat细胞凋亡主要是通过激活Caspase路径和线粒体路径来完成的。
BACKGROUND & OBJECTIVE: Functional monoclonal antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor have cytotoxic activity against tumor cells. We developed a functional monoclonal antibody against death receptor (DR5) 5 (mDRA-6). We investigated the molecular mechanism of caspase-induced apoptosis in Jurkat cells. METHODS: The DNA ladder of Jurkat cells was detected by agarose gel electrophoresis after mDRA-6 treatment. Cell viability was detected by MTT assay. Apoptosis was quantified by Annexin V-FITC / PI double staining flow cytometry. Caspase-10 , -9, -8, -3 and other inhibitors of mDRA-6-induced apoptosis; western blot detection of apoptotic signaling proteins Caspase-10, -9, -8, -3, poly ADP ribose (ADP-ribose polymerase), BH3-interacting domain death agonist (Bid), shortening of Bid (truncated Bid, tBid) and cytochrome c (Cyto C) Cracking situation. Results: The agarose gel electrophoresis showed that the DNA showed obvious ladder-shaped banding pattern. MDRA-6 had obvious apoptosis-inducing effect on Jurkat cells in a dose-effect relationship. The apoptotic rates of Jurkat cells treated with 2.0μg / mL mDRA-6 for 0.25h, 0.5h, 1h and 2h were 16.2%, 28.3%, 69.2% and 78.2% respectively. Caspase-8 inhibitor significantly inhibited the induction of mDRA-6 The inhibitory rates of Caspase-3 and Caspase-9 were 54.2% and 8.7%, respectively, while the inhibitory rates of Caspase-3 and Caspase-10 were 77.9% and 54.2%, respectively. 8, -3, and -9 showed prolonged proenzyme and increased activation fragments as time prolonged, degraded fragments of PARP also increased, Bid activated and degraded to tbid, Cyto C was released in a large amount, while Caspase-10 zymogen did not change significantly , No activated fragment appears. Conclusion: The apoptosis of Jurkat cells induced by mDRA-6 is mainly through the activation of Caspase pathway and mitochondrial pathway.