抗人DR5单克隆功能抗体mDRA-6诱导Jurkat细胞凋亡的Caspase路径机制研究

The Caspase-dependent mechanisms are involved in the induction of apoptosis of Jurkat cells by a novel agonist anti-human DR5 monoclonal antibody

目的:探讨抗人死亡受体5(DR5)功能性单克隆抗体(mDRA-6)对Jurkat细胞诱导凋亡的Caspase分子机制。方法:琼脂糖凝胶电泳检测mDRA-6作用后Jurkat细胞的DNA ladder;MTT法检测单克隆抗体(mDRA-6)对Jurkat细胞生长抑制作用的剂量-效果关系;Annexin V-FITC/PI双染流式细胞术定量分析mDRA-6对Jurkat细胞的凋亡作用;观察Caspase-10、9、8、3等抑制剂对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、Bid(tbid)、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细胞具有明显的生长抑制作用且呈量-效关系,Annexin V-FITC/PI双染流式细胞术检测显示,mDRA-6在2.0μg/ml浓度作用Jurkat细胞0.25、0.5、1、2小时,其凋亡率分别为16.17%、28.25%、69.23%、78.23%;Caspase-8抑制剂能明显抑制mDRA-6诱导的细胞凋亡,抑制率为77.85%,Caspase-3和Cas-pase-9抑制剂的抑制率分别为54.20%、8.74%,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,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细胞凋亡主要是激活了死亡受体信号传导途径上游的Caspase-8引起的,该细胞凋亡机制涉及Caspase-3、Caspase-9及Bid(tbid)、Cyto c等分子,而同样是凋亡的上游信号Caspase-10没有参与此凋亡过程。本研究为mDRA-6的人源化改造及后续的实验研究打下了基础。

Objective：To investigate Caspase-dependent mechanisms involved in induction of Jurkat cell apoptosis by a novel agonist anti-human death receptor5（DR5） monoclonal antibody（mDRA-6）.Methods：The DNA fragmentation in Jurkat cells by mDRA-6 was analyzed by agarose gel electrophoresis.Jurkat cells were incubated with mDRA-6 at different concentrations and then suppression of cell growth was determined by MTT assay.The apoptotic rate of Jurkat cells was detected by flow cytometry with Annexin V-FITC/PI double staining.The inhibitors for Caspase-10,9,8 and Caspase-3 were used to block the apoptotic pathway of Jurkat cells by mDRA-6 treatment.Furthermore,the changes of cleavage products of Caspase-10,9,8,3 and Bid（tbid）,Cyto c were analyzed by Western blot after mDRA-6 treat ment of Jurkat cells.Results：The mDRA-6 suppressed cell growth and cytotoxicity in a dose-dependment manner,and DNA fragmentation was observed in Jurkat cells under agarose gel electrophoresis.After mDRA-6 treatment at 2.0 μg/ml for 0.25 h,0.5 h,1 h,2 h,the rates of apoptotic frequency for Jurkat cells was 16.17%,28.25%,69.23%,78.23%,respectively by Annexin V-FITC/PI double staining.Caspase-8 inhibitor inhibited the apoptosis of Jurkat cells induced by mDRA-6,and the rate of apoptotic cells was 77.85%.Inhibitor of either Caspase-3 or Caspase-9 inhibited the apoptotisis of Jurkat cells by 54.20% and 8.74%,respectively,whereas Caspase-10 inhibitor had no effects.Active cleavage fragments of Caspase-8,9,3 and Bid（tbid） as well as Cyto c,were shown by Western blot and did not for cleavage products of Caspase-10.Conclusion：Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR5 ligation to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated.In this process Caspase-8 is involved as the initial trigger,and Caspase10 not.mDRA-6 may be a useful agent in investigating for anti-tumor therapy by using TRAIL/DR5 model.