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

作　　者：杜耀武[1] 刘广超[1] 王靖[1] 赵粤萍[1] 李淑莲[1] 蒋祺[1] 蔡静[1] 马远方[1] 

机构地区：[1]河南大学免疫学研究所河南大学医学院河南大学天然药物与免疫工程重点实验室,开封475001

出　　处：《中国免疫学杂志》2009年第1期48-53,79,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(No.30571697);河南省杰出人才创新基金项目(No.074200510014)

摘　　要：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.

关 键 词：死亡受体5 功能性抗体 JURKAT细胞 细胞凋亡 分子机制 

分 类 号：R392.11[医药卫生—免疫学]