抗人DR5功能性单克隆抗体诱导Jurkat细胞凋亡的线粒体途径的分子机制  

The mitochondrial-dependent molecular mechanisms for inducing apoptosis in Jurkat cells by a novel agonistic anti-human DR5 monoclonal antibody

作　　者：杜耀武[1] 柴立辉[1] 黄红莹[1] 白慧玲[1] 赵粤萍[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省级重点实验室,开封475001

出　　处：《中国免疫学杂志》2010年第1期3-7,共5页Chinese Journal of Immunology

基　　金：国家863重大专项子课题(No.2006AA02A254);河南省杰出人才创新基金项目(No.074200510014)的资助

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细胞诱导凋亡的线粒体途径的分子机制。方法:MTT法检测单克隆抗体(mDRA-6)对Jurkat细胞生长抑制作用的剂量-效果及时间-效果关系;JC-1单染流式细胞术定量分析技术对凋亡的Jurkat细胞线粒体膜电位的变化进行分析;免疫印迹技术检测凋亡细胞的Caspase-8、3、9及Bid、Bax、Bcl-2、Cytoc等凋亡相关蛋白的表达情况。结果:mDRA-6对Jurkat细胞抑制具有明显剂量-效果和时间-效果关系;流式细胞仪检测显示,2.0μg/ml浓度的mDRA-6作用15、30、60和120分钟时,Jurkat细胞线粒体膜电位改变率分别为20.14%、19.34%、21.11%、30.90%,呈逐渐增高趋势。免疫印迹技术检测显示,mDRA-6作用后,Jurkat细胞Caspase-8、9及Bid、Bax、Bcl-2、Cytoc等表达活性增加,并且Cytoc在线粒体内为递减而在胞浆呈递增的表达现象。结论:mDRA-6通过激活外源性膜受体,继而启动细胞线粒体途径导致Jurkat细胞凋亡。本研究为mDRA-6对白血病治疗奠定实验基础。Objective：To investigate mitochondrial-dependent molecular mechanisms of a novel agonistic anti-human death receptor 5 （DRS） monoclonal antibody（mDRA-6） inducing apoptosis in Jurkat cell. Methods： The dose-dependent and time-dependent cell growth suppression of mDRA-6 in Jurkat cells was determined by MTT assay. The measurement of the mitochondrial transmembrane potential（△ψm） of Jurkat cells was detected by flow cytometry with JC-1 single staining. Caspase-8,9 as well as Bid, Bax, Bcl-2 and Cyto c of apoptotic Jurkat cells were analyzed by Western blot after mDRA-6 treatment. Results： The mDRA-6 induced cell growth suppression and cytotoxicity in dose-depen- dent manner and time-dependent manner. After mDRA-6 treatment at 2.0μg/ml for15 min, 30 min, 60 min and 120 min, the change in △ψm were 20.14 %, 19.34 %, 21.11% and 30.90 % respectively by JC-1 single staining. Western blot revealed that the level of active fragments of Caspase-8,9 and Bid, Bax, Bcl-2 and Cyto c respectively, and the amount of Cyto c was increased in cytosol concomitant with the related atten- uation of Cyto c in mitochondfia. Conclusion： Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR.5 ligation to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated, and endogenic mitochondrial-dependent cell apoptosis pathway is activated. mDRA-6 may be a useful agent in investigating human leukemia therapy by using TRAIL/DRS.

关 键 词：死亡受体5 单克隆抗体 JURKAT细胞 细胞凋亡 

分 类 号：R392.11[医药卫生—免疫学]