mDRA-6诱导Jurkat细胞凋亡的外源性机制

目的:探讨抗人死亡受体5(DR5)单克隆功能抗体(mDRA-6)对人白血病Jurkat细胞诱导凋亡的外源性机制。方法:用显微镜观察mDRA-6作用后的Jurkat细胞形态学变化,采用Annexin V-FITC/PI双染流式细胞术定量分析技术对Jurkat细胞的凋亡进行定量分析;利用免疫印迹技术检测凋亡细胞的Caspase 10、Caspase 9、Caspase8、Caspase 7、Caspase 3等凋亡相关蛋白的表达及活化情况。结果:mDRA-6在5 mg/L浓度作用Jurkat细胞30min时就出现典型的细胞凋亡形态改变,Annexin V-FITC/PI双染流式细胞术检测显示,Jurkat细胞在30 min、60min1、20 min的凋亡率分别为30.20%、69.03%、79.55%。免疫印迹技术检测显示Caspase 9、Caspase 8、Caspase7、Caspase 3均呈现活化片断的表达,而Caspase 10无变化。结论:mDRA-6可通过细胞膜表面死亡受体信号传导途径诱导Jurkat细胞的凋亡,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。

Extrinsic mechanisms of the apoptosis of Jurkat cells induced by mDRA-6

Objective: To investigate extrinsic mechanisms of a novel agonistic anti-human death receptor 5(DR5) monoclonal antibody(mDRA-6)inducing apoptosis human leukemia Jurkat cell.Methods: After affected by mDRA 6,the apoptotic morphology changes of Jurkat cells were observed with microscope.The apoptotic rate of mDRA-6 on Jurkat cells was detected by flow cytometry with Annexin V-FITC / PI double staining.The apoptotic signals proteins active cleavage products of Caspase 10,Caspase 9,Caspase 8,Caspase 7 and Caspase 3 were analyzed by Westernand blotting after mDRA-6 treatment.Results: Jurkat cells displayed typical apoptotic morphology after mDRA-6 treatment for 30 min,When treated with 5.0 mg/L mDRA-6,the apoptosis rates of Jurkat cells were 30.20% at 0.5 h,69.2% at 1 h,and 79.55% at 2 h.Western blotting revealed active fragments of Caspase 9,Caspase 8,Caspase 7 and Caspase 3 respectively,and the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable.Conclusion: Apoptotic pathway induced by mDRA 6 in Jurkat cells can be initiated extrinsic signal transduction pathway of membrane death receptor pathway,followed by recruitment Fas associated death domain protein leaded to direct activation of the Caspase cascade,which may be a useful agent in investigating anti-tumor therapy by usingTRAIL/DR5.