抗人DR5单克隆抗体诱导HL-60细胞凋亡机制

探讨抗人DR5单克隆抗体(mDRA-6)诱导白血病细胞系HL-60细胞凋亡的可能机制。MTT法检测mDRA-6对HL-60细胞的生长增殖的影响,以及Caspase 8、10、3抑制剂对mDRA-6抑制HL-60细胞生长增殖的影响;琼脂糖凝胶电泳检测HL-60细胞DNA片断化降解;Western blot检测mDRA-6对HL-60细胞Caspase 8、10、3的激活改变。结果发现,mDRA-6呈时间、浓度依赖性地抑制HL-60细胞的生长增殖,10 mg/L的mDRA-6作用HL-60细胞6 h、8 h和10h,细胞增殖抑制率分别为23.96%、44.10%和50.28%;琼脂糖凝胶电泳显示,10 mg/L的mDRA-6作用6 h,HL-60细胞呈现凋亡细胞特有的DNA梯形条带;Western blot检测结果发现,mDRA-6作用HL 60细胞不同时间,Caspase 8均只有酶原条带出现,无激活片段产生,而Caspase 10和Caspase 3显示明显裂解片段产生,并随mDRA-6作用时间延长而增多;预先使用15μmol/L的Caspase 10及Caspase 3抑制剂孵育细胞1 h,能够使mDRA-6对HL-60细胞的生长抑制率分别降低64.15%(t=10.13、P<0.01)和53.69(t=8.93、P<0.01)%,而预先使用15μmol/L的Caspase 8抑制剂孵育细胞1 h,mDRA-6对HL-60细胞的生长抑制率仅降低4.40%(t=0.52、P>0.05)。以上实验结果提示,mDRA-6通过激活Caspase 10启动凋亡信号分子,诱导白血病HL-60细胞凋亡。

The mechanism of HL-60 cell apoptosis induced by anti-human DR5 monoclonal antibody

To study the mechanism of HL-60 cell apoptosis induced by anti-human DR5 monoclonal antibody,HL-60 cells were treated with mDRA-6 or / and Caspase inhibitors,and the inhibition ratio was determined using MTT assay.The DNA ladder of HL-60 cells treated with mDRA-6(lOmg/L) for 6 h was analyzed by 1%agrose gel electrophoresis.The expression of Caspase 8,Caspase 10 and Caspase 3 of HL-60 cells after treating with mDRA-6 were detected by Western blotting,respectively. It was demonstrated that the proliferation of HL-60 cell after treating with mDRA-6 was inhibited in a dose-dependent and time-dependent manner,the inhibition ratio of HL-60 cells treated by 10 jig/ml mDRA-6 for 6 h,8 h and 10 h were 23. 96%>44.10%and 50.28%,respectively.DNA fragmentation detection indicated that HL-60 cell apoptosis was induced by mDRA-6.Western blotting revealed that Caspase 10 and Caspase 3 in HL-60 cells treated with mDRA-6 were activated in a time-dependent manner,but Caspase 8 did not.The HL-60 cells were incubated with the inhibitors for Caspase 8,10 and 3 for 1 hour,respectively,and then the cells were treated with mDRA-6,HL-60 cell apoptosis induced by mDRA-6 were reduced by 4.40%(t = 0.52,P>0.05),64.15%(r= 10.13,P<0.01) and 53.69(r=8.93,P<0.01)%,respectively.Therefore,it is evident that Caspase 10 inhibits apoptosis of HL-60 cells pretreated with anti-human DR5 monoclonal antibody mDRA-6.