Cyclins/CDKs在Fas介导细胞凋亡过程中的变化及其机制  

Changes of Cyclins/CDKs in Fas-mediated apoptosis and the mechansim

作　　者：全晓明[1] 郭建敏[1] 

机构地区：[1]湖北省新华医院外科,武汉430015

出　　处：《中华实验外科杂志》2010年第11期1660-1662,共3页Chinese Journal of Experimental Surgery

摘　　要：目的 探讨Cyclins/CDKs在Fas介导的细胞凋亡过程中的作用机制.方法 以白血病细胞株（Molt-4和Jurkat细胞株）和健康人的外周血淋巴细胞为载体,用人重组Fas配体诱导稳定而又典型的细胞周期特异性凋亡模型 用Cyclin/DNA双参数流式细胞术检测Cyclins的表达 用Western blot法检测CDK1和CDK2的磷酸化位点以及进一步确认Cyclins的表达.结果 研究rhFasL诱导Jurkat、Molt-4细胞和进入细胞周期的PBL凋亡时,G1期的Cyclin D3在发生凋亡效应前明显升高、发生凋亡效应后明显下降,而Cyclin E则先明显下降后升高,Cyclin A和B1无明显变化 CDK2 Thr-160位点的磷酸化水平在发生凋亡效应前后均明显下降,CDK1 Thr-161、Tyr-15位点的磷酸化水平在发生凋亡效应后稍有下降 G0期淋巴细胞则不能被诱导出明显的细胞凋亡.结论 Fas介导的细胞凋亡的发生与细胞是否通过限制点进入细胞周期有关,细胞凋亡发生于晚G1期时G1期Cyclin E的表达下降以至不能磷酸化或者磷酸化CDK2不全,在检测点的监督下DNA受损的细胞不能通过G1/S交界进入S期而发生细胞凋亡.在凋亡发生后期Cyclins/CDK的变化与细胞被阻滞在G1期有关.Objective To study the changes of Cyclins/CDKs expression in Fas-mediated apoptosis, and the action mechanism in leukaemia cell lines and peripheral blood lymphocytes （PBL） in vitro.Methods The target cells--leukaemia cell lines and activated PBL stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, and the changes of Cyclins expression that related to the duration of inducing apoptosis was detected by Cyclin/DNA bi-parameter flow cytometry. Rb protein and the phosphorylation site of CDK1 and CDK2 were checked by Western blotting. Results Cyclin D3 expression was increased and Cyclin E expression decreased evidently, and the expression of Cyclins A and B1 had no significant change before the induction of Fas-mediated apoptosis in Jurkat, Molt-4 cell lines and PBL which entering cell cycle progression, till the late stage of inducing apoptosis in these cells the expression of Cyclin D3 was decreased, while Cyclin E increased. Meanwhile, apoptosis couldn＇ t be induced by rhFas ligand in G0-phase PBL. The level of CDK2 Thr-160 phosphorylation was decreased through the induction of Fas-mediated apoptosis evidently. The phosphorylation of CDK1 had no significant changes. Conclusion Fas-mediated apoptosis was located at late G1-phase and determined by whether the target cells had passed through the restriction point to cell division cycle or not. And the cell cycle specificity of Fas-mediated apoptosis was the result of the cells with DNA damage being blocked at late G1-phase,due to the decrease of Cyclin E expression and hypophosphorylation of CDK2 and under the surveillance of G1-phase check point. The changes of Cyclins expression at the late stage of Fas-mediated apoptosis is the result of cell arrest at G1-stage.

关 键 词：脱噬作用 流式细胞术 

分 类 号：R346[医药卫生—基础医学]