CAJ | 학술논문

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.
목적 탐토Cyclins/CDKs재Fas개도적세포조망과정중적작용궤제.방법 이백혈병세포주(Molt-4화Jurkat세포주)화건강인적외주혈림파세포위재체,용인중조Fas배체유도은정이우전형적세포주기특이성조망모형;용Cyclin/DNA쌍삼수류식세포술검측Cyclins적표체;용Western blot법검측CDK1화CDK2적린산화위점이급진일보학인Cyclins적표체.결과 연구rhFasL유도Jurkat、Molt-4세포화진입세포주기적PBL조망시,G1기적Cyclin D3재발생조망효응전명현승고、발생조망효응후명현하강,이Cyclin E칙선명현하강후승고,Cyclin A화B1무명현변화;CDK2 Thr-160위점적린산화수평재발생조망효응전후균명현하강,CDK1 Thr-161、Tyr-15위점적린산화수평재발생조망효응후초유하강;G0기림파세포칙불능피유도출명현적세포조망.결론 Fas개도적세포조망적발생여세포시부통과한제점진입세포주기유관,세포조망발생우만G1기시G1기Cyclin E적표체하강이지불능린산화혹자린산화CDK2불전,재검측점적감독하DNA수손적세포불능통과G1/S교계진입S기이발생세포조망.재조망발생후기Cyclins/CDK적변화여세포피조체재G1기유관.
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.