【摘要】 目的分析Fas诱导的B淋巴瘤细胞株MML-1凋亡过程中,PMA阻断MML-1进入细胞周期后细胞凋亡敏感性降低的原因。方法采用10 nmol/L PMA预处理MML-1细胞24 h,用流式细胞仪检测PMA阻断细胞周期前后50 ng/mL抗Fas抗体诱导的MML-1细胞凋亡率的变化。采用PI-Triton X和active caspase-3/8双染色、Western blotting技术分析PMA处理前后,经抗Fas抗体诱导的MML-1细胞内活化caspase-3/8的表达。通过Western blotting技术检测G1期细胞的凋亡相关蛋白caspase-3/8、Bcl-2、FLIP和Akt的表达。结果 PMA预处理MML-1细胞24 h后,G1期细胞比例显著升高,达93.77%。PMA预处理前,抗Fas抗体诱导6 h后的MML-1细胞凋亡率为56%;经PMA预处理,抗Fas抗体诱导6 h后的MML-1细胞凋亡率为14%。流式细胞术和Western blotting检测发现经PMA预处理,抗Fas抗体诱导后活化caspase-3和caspase-8蛋白的表达受到显著抑制。Westernblotting检测显示G1期细胞中凋亡抑制蛋白Bcl-2的表达上调。结论将MML-1细胞阻断在G1期能够抑制Fas诱导的细胞凋亡,可能与凋亡抑制分子Bcl-2在G1期的表达上调有关。更多还原

【Abstract】 Objective To investigate the mechanism of Fas-mediated apoptosis of MML-1 cells inhibited in G1 phase by PMA. Methods MML-1 cells were pretreated by 10 nmol/L PMA for 24 h,and the apoptosis rates of MML-1 cells induced by 50 ng/mL anti-Fas antibody were detected by flow cytometry before and after cell cycle arrest by PMA.The expression of active caspase-3/8 in anti-Fas antibody-induced MML-1 cells was detected by PI-Triton X and active caspase-3/8 double staining and Western blotting before and after PMA treatment.The expression of apoptosis related protein of caspase-3/8,Bcl-2,FLIP and Akt in cells of G1 phase was detected by Western blotting. Results Twenty-four hours after treatment of MML-1 cells by PMA,the percentage of cells in G1 phase significantly increased(93.77%).Before PMA treatment,the apoptosis rate of MML-1 cells was 56% after Fas-mediated apoptosis for 6 h.Whereas after PMA treatment,the apoptosis rate of MML-1 cells was 14% after Fas-mediated apoptosis for 6 h.It was revealed by flow cytometry and Western blotting that the expression of active caspase-3/8 was significantly inhibited after PMA treatment.Western blotting demonstrated that the expression of Bcl-2 was up-regulated in cells of G1 phase. Conclusion Fas-mediated apoptosis would be inhibited in MML-1 cells arrested in G1 phase,which might be related to the up-regulation of Bcl-2 in G1 phase.更多还原