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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。
OBJECTIVE: To analyze the reasons why PMA blocks the apoptosis-inducing effect of MML-1 after it enters the cell cycle during the apoptosis of Fas-induced B lymphoma cell line MML-1. Methods MML-1 cells were pretreated with 10 nmol / L PMA for 24 h. The apoptosis rate of MML-1 cells induced by 50 ng / mL anti-Fas antibody was detected by flow cytometry. The expression of activated caspase-3/8 in MML-1 cells induced by anti-Fas antibody before and after PMA treatment was analyzed by PI-Triton X and active caspase-3/8 double staining and Western blotting. The expression of apoptosis-related proteins caspase-3/8, Bcl-2, FLIP and Akt in G1 phase were detected by Western blotting. Results After pretreated with PMA for 24 h, the proportion of cells in G1 phase increased significantly, reaching 93.77%. Before PMA pretreatment, the apoptosis rate of MML-1 cells was 56% after anti-Fas antibody was induced for 6 h, and the apoptosis rate of MML-1 cells was 14% after anti-Fas antibody pretreatment for 6 h. Flow cytometry and Western blotting showed that pretreatment with PMA significantly inhibited the expression of caspase-3 and caspase-8 after anti-Fas antibody. Western blotting showed that the expression of apoptosis-suppressing protein Bcl-2 in G1 phase was up-regulated. Conclusion Blocking MML-1 cells in G1 phase can inhibit Fas-induced apoptosis, which may be related to the up-regulation of Bcl-2 in G1 phase.