肺炎链球菌溶血素经死亡受体/Fas途径和线粒体途径诱导RAW264.7细胞凋亡

目的:探讨肺炎链球菌溶血素(Pneumolysin,Ply)对小鼠RAW264.7细胞的增殖抑制和诱导凋亡的作用及机制。方法:Ply蛋白加入RAW264.7细胞培养上清与细胞共孵育。倒置显微镜观察Ply对RAW264.7细胞形态的影响。MTT法检测Ply对RAW264.7细胞的增殖抑制。Annexin V法检测细胞凋亡率。分光光度法检测Caspase-3、8、9活性。免疫细胞化学法检测到Bax、Fas、Bcl-2蛋白的表达。结果:Ply对小鼠RAW264.7细胞有明显的增殖抑制作用,呈剂量和时间依赖性;1μg/ml Ply处理RAW264.7细胞24小时后,可见典型的凋亡形态学改变;1μg/ml Ply处理RAW264.7细胞1小时和3小时后,细胞凋亡率分别为32.90%和51.56%(P<0.05);1μg/ml Ply蛋白处理RAW264.7细胞24小时,Caspase-3、8、9活性均比对照组升高(P<0.05);免疫细胞化学法检测到Bax、Fas表达较对照组增强,Bcl-2表达减弱(P<0.01)。结论:Ply可诱导小鼠RAW264.7细胞凋亡,诱导凋亡的机制可能是通过死亡受体/Fas途径和线粒体途径双重机制的介导实现。

Pneumolysin induced apoptosis in murine RAW264.7 cells through death receptor/Fas and mitochondrial pathway

Objective:To study the mechanism of proliferation inhibition and apoptosis of murine marcrophage-like cell line RAW264.7 cells induced by Pneumolysin(Ply).Methods:Protein Ply was added into culture supernatant of RAW264.7 cells and cocultured with the cells.The growth inhibition rate of RAW264.7 cells was measured by MTT.The apoptosis rate of RAW264.7 cells was examined by Annexin V-FITC/PI-stain.The activity of Caspase-3,8,9 was detected by spectrophotometry.The expressions of Bax,Fas,Bcl-2 were investigated by immunocytochemistry.Results:Ply presented the apparent proliferation inhibition on RAW264.7 cells in a time-and dose-dependent manner.The typical morphological features of apoptosis were observed after Ply treatment at 1 μg/ml for 24 h.And the apoptosis rate of RAW264.7 cells was 32.90% and 51.56% after Ply treatment at 1 μg/ml for 1 h and 3 h respectively(P<0.05).The activity of Caspase-3,8,9 was increased compared with control after Ply treatment at 1 μg/ml for 24 h(P< 0.05).The expression of Bax,Fas was increased,while the expression of Bcl-2 was reduced compared with control(P<0.01).Conclusion:Ply induced apoptosis in murine RAW264.7 cells through death receptor/Fas and mitochondrial pathway.