结核分枝杆菌分泌蛋白ESAT-6对小鼠巨噬细胞凋亡的诱导作用

Mycobacterium tuberculosis secreted protein ESAT-6 induces the apoptosis of mouse macrophages

【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenic target of 6 kD)对小鼠巨噬细胞凋亡的影响及机理。【方法】应用流式细胞仪分析稳定表达ESAT-6和经重组ESAT-6蛋白作用的小鼠RAW264.7细胞的凋亡;应用Caspase-3检测试剂盒检测caspase-3活性变化;进一步应用Western blotting分析稳转细胞系ESAT-6表达情况。【结果】RAW-EGFP-ESAT-6细胞系培养48 h后的凋亡率与caspase-3活性显著高于RAW264.7和RAW-EGFP细胞系(P<0.05);RAW-flag-ESAT-6细胞系培养48 h后的凋亡率与caspase-3活性显著高于RAW264.7和相应对照细胞系(P<0.01)。10μg/ml重组ESAT-6可显著诱导RAW264.7细胞凋亡,而5μg/ml重组ESAT-6无明显诱导细胞凋亡效果。稳转细胞系RAW-flag-ESAT-6细胞内flag-ESAT-6浓度大约只有500 ng/ml。【结论】胞内低水平表达结核分枝杆菌分泌蛋白ESAT-6可依赖caspase-3信号通路诱导小鼠RAW264.7细胞凋亡。

【Objective】To investigate the effects and the mechanism of Mycobacterium tuberculosis ESX-1 secreted protein ESAT-6 in inducing the apoptosis of mouse macrophages.【Methods】The apoptosis of RAW264.7 stably expressing ESAT-6 or treated by rESAT-6 was analyzed by flow cytometry,and the caspase-3 activity was detected by caspase-3 detection kit. Further,the expression of flag-ESAT-6 protein in RAW-flag-ESAT-6 was analyzed by Western blotting.【Results】Flow cytometric analysis showed that the percentage of apoptosis of RAW-EGFP-ESAT-6 and RAW-flag-ESAT-6 cell lines and their caspase-3 activity were both significantly higher than that of wild type cells and control cells. The apoptosis of RAW264.7 could be induced by 10 μg/ml rESAT-6, but the concentration of flag-ESAT-6 protein in RAW-flag-ESAT-6 was only about 500 ng/ml.【Conclusion】Low expression of ESAT-6 in cytoplasm can induce the apoptosis of RAW264.7 by caspase-3 pathway.