结核分枝杆菌ESX-1分泌蛋白ESAT-6增强巨噬细胞吞噬功能

ESX-1 secreted protein ESAT-6 of Mycobacterium tuberculosis enhances the phagocytosis of RAW264.7 macrophages

摘要:

摘要:【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6 (early secreted antigenic target of 6 kDa)对巨噬细胞吞噬功能的影响。【方法】用重组质粒pFLAG-ESAT-6和pFLAG-EGFP 转染RAW264.7细胞，经G418筛选，PCR、RT-PCR和Western blot鉴定，获得稳定表达flag-ESAT-6和flag-EGFP的RAW细胞系，然后用流式细胞术观察各稳转细胞系吞噬荧光微球的能力，并用共聚焦显微镜和菌落计数法检测稳转细胞系吞噬大肠杆菌(Escherichia coli)的能力。【结果】获得了稳定表达flag-ESAT-6 的RAWE6细胞系和表达flag-EGFP的RAW-EGFP细胞系;流式细胞术检测结果表明RAW-E6吞噬荧光微球的能力显著强于野生型细胞系RAW264.7和对照细胞系RAW-EGFP;菌落计数和激光共聚焦分析表明RAW-E6细胞系吞噬E.coli的能力也显著强于RAW264.7和RAW-EGFP。【结论】通过胞内表达发现结核分枝杆菌分泌蛋白ESAT-6能够增强巨噬细胞的吞噬功能，这将为深入理解结核分枝杆菌的致病机制提供新的思路。

关键词:关键词:结核分枝杆菌(Mycobacterium tuberculosis)，ESAT-6，巨噬细胞，吞噬作用

Abstract:

Abstract:［Objective］ To investigate the effects of Mycobacterium tuberculosis ESX-1 protein early secreted antigenic target of 6 kDa(ESAT-6) in modulating phagocytosis of RAW264. 7 cells.［Methods］RAW264.7 cells were transfected with recombinant plasmids pFLAG-ESAT-6 and pFLAG-EGFP by liposome． After screening with a high level of G418，the macrophage cell lines stably expressing flag-ESAT-6 or flag-EGFP proteins were obtained．The cell lines were further identified by PCR，RT-PCR and western blot．The phagocytosis of those cell lines was analyzed for ingested fluorescent beads by flow cytometry and for phagocytized Escherichia coli(E.coli) by colony count and confocalmicroscopy.［Results］We established successfully RAW-E6 cell line stably expressing flag-ESAT-6 and RAW-EGFP cell line stably expressing flag-EGFP． Flow cytometric analysis shows that the percentage of phagocytosis of RAW-E6 was higher than that of RAW264.7 and RAW-EGFP. Colony count and confocal microscopy test also show that RAW-E6 had higher phagocytosis ability than RAW264.7 and RAW-EGFP.［Conclusion］The secreted protein ESAT-6 can enhance the phagocytosis of macrophages，which provides new evidence to understand the pathogenesis of Mycobacterium tuberculosis.

Key words:Keywords: Mycobacterium tuberculosis，ESAT-6，macrophage，phagocytosis