论文部分内容阅读
【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenictarget 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RAW-E6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。
【Objective】 The purpose of this study was to investigate the effect of mycobacterium tuberculosis secreted protein (ESAT-6) on phagocytosis of macrophages. 【Methods】 RAW264.7 cells were transfected with the recombinant plasmids pFLAG-ESAT-6 and pFLAG-EGFP, and then the RAW264.7 cells stably expressing flag-ESAT-6 and flag-EGFP were screened by G418, PCR, RT-PCR and Western blot Cell lines. The ability of each stable cell line to phagocytize the fluorescent microspheres was observed by flow cytometry. The ability of the stable cell lines to phagocytize Escherichia coli was detected by confocal microscopy and colony counting. 【Result】 The RAW-E6 cell line stably expressing flag-ESAT-6 and the RAW-EGFP cell line expressing flag-EGFP were obtained. The results of flow cytometry showed that RAW-E6 phagocytose fluorescent microspheres significantly stronger than wild-type Cell line RAW264.7 and the control cell line RAW-EGFP; colony counting and laser confocal analysis showed that RAW-E6 cell line phagocytosis of E. coli was also significantly stronger than RAW264.7 and RAW-EGFP. 【Conclusion】 It is found that ESAT-6 secreted by Mycobacterium tuberculosis can enhance the phagocytosis of macrophages by intracellular expression, which will provide a new idea for understanding the pathogenesis of Mycobacterium tuberculosis.