结核分枝杆菌eis基因对鼠巨噬细胞Raw264．7白噬影响的研究

目的探讨结核分枝杆菌eis基因对巨噬细胞自噬的影响。方法将鼠巨噬细胞Raw264．7以自噬体荧光表达质粒GFP-LC3转染，将含eis基因的重组耻垢分枝杆菌MS—pmv261-eis与不含eis基因的耻垢分枝杆菌MS—pmv261分别感染宿主巨噬细胞，透射电镜下观察自噬小体形成情况，荧光显微镜下观察自噬荧光并计数，Westernblot检测如基因表达的蛋白及自噬蛋白LC3-Ⅱ的表达水平。结果结核分枝杆菌eis基因可抑制感染宿主细胞自噬小体的形成，并显著抑制自噬荧光小点形成（P〈0．05），显著降低了自噬蛋白LC3-Ⅱ表达水平。结论结核分枝杆菌e曲基因对Raw264．7细胞自噬有抑制作用。

Mycobacterium tuberculosis eis gene inhibited autophage in mice macrophage cell line Raw264.7

Objective To identify the role of eis gene of Mycobacterium tuberculosis in the process of autophage of mice macrophage cell line Raw264.7. Methods Raw264.7 cells were transfected with GFP-LC3, a fluorescent autophagosome expression plasmid. 12 hours later, the cells were infected with MS-pmv261-eis for 6 hours. MS- pmv261 was used as the control. The formation of autophagic multi-membrane was examined by TEM ; the Autophagy fluorescent dots were observed under the fluorescence microscope. We further detected the EIS protein and autophagic protein LC3-II by Western blotting. Results EIS protein decreased the fluorescene dots of autophage and mutli-membrane （P 〈 0.05 ）. Moreover, the EIS protein decreased the formation of autophagic protein as indicated by Western blot result. Conclusion The eis gene of Mycobacterium tuberculosis can inhibit the autophage of mice macrophage, which plays a key role in the persistency of tuberculosis.