不同毒力的分枝杆菌感染促进小鼠RAW264.7巨噬细胞自噬及mTOR和AKT磷酸化

Different virulence Mycobacterium promotes RAW264.7 macrophage autophagy and phosphorylation of mTOR and AKT

目的探讨不同毒力分枝杆菌感染对巨噬细胞自噬水平的影响。方法分别用表达绿色荧光蛋白(GFP)的结核分枝杆菌标准株H37Rv、卡介苗(BCG)和耻垢分枝杆菌Ms1-2c株(Ms)感染小鼠RAW264.7巨噬细胞,流式细胞术检测巨噬细胞的自噬水平,激光共聚焦显微镜观察细胞内吞噬体与溶酶体的共定位,Western blot法检测微管相关蛋白1轻链3(LC3)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化的mTOR(p-mTOR)、蛋白激酶B(AKT)和磷酸化的AKT(p-AKT)蛋白表达。结果与对照组相比,RAW264.7巨噬细胞感染H37Rv、BCG和Ms 24 h后,感染组的自噬水平均显著增加,Ms感染组的自噬水平最高;各感染组的LC3-Ⅱ蛋白表达均上调,H37Rv、BCG和Ms感染的细胞内吞噬体与溶酶体的共定位率分别为(11.33±0.88)%、(18.33±0.88)%和(48.67±0.66)%,Ms感染组的分枝杆菌与溶酶体共定位最明显。分枝杆菌感染巨噬细胞后,mTOR和AKT表达无明显改变,但其磷酸化水平均显著升高。结论分枝杆菌感染诱导巨噬细胞发生自噬,同时促进mTOR和AKT的磷酸化。

Objective To explore the change of autophagy levels of the macrophages infected by Mycobacterium with different virulence.Methods RAW264.7 cells were infected with Mycobacterium tuberculosis standard strain(H37Rv),Bacille Calmette Guérin(BCG)and Mycobacterium smegmatis(Ms).The cell autophagy was detected by flow cytometry and the colocalization of phagosomes and lysosomes was detected by confocal laser scanning microscopy.Meanwhile,the autophagy-associated protein LC3,mTOR,p-mTOR,AKT and p-AKT were detected by Western blotting.Results At 24 hours after RAW264.7 cells were infected by Mycobacterium with different virulence,flow cytometry showed that the level of autophagy was significantly up-regulated by H37Rv,BCG and Ms infection,and the highest level was in the Ms infection group.The level of LC3-II was significantly up-regulated after H37Rv,BCG and Ms infection,and the tendency was consistent with the result of flow cytometry.The colocalization rates of phagosomes and lysosomes after H37Rv,BCG and Ms infection were(11.33±0.88)%,(18.33±0.88)%and(48.67±0.66)%.The expression of mTOR and AKT had no significant changes after H37Rv,BCG and Ms infection,but the phosphorylation level significantly increased,which meant that PI3K-AKT-mTOR was activated.Conclusion Mycobacterium infection can induce the autophagy in macrophages and promote the phosphorylation of mTOR and AKT.