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目的研究mTOR在结核杆菌毒力因子ESAT6诱导的自噬抑制以及促进BCG增殖中的作用。方法 PCMV-HA-ESAT6质粒转染Raw264.7细胞,用蛋白免疫印迹检测LC3、P62、P-mTOR和P-70S6K表达水平;用mTOR阻断剂Torin1联合ESAT6转染以及分别作用于Raw264.7细胞后,免疫印迹检测P62和P-mTOR表达水平,LysoTracker Red染色观察溶酶体变化,BCG增殖实验计数各组菌落数。结果 ESAT6转染细胞后,细胞P62、P-mTOR和P-70S6K表达水平显著增高,LC3I完成向LC3II的转化;联合Torin1的ESAT6转染组和Torin1处理组的P-mTOR和P62无显著变化,溶酶体无变化,BCG菌落数减少。结论 ESAT6诱导的自噬抑制和BCG的增殖依赖于mTOR的活化。
Objective To investigate the role of mTOR in the inhibition of autophagy induced by Mycobacterium tuberculosis virulence factor ESAT6 and the promotion of BCG proliferation. Methods Raw264.7 cells were transfected with plasmid PCMV-HA-ESAT6 and the expressions of LC3, P62, P-mTOR and P-70S6K were detected by Western blotting. The mTOR inhibitor Torin1 and ESAT6 were transfected into Raw264.7 cells, After the cells, the expression of P62 and P-mTOR was detected by immunoblotting, the changes of lysosomes were observed by LysoTracker Red staining, and the number of colonies in each group was counted by BCG proliferation assay. Results The expression of P62, P-mTOR and P-70S6K in ESAT6 transfected cells were significantly increased and LC3I was transformed into LC3II. There was no significant change in P-mTOR and P62 between ESAT6-transfected group and Torin1-treated group, No changes in lysosomes, BCG colonies decreased. Conclusion ESAT6-induced autophagy inhibition and BCG proliferation depend on the activation of mTOR.