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目的研究结核分枝杆菌分泌性酸性磷酸酶(Sap M)对小鼠巨噬细胞自噬的影响。方法分别用Sap M、渥曼青霉素、饥饿处理GFP-LC3-Raw264.7细胞。荧光显微镜下观察自噬体的形成,Western blot法检测微管相关蛋白1轻链3(LC3)Ⅱ水平;再用Sap M处理GFP-LC3-Raw264.7细胞后加氯喹,Western blot法检测LC3Ⅱ水平。结果 Sap M和饥饿均导致GFP-LC3斑点增多和LC3Ⅱ水平增高,在Sap M处理的细胞中加入氯喹并没有引起LC3Ⅱ的进一步升高。结论 Sap M可阻断自噬体与溶酶体的融合,从而抑制小鼠巨噬细胞自噬。
Objective To study the effect of Mycobacterium tuberculosis secreted acid phosphatase (Sap M) on macrophage autophagy in mice. Methods GFP-LC3-Raw264.7 cells were treated with Sap M, wortmannin, starvation respectively. The formation of autophagosomes was observed under a fluorescence microscope. The level of microtubule-associated protein 1 light chain 3 (LC3) Ⅱ was detected by Western blotting. The GFP-LC3-Raw264.7 cells were treated with chloroquine by Sap M and detected by Western blot Level. Results Both Sap M and starvation resulted in increased GFP-LC3 plaque and increased LC3II levels. The addition of chloroquine to Sap M-treated cells did not cause any further increase in LC3II. Conclusion Sap M can block the fusion of autophagosomes and lysosomes and thus inhibit the autophagy of mouse macrophages.