非经典型自噬替代经典型自噬影响单核-巨噬细胞分化的研究

Canonical autophagy can be replaced by alternative autophagy in monocytemacrophage differentiation

目的:研究经典型自噬缺失时,非经典型自噬是否在小鼠单核-巨噬细胞分化中起作用。方法:将Atg7小鼠与Lyz-Cre小鼠杂交获得单核巨噬细胞自噬特异性敲除的小鼠模型Atg7f/f;Lyz-Cre小鼠,使用C57BL/6小鼠作为对照组。应用磁珠分选的方法富集单核细胞并加因子诱导分化,流式细胞术检测单核-巨噬分化比例(Ly6C-F4/80)及细胞凋亡比例;实时荧光定量PCR方法检测非经典型自噬关键基因Vps34,Beclin1和ULK1 mRNA表达水平;Western Blot方法检测Vps34,beclin1,ULK1以及非经典型自噬关键蛋白Rab9的蛋白表达水平;使用非经典型自噬抑制剂Brefeldin A(BFA)处理单核细胞,流式细胞术检测单核-巨噬分化比例;使用共聚焦显微镜及电镜观察自噬情况。结果:与野生型小鼠相比,Atg7f/f;Lyz-Cre小鼠单核-巨噬细胞分化能力明显降低,凋亡比率显著上升(P均<0.05)。共聚焦及电镜结果显示Atg7f/f;Lyz-Cre小鼠经典型自噬被破坏,但仍存在自噬响应。而非经典型自噬关键因子Vps34,Beclin1,ULK1及Rab9蛋白表达水平均上调(P均<0.05)。非经典型自噬抑制剂BFA能抑制Atg7f/f;Lyz-Cre小鼠单核-巨噬分化但不影响野生型小鼠。结论:经典型自噬对小鼠单核-巨噬细胞分化起重要作用,而当其被破坏时,非经典型自噬活性上调以维持单核-巨噬细胞分化。

To study the effects of alternative autophagy on monocyte-macrophage flox/floxdifferentiation in the absence of canonical autophagy. METHODS：Atg7 mice were crossed with Lyz-Cre mice to assess deletion of autophagy in monocytes and macrophages. C57BL/6 mice were used as control group. The monocytes were harvested from wild type and Atg7f/f;Lyz-Cre mice. Monocyte-macrophage differentiation was induced by M-SCF. The percentage of Ly6C-F4/80 cells and apoptosis were analyzed with or without Brefeldin A （BFA） treatment. The key members of alternative autophagy including Vps34,Beclin1,ULK1 and Rab9 were evaluated. The autophagy activity was monitored by confocal and transmission electron microscope. RESULTS：Monocyte-macrophage differentiation in Atg7f/f;Lyz-Cre mice was significantly crippled with elevated apoptosis（P〈0.05）. Confocal and transmission electron microscope results showed that the deletion of Atg7 in myeloid cells impaired the canonical autophagy while autophagy response was still preserved. The expression level of key members in alternative autophagy such as Vps34,Beclin1,ULK1 and Rab9 were elevated（all P〈0.05）. BFA,the inhibitor of alternative autophagy,inhibited monocyte-macrophage differentiation of Atg7f/f;Lyz-Cre mice rather than wild type mice. CONCLUSION：Canonical autophagy,which is important in monocyte-macrophage differentiation,could be replaced by alternative autophagy.