【摘要】 目的研究NADPH氧化酶(NOX)参与烟曲霉孢子诱导巨噬细胞自噬及其作用机制。方法取对数生长期的RAW264.7细胞,随机分为3组:以烟曲霉孢子刺激的处理组、烟曲霉孢子联合NOX抑制剂氯化二亚苯基碘鎓(DPI)的处理组和无处理的空白组。细胞作用2 h后荧光显微镜观察细胞内二氯荧光黄亮度以检测活性氧(ROS)水平,免疫印迹法检测微管相关蛋白轻链3(LC3)的表达量,免疫荧光技术观察LC3的表达与定位。结果烟曲霉孢子刺激后RAW264.7细胞的ROS与LC3BⅡ表达水平显著升高,同时LC3呈斑点状聚集并与烟曲霉孢子共定位;以DPI抑制后ROS与LC3 BⅡ表达量与空白组相比无明显变化,并且LC3在细胞内弥散分布。结论烟曲霉感染时NOX通过产生ROS活化巨噬细胞自噬。更多还原

【Abstract】 Objective To investigate whether or not NADPH oxidase( NOX) participates in Aspergillus fumigatus induced macrophage autophagy in RAW264. 7 cells and explore the possible mechanism. Methods RAW264. 7 cells in the exponential growth phase were randomized into three groups: Aspergillus fumigatus conidia( MOI 5 ∶ 1) treated group,Aspergillus fumigatus conidia plus NOX inhibitor diphenyleneiodonium chloride( DPI) treated group; negative control group( untreated). The levels of intracellular reactive oxygen species( ROS) were measured with dichiorodihydrofluorescein diacetate( DCFH-DA) staining by fluorescence microscope. The expression of microtubule-associated protein light chain-3( LC3) protein in RAW264. 7 cells was detected by Western blotting. Then the expression and positioning of LC-3 were observed by immunofluorescence staining. Results After stimulated with conidia of Aspergillus fumigatus,RAW264. 7 cells showed significant increases of ROS and LC3BⅡ. Interestingly,LC3 was recruited to phagosomes which contained Aspergil us fumigatus conidia. After the NOX inhibitor DPI blocked the ROS production,the expressions of ROS and LC3 B Ⅱ had no significant increases as compared with the negative control group,and the distribution of LC3 was diffuse in cytoplasm.Conclusion NOX induces macrophage autophagy by producing ROS in Aspergillus fumigatus infection.更多还原