不同形态的烟曲霉对巨噬细胞自噬水平的影响

The impact of different shapes of Aspergillus fumigatus on the level of macrophage autophagy

目的探究烟曲霉静息孢子、膨胀孢子以及菌丝对巨噬细胞自噬水平的影响。方法培养烟曲霉并收获静息孢子,在沙保弱液体培养基中振荡不同时间获得膨胀孢子及菌丝。以3种形态的烟曲霉分组处理RAW264.7细胞,免疫印迹法检测LC3BⅡ蛋白的表达量,逆转录PCR检测自噬相关蛋白Atg5、Atg7、Atg12 mRNA的转录水平。观察不同形态的烟曲霉刺激GFP-LC3B-RAW264.7细胞后GFP-LC3B的表达与定位。结果膨胀的烟曲霉孢子及菌丝刺激巨噬细胞后LC3BⅡ表达水平升高;Atg5与Atg12 mRNA的转录均明显增高,Atg7转录水平无显著变化;膨胀孢子诱导LC3B呈斑点样聚集并与之共定位,烟曲霉菌丝刺激后自噬体增多。结论烟曲霉膨胀孢子与菌丝能显著提高巨噬细胞自噬水平,静息的分生孢子不能引起巨噬细胞自噬功能的应答。

Objective To investigate the effect of resting spores, swollen spores and hyphae of Aspergillus fumigatus on the level of macrophage autophagy. Methods Resting spores of Aspergillus fumigatus were obtained from Sabouraud agar medium and swollen spores and hyphae of Aspergillus fumigatus were reaped by shaking Sabouraud liquid medium for different time period. RAW264.7 cells were treated with three different shapes of Aspergillus fumigatus in the three groups. Then the expression of LC3B II protein in RAW264.7 cells were detected by western blot assay. The levels of mRNA transcription of AtgS, Atg7 and Atgl2 were evaluated by RT-PCR. The expression and positioning of GFP-LC3B were observed after using the three different shapes of Aspergillusfumigatus to stimulate GFP-LC3B-RAW264.7 cells. Results After stimulated with swollen spores and hyphae of Aspergillusfumigatus, RAW264.7 cells showed significant increases of LC-3B II amount and transcriptional levels of Atg5 and Atgl2, but the transcriptional level of Atg7 had no significant difference. Interestingly, the number of autophagosomes was increased, which was demonstrated by the co-localization of GFP-LC3 and swollen spores on autophago-somes. Conclusion Swollen spores and hyphae of Aspergillus fumigatus can obviously advance the levels of auto- phagy of macrophages; resting spores can not cause the autophagic response of macrophages.