烟曲霉孢子诱导M1型巨噬细胞极化的机制研究

The mechanism of M1 macrophages polarization induced by Aspergillus fumigatus spores

目的探讨烟曲霉孢子诱导M1型巨噬细胞极化的分子机制。方法分离外周血单核细胞,体外经PMA刺激后诱导成M0巨噬细胞,经烟曲霉活化孢子刺激培养2 d后检测巨噬细胞表型和特征基因表达,确定其极化类型。流式细胞术(Flow Cytometry,FCM)、实时定量反转录聚合酶链式反应(Real-Time Quantitative Reverse Transcription,qRT-PCR)确定巨噬细胞极化状态,免疫共沉淀(Western-Blotting)法检测Dectin-1/2分子及其下游蛋白在巨噬细胞表达的差异。结果烟曲霉孢子被激活后,可以诱导巨噬细胞向M1型极化,同时上调巨噬细胞表面Dectin-1/2分子的表达,继而激活Syk/CARD9/NF-κB炎症信号通路;同时Syk/STAT1信号通路被激活后进一步促进M1型巨噬细胞极化。结论Dectin/Syk/CARD9信号通路在烟曲霉孢子诱导巨噬细胞M1极化中发挥重要的作用,可为烟曲霉相关疾病的诊断和干预提供重要参考和理论依据。

Objective To explore the molecular mechanism of M1macrophage polarization induced by Aspergillus fumigatus spores.Methods Separate and purify monocytes from peripheral blood and induce them into M0macrophages by PMA in vitro.Detect the phenotype and characteristic gene expression of macrophages to determine their polarization type after being cultured with Aspergillus fumigatus swollen spores for 2days.Flow cytometry(FCM)and realtime quantitative reverse transcription polymerase chain reaction(qRT PCR)were used to determine the polarization status of macrophages,WB experiment was used to detect the expression of Dectin-1/Dectin-2molecules and downstream proteins in macrophages stimulated by Aspergillus fumigatus swollen spores.Results The swollen spores of Aspergillus fumigatus induce M1macrophages polarization.Aspergillus fumigatus swollen spores can significantly increase the gene and protein expression levels of Dectin-1and Dectin-2on the surface of macrophages,and cause phosphorylation of Syk,which in turn induces downstream CARD9expression and activates NF-κB signaling pathway;Meanwhile,Syk phosphorylation activates the STAT1signaling pathway,further promote M1macrophages polarization and inflammatory cytokines secretion.Conclusion The Dectin/Syk/CARD9signaling pathway plays an important role in the induction of M1polarization of macrophages by Aspergillus fumigatus spores,providing important reference and theoretical basis for the diagnosis and intervention of Aspergillus fumigatus related diseases.