烟曲霉感染相关microRNAs在肺泡巨噬细胞炎症反应通路中的作用研究

The balance between pro- and anti-inflammation plays an important role in treating invasive Aspergillus fungi infection. In addition to executing its primary phagocytic function upon excountering the AP, AMs are thought to play the pivotal role in coordinating the host inflammatory response. However，the mechanism of how to avoid over-production of pro-inflammatory cytokines in AMs is still unknown. We have found previously that the expression of miR-146a、155 have increased in mouse alveoli marcrophages infected by aspergillus conidia, and the corresponding oligonucleotide restrained the expression of IL-1β、TNF-a, which indicated that the two miRNAs may involved in the regulation of signal transduction in AMs. Thus we plan to establish the aspergillus infected cell model, screening and evaluating the corresponding miRNA induced by aspergillus infection through miRNA assay, northern blotting and qPCR, predicting the target gene, assaying the expression of miRNA,IL-1β and TNF-a after blocking the NF-κB signal pathway, analyzing the negative regulation of the target miRNA through over-expression and inhibition of the corresponding miRNA.We try to investigate the mechanism of how the target miRNA regulate the signal transduction,then clarifying further the mechanism molecularly in aspergillus infection.

平衡促炎/抗炎反应以避免机体二次损伤是侵袭性曲霉感染治疗成功的重要因素。肺泡巨噬细胞（AMs）在此过程中起免疫调节作用。但如何通过负反馈通路调控信号转导，抑制AMs产生过多促炎因子的具体机制尚不清楚。我们前期研究发现烟曲霉（AP）感染的小鼠AMs中miR-146a、155表达上调，并伴有IL-1β、TNF-a升高，提示这两种miRNA可能参与对AMs胞内信号转导通路的调控。本项目拟建立AP感染小鼠AMs模型，通过miRNA芯片、免疫印迹和qPCR技术，筛选并鉴定烟曲霉感染诱导的miRNA；通过生物信息学预测和报告载体实验鉴定其靶基因；分别阻断NF-κB通路蛋白后测定miRNA及IL-1β、TNF-a表达；并通过miRNA过表达和抑制实验分析其负性调控功能。探讨AP感染、miRNA变化对信号转导通路的作用机制，对进一步阐明曲霉感染的分子致病机制具有重要意义。

背景：平衡促炎/抗炎反应以避免机体二次损伤是侵袭性曲霉感染治疗成功的重要因素。肺泡巨噬细胞能激活包括MAPK及NF-κB在内的多种信号转导途径，介导促炎因子平衡促炎/抗炎反应以避免机体二次损伤是侵袭性曲霉感染治疗成功的重要因素。肺泡巨噬细胞能激活包括MAPK及NF-κB在内的多种信号转导途径，介导促炎因子的上调/下调而清除病原菌。但如何通过miRNA调控信号转导的具体机制尚不清楚。.目的：建立烟曲霉感染小鼠肺泡巨噬细胞模型，通过miRNA芯片、免疫印迹和定量PCR技术，筛选并鉴定烟曲霉感染诱导的miRNA；通过生物信息学预测和报告载体实验鉴定其靶基因；使用拮抗剂分别阻断P38MAPK、ERK、JUK及NF-κB亚基IKKB后测定miRNA及IL-1β、TNF-a表达；并通过miRNA过表达和抑制实验分析其负性调控功能。.结果：在烟曲霉孢子及LPS刺激后，有21种miRAN有差异表达。其中2种miRNA（miR-148a-5p 、miR-155）表达上调超过2倍或以上，2种miRNA（miR-146a-5p、miR-192-5p）下调超过2倍或以上。对信号通路的研究发现NF-κB参与4种miRNA的转录，而P38MAPK通路还参与miR-148a-5p的转录。IL-1β、TNF-α能诱导miR-148a-5p 、miR-155表达而抑制miR-146a-5p、miR-192-5p表达。.结论：miR-148a-5p 、miR-155、miR-146a-5p、miR-192-5p在肺泡巨噬细胞对烟曲霉刺激的免疫反应中起重要的作用。