MHC-Ⅱ参与的TLR2信号通路在肺炎支原体感染山羊肺泡巨噬细胞中的分子机制研究

Caprine mycoplasma pneumonia is a contagious disease caused by mycoplasma mycoides subsp. capri. Both of TLR2 and MHC-Ⅱinvolved in the process of mycoplasma infection. It has been demonstrated that MHC class II molecules promote TLR-triggered innate immune responses to the microbial invasion. However, the mechanism of this phenomenon has not been well studied in caprine mycoplasma pneumonia. The present study, therefore, will be carried out to study the molecular mechanism of MHC involved in the TLR2 signaling pathways function in the process of alveolar macrophage responses to the pneumonia mycoplasma. According to the respective gene character of MHC-Ⅱand TLR2,RNAi method and CRISPR/Cas9 system will be used to knock-down MHC-Ⅱ and to knock-out TLR2,respectively, in the caprine alveolar macrophage. Real-time PCR will be used to detect the expression of MHC-Ⅱ，TLR2 and genes located in the TLR2 pathways, in the two groups. In addition, after infected by pneumonia mycoplasma, the adhesion characteristics, and the cell apoptosis in the two groups will be detected by indirect immunofluorescence method and flow cytometry, respectively. Real-time PCR will be applied to analyze the expression of genes located in the TLR2 signaling pathway, in the two group infection by pneumonia mycoplasma. Taking together, we will reveal the molecular mechanism that, MHC involved in the TLR2 signaling pathways function in the process of alveolar macrophage responses to the pneumonia mycoplasma, with the hope to provide the basis for breeding disease-resistance goat.

山羊支原体肺炎是由肺炎支原体引发的一种高度接触性传染病。研究表明，MHC-Ⅱ分子和TLR2均与支原体感染有关，近年来，研究发现，MHC-Ⅱ类分子参与的TLR2信号通路能够增强机体抵抗外源微生物侵入。而这种机制在山羊支原体肺炎上的研究尚未见报道。本研究拟通过分离培养山羊肺泡巨噬细胞，依据MHC-Ⅱ和TLR2各自的基因结构特性，分别采用RNA干扰和CRISPR/Cas9技术获得MHC-Ⅱ基因表达抑制细胞和TLR2基因敲除细胞，采用Real-time PCR检测MHC-Ⅱ、TLR2及其通路相关基因在组间的差异表达；同时采用流式细胞术、ELISA、 Real-time PCR和间接免疫荧光技术研究在支原体感染下各组细胞生长速度、凋亡率及相关细胞因子的变化，检测各组中支原体黏附能力。初步阐明MHC-Ⅱ参与的TLR2信号调控在巨噬细胞应答山羊肺炎支原体感染的机制，为应用于山羊分子抗病育种打下基础。

山羊支原体肺炎是由肺炎支原体引发的一种高度接触性传染病。研究表明，MHC-II分子和TLR2 均与支原体感染有关，近年来，研究发现，MHC-II类分子参与的TLR2信号通路能够增强机体抵抗外源微生物侵入。而这种机制在山羊支原体肺炎上的研究尚未见报道。 .项目的工作内容主要是解析了 MHC-II类分子参与的 TLR2 信号通路相关基因在肺炎支原体感染山羊肺泡巨噬细胞后的分子机制。具体包括： CRISPR/Cas9基因敲除系统TLR构建, MHC II类分子和TLR2的RNAi载体，山羊肺泡巨噬细胞、单核／巨噬细胞分离培养，山羊肺炎支原体的分离鉴定以及对巨噬细胞的感染，同时采用荧光定量PCR方法对TLR2及其信号通路基因在支原体MALP-2感染山羊巨噬细胞后基因表达检测。.项目的研究结论包括：1）发现MHC受抑制后，TLR2信号通路有关基因表达下降，初步揭示并阐明MHC II类分子参与巨噬细胞TLR2信号通路的免疫调控表达。2）发现TLR2和MHC在支原体感染巨噬细胞中发挥重要的作用；3）进一步发现在TLR2基因表达被抑制后,其下游基因myD88, NF-kB基因表达均出现下调,而C IITA, MHC-DRA/DRB, TLR4基因表达显著上升，推测MHC在抵抗支原体感染过程中发挥重要作用。综上，结合细胞凋亡和从分子基因手段检测发现MHC II类分子和TLR2在巨噬细胞体外培养体系应答山羊肺炎支原体感染过程中发挥重要的作用。.项目原计划共发表学术论文 3-4 篇,其中 SCI 收录论文2篇。实际已发表学术论文6篇，其中SCI期刊收录论文4篇；以本项目为依托，申请人又获得了安徽省农业科学院学科建设项目1项；有2项国家发明专利正在实审阶段，且依托本项目的实施，项目申请人获得了单位公派赴美UCLA进修的机会；申请人及团队成员共3人职称分别得到了晋升晋级。