自噬在绵羊肺炎支原体感染巨噬细胞中的作用及调控机理研究

Contagious ovine pleuropneumoniae, caused by Mycoplasma ovipneumoniae, is the highly contagious disease. The study on the pathogenic mechanisms of M.ovipneumoniae is lagging behind, especially the relationship between M.ovipneumoniae with the host cell autophagy has not been reported.In the project, the murine macrophage cell line and primary mouse macrophages will be infected with M.ovipneumoniae Y98(the type strain)respectively in vitro.Then,using laser scanning confocal microscope to observe the formation of autophagysome, and western blot to detect autophagy-related proteins, we can know the formation mechanism and funtion of autophagy during M.ovipneumoniae infection;the influence of macrophage autophagy on killing mycoplasma ovipneumoniae and the effect of anti-mycoplasma infection immune factor IFN-γ on macrophage autophagy will be studied further;Using Y98 infecting autophage-inhibitted mice, we will study the impact of autophagy on immune response by M.ovipneumoniae in vivo.In the project, we will explore the role of autophagy in mycoplasma ovipneumoniae infection in macrophages and its regulation mechanism and in immune response elicited by M.ovipneumoniae infection preliminary in vivo. The results will be beneficial to elucidate the pathogenesis of M.ovipneumoniae and the prevention and treatment against contagious ovine pleuropneumoniae.

绵羊传染性胸膜肺炎，是由绵羊肺炎支原体引起的高度接触性传染病。有关绵羊肺炎支原体致病机制的研究相对滞后，尤其是其与宿主细胞自噬之间的关系目前尚未见报道。本项目拟在体外条件下，以绵羊肺炎支原体标准株Y98株分别感染鼠巨噬细胞系及鼠原代巨噬细胞，利用激光共聚焦扫描显微镜观察自噬体的形成、western blot检测自噬相关蛋白，探讨自噬在支原体感染过程中的形成机制；进一步检测巨噬细胞自噬对绵羊肺炎支原体的清除的影响；同时检测抗支原体感染免疫因子IFN-γ对巨噬细胞自噬的影响；并以支原体感染自噬抑制小鼠，检测体内条件下，自噬对该菌清除的影响。通过本项课题的开展，对自噬在绵羊肺炎支原体感染巨噬细胞中的作用及调控机制进行初步研究，并探讨体内条件下，自噬对支原体感染引发的免疫应答的影响。研究结果将有益于阐明绵羊肺炎支原体的致病机制及针对绵羊传染性胸膜肺炎的预防与治疗。

绵羊传染性胸膜肺炎，是由绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)引起的高度接触性传染病。自噬在抗感染免疫过程中发挥着重要的功能，但其在MO感染宿主巨噬细胞及肺脏上皮细胞中的作用机制尚无报道，本课题就此问题进行了研究。实验中以MO Y98株感染巨噬细胞Raw264.7及肺脏上皮细胞TC-1，利用免疫电镜观察发现了自噬体及自噬溶酶体结构，初步证明了MO能够诱导Raw264.7及TC-1发生自噬；通过GFP-mRFP-LC3串联荧光腺病毒，检测到了从自噬体到自噬溶酶体的完整自噬流；qPCR及Western blot检测到自噬相关因子p62、Atg5、Atg7、LC3量等随感染时间的延长其表达均显著升高。以上结果表明MO 感染可以诱导Raw264.7及TC-1发生自噬。利用RNAi沉默p62、Atg7作用于Raw264.7及TC-1，发现胞内MO 繁殖能力增强，表明Raw264.7自噬及TC-1自噬均对MO 的清除具有重要作用。进一步研究发现胞内受体NOD2通过JNK-Bcl-2/Beclin1途径参与MO诱导的RAW264.7细胞自噬，并参与细胞对MO的清除；MO 对绵羊原代巨噬细胞自噬具有抑制作用，自噬的激活亦有助于该细胞对MO 的清除，认为抑制自噬可能是MO在宿主体内生存并造成持续感染的机制之一。本研究为MO 与宿主细胞的互作机制以及胞内受体NOD2对无肽聚糖组分病原菌的识别机制提供了新的理论依据。