绵羊肺炎支原体荚膜多糖对支气管上皮细胞炎性反应与氧化损伤的作用机制研究

发布时间：2018-03-23 06:09

本文选题：绵羊肺炎支原体　切入点：荚膜多糖　出处：《宁夏大学》2017年博士论文　论文类型：学位论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneuooniae,MO)是从绵羊或山羊支气管中分离得到的一种最常见的病原菌,主要引起羊支原体肺炎。患病羊临床上以渐进性消瘦、高热、气喘、持续性咳嗽、肺和胸膜发生浆液性和纤维素性炎症为特征。而与其他支原体相比,目前对于绵羊肺炎支原体致病机制的研究还非常有限,但普遍认为准确的黏附作用应当是决定其是否能够成功感染的关键。绵羊肺炎支原体无细胞壁结构,感染宿主机体时主要通过膜外的荚膜样物质介导菌体与宿主细胞间的相互作用。已有报道证实荚膜多糖(capsularpolysaccharides,CPS)是位于绵羊肺炎支原体菌体最外层的一种重要毒力因子,其在菌体抗干燥、粘附、侵入、传播以及抵抗宿主细胞的固有免疫和特异性免疫防御机制等方面的作用正被逐渐认识。本研究在成功提取并纯化了绵羊肺炎支原体模式株Y98荚膜多糖(CPS)的基础上,制备了CPS的多克隆抗体,并利用绵羊支气管原代上皮细胞气液相(air-liquid interface,ALI)培养模型,对CPS诱发免疫学炎性反应与氧化损伤过程中的分子机制进行了探究。研究首先发现,绵羊肺炎支原体CPS刺激ALI支气管原代上皮细胞后诱导了 TLRs级联信号通路介导的免疫学炎性反应的发生。TLRs 信号中 MyD88、IRAKs、TRAF6、p-NF-κB、p-AP-1、IRF5 与 TRAF3、TBK1、IRF3等关键通路蛋白与核转录因子的表达量在CPS刺激后均呈现出明显的上调趋势,表明TLRs级联的MyD88依赖性与MyD88非依赖性信号转导通路在支气管上皮细胞响应CPS刺激的过程中均扮演重要角色。研究同时发现CPS抗体的预处理可以显著下调由绵羊肺炎支原体感染所诱导的炎性相关因子(包括促炎性细胞因子IL1β、IL6、IL8和TNFα等,抑炎性细胞因子IL10和TGFβ等)的高表达,提示CPS是绵羊肺炎支原体的一个重要毒力因子,其致病机制可能是通过诱导支气管周围大量炎症相关因子的释放,从而实现对宿主免疫学炎症反应的调控。此外,分子机制研究表明绵羊肺炎支原体CPS可以通过线粒体介导的内源性途径与死亡受体介导的外源性途径同时诱导支气管上皮细胞的凋亡,JNK/P38MAPK信号通路在此过程中起重要的促凋亡作用。值得注意的是,CPS诱发宿主细胞凋亡的过程伴随ROS的高表达,ROS清除剂NAC的预处理可以明显抑制这一现象的发生,因此提示ROS的过量产生可能是诱导绵羊支气管原代上皮细胞凋亡的直接因素之一。以上的研究结果揭示,TLRs信号介导的炎性反应与ROS介导的细胞凋亡在支气管上皮细胞应答绵羊肺炎支原体荚膜多糖刺激的免疫调控机制中发挥了关键性的作用。这些发现为进一步研究绵羊肺炎支原体的主要毒力因子及其致病机理、阐明宿主机体的免疫应答调控机制提供了新的思路。
[Abstract]:Mycoplasma pneumoniae (Mycoplasma ovipneuooniae MO) is one of the most common pathogens isolated from sheep or goats in bronchial, mainly caused by mycoplasma pneumonia. The prevalence of sheep sheep clinically with progressive weight loss, fever, persistent cough, asthma, lung and pleura had serous and fibrinous inflammation is characterized. Compared with other current research on Mycoplasma pneumoniae, Mycoplasma pneumoniae pathogenesis is still very limited, but it is generally believed that adhesion should be accurate to determine whether the key to success. No infection of Mycoplasma pneumoniae infection in the host cell wall structure, when the capsule like substances mainly through the membrane dielectric guide interaction with host cell between the cells. It has been reported that the capsular polysaccharide (capsularpolysaccharides, CPS) is an important virulence factor in Mycoplasma pneumoniae bacteria in the outer. Bacterial resistance to dry, adhesion, invasion, spread and resistance of host cell innate and specific immune defence mechanism function is being increasingly recognized. This study successfully extracted and Mycoplasma pneumoniae strain Y98 purified capsular polysaccharide (CPS) on the basis of the polyclonal antibody against CPS was prepared. And the use of primary bronchial epithelial cells (air-liquid, interface, ALI phase) culture model and molecular mechanism of CPS induced immunological inflammatory reaction and oxidative damage in the process were studied. Firstly, MyD88,.TLRs signal in mycoplasma pneumonia of sheep CPS immunological inflammatory stimuli ALI primary bronchial epithelial cells after the induction of TLRs cascade signaling mediated by IRAKs, TRAF6, p-NF- and IRF5 K B, p-AP-1, TRAF3, TBK1, IRF3 protein expression in the key pathway of nuclear transcription factor were found after CPS stimulation The upward trend is obvious, that plays an important role in TLRs cascade MyD88 dependent and MyD88 dependent signal transduction pathway in bronchial epithelial cells in response to CPS stimulation in the process. The study also found that CPS antibody pretreatment can significantly down regulate inflammatory factors induced by Mycoplasma pneumoniae infection (including pro-inflammatory cytokine IL1 beta, IL6, IL8 and TNF etc, suppression of inflammatory cytokines IL10 and TGF P) high expression, suggesting that CPS is an important virulence factor of Mycoplasma pneumoniae and its pathogenic mechanism may be through the relevant factor induced bronchial inflammation around massive release, so as to realize the control of the host the immunological inflammatory reaction. In addition, the molecular mechanism study indicated that exogenous endogenous pathway pathway Mycoplasma pneumoniae CPS can be mediated by mitochondria and death receptor mediated and induced by gas The apoptosis of tubular epithelial cells, JNK/P38MAPK signaling pathway in this process apoptosis is important. It is worth noting that the process of CPS induced apoptosis of host cells with high expression of ROS, ROS scavenger NAC pretreatment can significantly inhibit the occurrence of this phenomenon, therefore excessive production of ROS may be one of the direct factors of the original generation of sheep bronchial epithelial cell apoptosis. These results revealed that the apoptosis of inflammatory reaction and ROS mediated TLRs signaling mediated by play a key role in the regulation of immune response mechanism of bronchial epithelial cells of Mycoplasma pneumoniae capsular polysaccharide stimulation. These findings as the main virulence factors and its pathogenic mechanism further Mycoplasma ovipneumoniae, provides a new way to regulate the immune response mechanism of the host organism.

【学位授予单位】：宁夏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.62

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