自噬在巨噬细胞抗绵羊肺炎支原体感染中作用的研究

发布时间：2018-03-23 02:26

本文选题：绵羊肺炎支原体　切入点：巨噬细胞　出处：《宁夏大学》2017年硕士论文　论文类型：学位论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)引起的绵羊传染性胸膜肺炎(Contagious ovine pleuropneumonia)是威胁养羊业的重要疾病之一。该疾病遍布我国多个地区,尤其在西部畜牧业发达的地区,造成了巨大的经济损失。自噬(autophagy)是真核细胞对抗应激、维护细胞内环境稳定的一种保守手段,依赖溶酶体的细胞分解代谢过程,用以降解受损或衰老的蛋白质、细胞器等细胞结构。另外,根据研究报道可知自噬在机体抗感染免疫过程中也扮演着重要角色。近年来多项研究表明,MO可对宿主细胞造成极大危害,但MO在感染细胞的过程中是否介导了细胞自噬的发生,以及自噬对MO起什么样的作用目前尚不明确。本实验以小鼠巨噬细胞RAW264.7为研究对象,通过建立MO-Y98感染的细胞模型,从形态观察、基因水平、蛋白水平上检测MO感染后的RAW264.7是否可介导自噬的发生以及重要自噬相关因子的变化,并利用RNAi技术研究巨噬细胞自噬对MO清除的影响。实验结果如下:(1)将MO用Dil染料染色后感染RAW264.7,可见MO聚集在细胞核的周围,表明MO可被巨噬细胞吞噬。(2)免疫荧光结果显示,MO感染RAW264.7 12h后,可见LC3、Atg16L绿色荧光斑点聚集在细胞核周围,观察到自噬体。GFP-mRFP-LC3串联荧光蛋白腺病毒监测MO感染的RAW264.7自噬流的形成情况,发现感染6h自噬体出现,12h、24h自噬溶酶体出现,12h时自噬体数量最多,表明MO诱导RAW264.7后可形成完整自噬流,且在12h时自噬流最为强烈。(3)免疫电镜结果显示,MO感染RAW264.7 12h时,可观察到具有双层膜结构的自噬体和单层膜结构的自噬溶酶体。(4)qPCR和Western blot结果显示,MO感染RAW264.7 6h、12h、24h后,自噬相关因子LC3、p62、Atg5、Atg7在转录水平和蛋白水平明显升高,与同期对照相比,差异性显著。(5)将自噬抑制剂巴弗洛霉素Al加入感染MO的RAW264.7,CCU检测发现在作用18h时,实验组CCU值显著高于同期对照组。结果显示,MO诱导RAW264.7自噬,并参与巨噬细胞对MO的清除。(6)利用RNAi技术沉默RAW264.7自噬关键因子p62和Atg7,感染MO后,进行CCU检测,发现RAW264.7中MO数量增加,表明巨噬细胞自噬被抑制后,降低了对MO的清除能力。综上所述,MO可被RAW264.7吞噬;感染MO后,RAW264.7胞内形成自噬体和自噬溶酶体结构,并且自噬相关调控因子表达上调,表明MO可诱导RAW264.7发生自噬;发现RAW264.7自噬下调后对MO清除能力下降,表明巨噬细胞自噬对MO的清除具有重要作用。本实验证明了MO感染可诱导巨噬细胞发生自噬,且初步研究了自噬在MO诱导的巨噬细胞中的作用机制,为MO与巨噬细胞之间的相互作用提供了理论基础,为进一步揭示MO的致病机制提供新的思路。
[Abstract]:Mycoplasma ovis pneumoniae (MOA) is one of the most important diseases that threaten the sheep industry, especially in the western regions where animal husbandry is developed. Autophagyis is a conservative way for eukaryotic cells to fight stress and maintain stability in the cell environment, relying on lysosomal cell catabolism to degrade damaged or aging proteins. In addition, autophagy also plays an important role in the process of anti-infection immunity. However, it is not clear whether MO mediates autophagy in the process of infected cells, and what role autophagy plays on MO. In this study, the murine macrophage RAW264.7 was used as the research object, and the cell model of MO-Y98 infection was established. Morphologic observation, gene level and protein level were used to detect whether RAW264.7 mediated autophagy and the changes of important autophagy related factors after MO infection. RNAi technique was used to study the effect of macrophage autophagy on the removal of MO. The results were as follows: 1) MO was stained with Dil dye and infected with RAW264.7. The results indicated that MO could be phagocytized by macrophages. The immunofluorescence results showed that LC3Ag16L green fluorescent spots gathered around the nucleus 12 h after RAW264.7 infection. The autophagy. GFP-mRFP-LC3 tandem fluorescent protein adenovirus was observed to monitor the formation of RAW264.7 autophagy in MO infection. It was found that the number of autophagy was the highest at 12h and 24h after infection, indicating that MO-induced RAW264.7 could form complete autophagy, and at 12h, the most intense autophagy was observed. The results of immunomicroscopy showed that MO-infected RAW264.7 at 12h. It was observed that autophagy with double-layer membrane structure and autophagy lysosome with monolayer structure. The results of QPCR and Western blot showed that the transcription level and protein level of autophagy related factor LC3p62Ag5Ag7 were significantly higher than those of control group. The results showed that the CCU value of the experimental group was significantly higher than that of the control group after 18 hours of exposure. The results showed that the RAW264.7 autophagy induced by Vavromycin Al was significantly higher than that of the control group. RNAi technique was used to silence the key factors of RAW264.7 autophagy p62 and Atg7. After infection with MO, the CCU was detected. The results showed that the number of MO in RAW264.7 was increased, indicating that macrophage autophagy was inhibited. In conclusion, MO can be swallowed by RAW264.7, autophagy and lysosomal structure are formed in RAW264.7 cells after infection with MO, and the expression of autophagy related regulatory factors is up-regulated, which indicates that MO can induce autophagy in RAW264.7. It was found that the ability of RAW264.7 autophagy to clear MO was decreased after down-regulation, which indicated that macrophage autophagy played an important role in the clearance of MO. This study demonstrated that MO infection could induce macrophage autophagy. The mechanism of autophagy in MO-induced macrophages was studied, which provided a theoretical basis for the interaction between MO and macrophages, and provided a new idea for further revealing the pathogenesis of MO.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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