本文選題：綿羊肺炎支原體　切入點：巨噬細胞　出處：《寧夏大學》2017年碩士論文　論文類型：學位論文

【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae,MO)引起的綿羊傳染性胸膜肺炎(Contagious ovine pleuropneumonia)是威脅養(yǎng)羊業(yè)的重要疾病之一。該疾病遍布我國多個地區(qū),尤其在西部畜牧業(yè)發(fā)達的地區(qū),造成了巨大的經(jīng)濟損失。自噬(autophagy)是真核細胞對抗應激、維護細胞內環(huán)境穩(wěn)定的一種保守手段,依賴溶酶體的細胞分解代謝過程,用以降解受損或衰老的蛋白質、細胞器等細胞結構。另外,根據(jù)研究報道可知自噬在機體抗感染免疫過程中也扮演著重要角色。近年來多項研究表明,MO可對宿主細胞造成極大危害,但MO在感染細胞的過程中是否介導了細胞自噬的發(fā)生,以及自噬對MO起什么樣的作用目前尚不明確。本實驗以小鼠巨噬細胞RAW264.7為研究對象,通過建立MO-Y98感染的細胞模型,從形態(tài)觀察、基因水平、蛋白水平上檢測MO感染后的RAW264.7是否可介導自噬的發(fā)生以及重要自噬相關因子的變化,并利用RNAi技術研究巨噬細胞自噬對MO清除的影響。實驗結果如下:(1)將MO用Dil染料染色后感染RAW264.7,可見MO聚集在細胞核的周圍,表明MO可被巨噬細胞吞噬。(2)免疫熒光結果顯示,MO感染RAW264.7 12h后,可見LC3、Atg16L綠色熒光斑點聚集在細胞核周圍,觀察到自噬體。GFP-mRFP-LC3串聯(lián)熒光蛋白腺病毒監(jiān)測MO感染的RAW264.7自噬流的形成情況,發(fā)現(xiàn)感染6h自噬體出現(xiàn),12h、24h自噬溶酶體出現(xiàn),12h時自噬體數(shù)量最多,表明MO誘導RAW264.7后可形成完整自噬流,且在12h時自噬流最為強烈。(3)免疫電鏡結果顯示,MO感染RAW264.7 12h時,可觀察到具有雙層膜結構的自噬體和單層膜結構的自噬溶酶體。(4)qPCR和Western blot結果顯示,MO感染RAW264.7 6h、12h、24h后,自噬相關因子LC3、p62、Atg5、Atg7在轉錄水平和蛋白水平明顯升高,與同期對照相比,差異性顯著。(5)將自噬抑制劑巴弗洛霉素Al加入感染MO的RAW264.7,CCU檢測發(fā)現(xiàn)在作用18h時,實驗組CCU值顯著高于同期對照組。結果顯示,MO誘導RAW264.7自噬,并參與巨噬細胞對MO的清除。(6)利用RNAi技術沉默RAW264.7自噬關鍵因子p62和Atg7,感染MO后,進行CCU檢測,發(fā)現(xiàn)RAW264.7中MO數(shù)量增加,表明巨噬細胞自噬被抑制后,降低了對MO的清除能力。綜上所述,MO可被RAW264.7吞噬;感染MO后,RAW264.7胞內形成自噬體和自噬溶酶體結構,并且自噬相關調控因子表達上調,表明MO可誘導RAW264.7發(fā)生自噬;發(fā)現(xiàn)RAW264.7自噬下調后對MO清除能力下降,表明巨噬細胞自噬對MO的清除具有重要作用。本實驗證明了MO感染可誘導巨噬細胞發(fā)生自噬,且初步研究了自噬在MO誘導的巨噬細胞中的作用機制,為MO與巨噬細胞之間的相互作用提供了理論基礎,為進一步揭示MO的致病機制提供新的思路。
[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.
【學位授予單位】：寧夏大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.26

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