本文選題：豬血凝性腦脊髓炎病毒 + 自噬　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：豬血凝性腦脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV)屬于β冠狀病毒屬的成員,是造成臨床上感染病豬出現(xiàn)典型腦脊髓炎癥狀的主要病原體之一。PHEV主要危害仔豬,尤其是哺乳期仔豬感染后,死亡率可達(dá)20~100%。PHEV感染機(jī)體后主要導(dǎo)致神經(jīng)損傷。在腦組織中,神經(jīng)細(xì)胞是該病毒復(fù)制的場(chǎng)所,未見(jiàn)其感染膠質(zhì)細(xì)胞的證據(jù)。通常情況下,病毒對(duì)細(xì)胞的入侵會(huì)激發(fā)細(xì)胞的免疫應(yīng)激反應(yīng),旨在清除外源入侵的病原體以維持細(xì)胞穩(wěn)態(tài),但病毒也可利用細(xì)胞內(nèi)的一些成分為其復(fù)制提供原材料,進(jìn)而造成機(jī)體的損傷。細(xì)胞自噬是病原體侵害細(xì)胞過(guò)程中發(fā)揮上述作用的一個(gè)重要過(guò)程�，F(xiàn)已證實(shí),細(xì)胞自噬與腫瘤、神經(jīng)退行性疾病、代謝相關(guān)疾病、免疫性疾病等發(fā)病過(guò)程密切相關(guān),而且在多種病毒、細(xì)菌等病原體感染細(xì)胞過(guò)程中均有細(xì)胞自噬誘導(dǎo)或抑制現(xiàn)象的發(fā)生。如前所述,PHEV在體內(nèi)主要感染神經(jīng)細(xì)胞,其感染過(guò)程是否能誘導(dǎo)神經(jīng)細(xì)胞的自噬現(xiàn)象,目前尚未見(jiàn)報(bào)道。因此,為了明確PHEV是否能夠誘導(dǎo)神經(jīng)細(xì)胞發(fā)生自噬,我們通過(guò)一系列自噬的相關(guān)實(shí)驗(yàn)對(duì)該過(guò)程進(jìn)行研究。將PHEV感染小鼠神經(jīng)瘤母細(xì)胞(N2a細(xì)胞),通過(guò)透射電鏡觀察發(fā)現(xiàn),在胞漿內(nèi)有典型的雙層或單層膜結(jié)構(gòu)的自噬體生成。此外,利用間接/直接免疫熒光以及Western blot實(shí)驗(yàn),發(fā)現(xiàn)PHEV能夠促使GFP-LC3在N2a細(xì)胞內(nèi)形成熒光聚點(diǎn),并促進(jìn)LC3蛋白的型別轉(zhuǎn)化,說(shuō)明PHEV感染能夠誘導(dǎo)神經(jīng)細(xì)胞自噬的發(fā)生。然而,在PHEV感染的過(guò)程中,自噬底物蛋白p62蛋白并沒(méi)有發(fā)生降解,同時(shí)利用氯喹(CQ)處理后,PHEV的感染也顯著下調(diào)。隨后,針對(duì)自噬溶酶體降解的過(guò)程我們進(jìn)行了進(jìn)一步的研究,在串聯(lián)標(biāo)記的熒光報(bào)告基因mRFP-GFP-LC3(ptfLC3)轉(zhuǎn)染N2a細(xì)胞,以及利用酸性晚期內(nèi)體和溶酶體標(biāo)記物L(fēng)ysoTracker標(biāo)記N2a細(xì)胞中,我們發(fā)現(xiàn)PHEV感染組中,自噬體并沒(méi)有與溶酶體融合,ptfLC3熒光報(bào)告質(zhì)粒顯示未淬滅,并且GFP-LC3與LysoTracker也未見(jiàn)共定位的現(xiàn)象。同時(shí),針對(duì)溶酶體相關(guān)膜蛋白1(LAMP1)的表達(dá)情況進(jìn)行分析表明,PHEV感染導(dǎo)致LAMP1發(fā)生了異常積累,并隨感染時(shí)間的延長(zhǎng)而加劇,進(jìn)一步說(shuō)明PHEV抑制了自噬體與溶酶體的融合。根據(jù)上述研究結(jié)果,說(shuō)明PHEV能夠誘導(dǎo)自噬并引起一種不完全的自噬效應(yīng)。為了進(jìn)一步明確PHEV誘導(dǎo)神經(jīng)細(xì)胞自噬的機(jī)制,我們利用酵母雙雜交系統(tǒng)篩選PHEV在誘導(dǎo)自噬過(guò)程中的互作蛋白。經(jīng)過(guò)初步篩選以及測(cè)序鑒定,得到14個(gè)候選的互作基因,對(duì)結(jié)果進(jìn)行分析,確定Snapin蛋白可能參與PHEV誘導(dǎo)自噬的過(guò)程。Snapin蛋白是一種新型的小分子蛋白,其不僅在囊泡運(yùn)輸機(jī)制中發(fā)揮轉(zhuǎn)運(yùn)功能,并且在晚期內(nèi)體的降解以及自噬溶酶體形成過(guò)程中發(fā)揮重要作用。隨后,我們首先利用酵母雙雜交回轉(zhuǎn)實(shí)驗(yàn)、免疫共沉淀以及直接/間接免疫熒光等方法,驗(yàn)證了二者之間的相互作用。在細(xì)胞中過(guò)表達(dá)Snapin蛋白后感染PHEV,病毒的復(fù)制并沒(méi)有發(fā)生明顯的改變,而利用siRNA干擾細(xì)胞內(nèi)源性Snapin蛋白的表達(dá)后感染PHEV,卻發(fā)現(xiàn)病毒的復(fù)制量受到明顯抑制。同時(shí),對(duì)LAMP1進(jìn)行檢測(cè)發(fā)現(xiàn),自噬溶酶體的積累與對(duì)照組相比明顯增多。該結(jié)果進(jìn)一步證實(shí)了Snapin蛋白參與了PHEV感染的過(guò)程,并很有可能與PHEV誘導(dǎo)不完全自噬效應(yīng)有關(guān)。上述結(jié)果不僅為PHEV致神經(jīng)損傷的機(jī)制研究奠定了基礎(chǔ),而且將為其能夠作為神經(jīng)退行性疾病模型提供重要的理論依據(jù)。
[Abstract]:Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus beta coronavirus, which is one of the main pathogens causing the symptoms of typical encephalomyelitis in clinical infected pigs,.PHEV is the main harm to piglets, especially after suckling piglets infection, the mortality rate can reach 20~100%.PHEV infection body. In the brain tissue, nerve cells are the sites for the replication of the virus, and there is no evidence for the infection of the glial cells. Usually, the invasion of the virus can stimulate the immune response of the cells to remove the alien invasive pathogens to maintain the cell homeostasis, but the virus can also make use of some of the cells in the cells. Cell autophagy is an important process in the process of pathogen invasion. Autophagy has been confirmed that cell autophagy is closely related to cancer, neurodegenerative diseases, metabolic related diseases, immune diseases, and many viruses, bacteria and other diseases. In the process of infection, there are autophagy induced or inhibited by cells. As mentioned earlier, PHEV is mainly infected with nerve cells in the body. It is not reported that the infection process can induce the autophagy of the nerve cells. Therefore, in order to determine whether PHEV can induce autophagy in neural cells, we have passed a series of autophagy. PHEV infected mice neuroma cells (N2a cells) were detected by transmission electron microscopy, and there was a typical bilayer or monolayer autophagic formation in the cytoplasm. Furthermore, using indirect / direct immunofluorescence and Western blot experiments, it was found that PHEV could induce GFP-LC3 in N2a cells. The formation of fluorescent spots and the transformation of the type of LC3 protein showed that PHEV infection could induce autophagy in neural cells. However, the autophagic protein p62 protein did not degrade in the process of PHEV infection, and the infection of PHEV was significantly reduced with chloroquine (CQ) treatment. Subsequently, the process of degradation of autophagic lysosomes We further studied the transfection of N2a cells in tandem labeled mRFP-GFP-LC3 (ptfLC3), and in N2a cells labeled with acid late endosome and lysosomal marker LysoTracker, we found that the autophagosome was not fused with the lysosome in the PHEV infection group, and the ptfLC3 fluorescent report plasmid showed no quenching and GFP. There was no co localization between -LC3 and LysoTracker. At the same time, the analysis of the expression of lysosome related membrane protein 1 (LAMP1) showed that PHEV infection resulted in abnormal accumulation of LAMP1 and increased with the prolongation of the infection time. It further indicated that PHEV inhibited the fusion of autophagosomes and lysosomes. According to the results of these studies, PHEV It can induce autophagy and cause an incomplete autophagy effect. In order to further clarify the mechanism of PHEV induction of autophagy, we use yeast two hybrid system to screen the interaction protein of PHEV during the induction of autophagy. After preliminary screening and sequencing identification, 14 candidate genes were obtained, and the results were analyzed and S was determined. Napin protein may be involved in the process of PHEV induced autophagy,.Snapin protein is a new small molecular protein, which not only plays the transport function in the vesicle transport mechanism, but also plays an important role in the degradation of late endosomes and the formation of autophagosomes. Then, we first used yeast two hybrid rotation experiment and immunization. The interaction between the two was verified by the methods of amylin and direct / indirect immunofluorescence. After overexpressing Snapin protein in the cells, PHEV was infected, and the replication of the virus did not change obviously, while siRNA interfered with the expression of endogenous Snapin protein to infect PHEV, but it was found that the replication of the virus was significantly inhibited. The detection of LAMP1 showed that the accumulation of autophagic lysosomes increased significantly compared with those of the control group. The results further confirmed that Snapin protein was involved in the process of PHEV infection and was likely to be related to the induction of incomplete autophagy by PHEV. These results not only laid the foundation for the mechanism of PHEV induced nerve damage, but also could be used for it. It provides important theoretical basis for neurodegenerative disease models.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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