Snapin在猪血凝性脑脊髓炎病毒诱导神经细胞自噬中的功能研究

发布时间：2018-05-22 14:40

本文选题：猪血凝性脑脊髓炎病毒 + 自噬　；参考：《吉林大学》2017年博士论文

【摘要】：猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV)属于β冠状病毒属的成员,是造成临床上感染病猪出现典型脑脊髓炎症状的主要病原体之一。PHEV主要危害仔猪,尤其是哺乳期仔猪感染后,死亡率可达20~100%。PHEV感染机体后主要导致神经损伤。在脑组织中,神经细胞是该病毒复制的场所,未见其感染胶质细胞的证据。通常情况下,病毒对细胞的入侵会激发细胞的免疫应激反应,旨在清除外源入侵的病原体以维持细胞稳态,但病毒也可利用细胞内的一些成分为其复制提供原材料,进而造成机体的损伤。细胞自噬是病原体侵害细胞过程中发挥上述作用的一个重要过程。现已证实,细胞自噬与肿瘤、神经退行性疾病、代谢相关疾病、免疫性疾病等发病过程密切相关,而且在多种病毒、细菌等病原体感染细胞过程中均有细胞自噬诱导或抑制现象的发生。如前所述,PHEV在体内主要感染神经细胞,其感染过程是否能诱导神经细胞的自噬现象,目前尚未见报道。因此,为了明确PHEV是否能够诱导神经细胞发生自噬,我们通过一系列自噬的相关实验对该过程进行研究。将PHEV感染小鼠神经瘤母细胞(N2a细胞),通过透射电镜观察发现,在胞浆内有典型的双层或单层膜结构的自噬体生成。此外,利用间接/直接免疫荧光以及Western blot实验,发现PHEV能够促使GFP-LC3在N2a细胞内形成荧光聚点,并促进LC3蛋白的型别转化,说明PHEV感染能够诱导神经细胞自噬的发生。然而,在PHEV感染的过程中,自噬底物蛋白p62蛋白并没有发生降解,同时利用氯喹(CQ)处理后,PHEV的感染也显著下调。随后,针对自噬溶酶体降解的过程我们进行了进一步的研究,在串联标记的荧光报告基因mRFP-GFP-LC3(ptfLC3)转染N2a细胞,以及利用酸性晚期内体和溶酶体标记物LysoTracker标记N2a细胞中,我们发现PHEV感染组中,自噬体并没有与溶酶体融合,ptfLC3荧光报告质粒显示未淬灭,并且GFP-LC3与LysoTracker也未见共定位的现象。同时,针对溶酶体相关膜蛋白1(LAMP1)的表达情况进行分析表明,PHEV感染导致LAMP1发生了异常积累,并随感染时间的延长而加剧,进一步说明PHEV抑制了自噬体与溶酶体的融合。根据上述研究结果,说明PHEV能够诱导自噬并引起一种不完全的自噬效应。为了进一步明确PHEV诱导神经细胞自噬的机制,我们利用酵母双杂交系统筛选PHEV在诱导自噬过程中的互作蛋白。经过初步筛选以及测序鉴定,得到14个候选的互作基因,对结果进行分析,确定Snapin蛋白可能参与PHEV诱导自噬的过程。Snapin蛋白是一种新型的小分子蛋白,其不仅在囊泡运输机制中发挥转运功能,并且在晚期内体的降解以及自噬溶酶体形成过程中发挥重要作用。随后,我们首先利用酵母双杂交回转实验、免疫共沉淀以及直接/间接免疫荧光等方法,验证了二者之间的相互作用。在细胞中过表达Snapin蛋白后感染PHEV,病毒的复制并没有发生明显的改变,而利用siRNA干扰细胞内源性Snapin蛋白的表达后感染PHEV,却发现病毒的复制量受到明显抑制。同时,对LAMP1进行检测发现,自噬溶酶体的积累与对照组相比明显增多。该结果进一步证实了Snapin蛋白参与了PHEV感染的过程,并很有可能与PHEV诱导不完全自噬效应有关。上述结果不仅为PHEV致神经损伤的机制研究奠定了基础,而且将为其能够作为神经退行性疾病模型提供重要的理论依据。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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