猪传染性胃肠炎病毒感染诱导肠上皮细胞线粒体自噬的研究

发布时间：2018-05-02 07:16

本文选题：TGEV + IPEC-J2细胞　；参考：《南京农业大学》2016年博士论文

【摘要】：猪传染性胃肠炎(Porcine transmissible gastroenteritis, TGE)是由猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV)引起的一种高度接触性、急性消化道传染病。TGEV感染的靶细胞是小肠上皮细胞。小肠上皮作为营养吸收的主要位点细胞内含有大量的线粒体。有一些病毒专门以线粒体为调控靶点,促进自身在细胞内复制和存活;有一些病毒通过与自噬受体相互作用、引起细胞应激等方式诱导自噬,增加病毒复制;还有一些病毒通过激活线粒体自噬受体、诱导线粒体损伤等方式引起线粒体自噬,促进病毒的持续感染。目前,关于TGEV与线粒体自噬相互作用的研究还未见报道。因此,为了探究线粒体自噬在TGEV感染中的作用和意义,我们应用猪空肠上皮细胞IPEC-J2作为TGEV的感染模型,首先研究了 TGEV对线粒体损伤和线粒体自噬的影响;其次研究了线粒体自噬对TGEV复制的影响;再次研究了细胞氧化应激对TGEV诱导线粒体自噬的影响;最后通过对TGEV的结构蛋白和非结构蛋白的过表达,寻找诱导线粒体自噬的关键蛋白。具体内容如下:1、TGEV感染IPEC-J2细胞诱导线粒体损伤和自噬的研究在病毒感染过程中,某些病毒能够通过引起线粒体损伤和自噬促进自身的持续感染。首先,为了研究线粒体在TGEV感染IPEC-J2细胞过程中是否存在损伤和自噬,流式细胞术检测了 TGEV感染后细胞内的线粒体损伤程度和线粒体数量变化,并通过透射电镜观察了 TGEV感染后细胞线粒体的结构变化。结果发现,TGEV感染12 h和24 h后细胞内的线粒体膜电位明显降低(P 0.01 ),线粒体形态出现明显肿胀变形,嵴消失,并观察到明显的双层膜包裹的线粒体。其次,为了确定TGEV感染是否激活细胞自噬清除受损的线粒体,通过Western-blot和流式细胞术检测TGEV感染后自噬相关蛋白Beclin-1和LC3-Ⅱ的变化以及线粒体数量的变化。结果证明,自噬蛋白Beclin-1和LC3-Ⅱ在TGEV感染24h到48h之间含量都明显增加(P0.01),感染12 h和24h后线粒体数量又明显减少(P0.01),但在感染48 h后恢复。为了进一步证明TGEV感染诱导线粒体自噬发生,通过共聚焦显微镜观察IPEC-J2细胞内线粒体和自噬体是否存在共定位现象。试验结果表明,TGEV感染12h后可见明显的线粒体与自噬体的共定位,随着感染时间延长,共定位现象更加明显。最后为了确定TGEV引起的线粒体自噬是否完全,通过检测p62/SQSTM1蛋白变化以及线粒体与溶酶体的融合现象,证明TGEV感染24h (P0.05)和48h (P0.01)能促进p62/SQSTM1蛋白的降解,促进线粒体和溶酶体的融合,诱导产生完全的线粒体自噬。以上结果显示TGEV感染IPEC-J2细胞能引起线粒体的损伤和线粒体自噬。2、线粒体自噬对TGEV复制和细胞凋亡影响的研究线粒体自噬可以抑制细胞凋亡,促进病毒的复制;也可以降低线粒体数量,减少能量产生,抑制病毒的复制。为了确定线粒体自噬是否影响TGEV的复制,通过自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)、自噬促进剂雷帕霉素(Rapamycin)和线粒体自噬促进剂羰基氰化物间氯苯腙(carbonylcyanide-m-chlorophenylhydrazone,CCCP)处理IPEC-J2细胞改变细胞自噬水平,检测自噬抑制或激活对TGEV的滴度和N蛋白表达的影响。试验结果表明,自噬抑制剂3-MA能显著降低TGEV感染24h和48h的滴度(P0.01),并且细胞凋亡明显增加(P0.01);自噬促进剂Rapamycin和线粒体自噬促进剂CCCP都能增加TGEV感染24 h和48 h后滴度(P 0.01 ),CCCP对TGEV感染24h (P0.05)和48h(P0.01)后N蛋白的表达有显著促进作用,雷帕霉素对TGEV感染48 h(P 0.05)后N蛋白的表达有显著促进作用。为了排除药物处理的非特异性作用,通过RNA干扰技术抑制自噬蛋白ATG5的表达,同样得到了与3-MA相似的结果。以上结果显示线粒体自噬通过抑制细胞凋亡促进TGEV的复制。3、线粒体氧化应激对TGEV诱导线粒体自噬影响的研究线粒体的氧化应激是介导线粒体自噬的主要途径。为了确定TGEV引起的线粒体自噬是否与线粒体氧化应激有关,通过流式细胞术检测了 TGEV感染后细胞的活性氧(reactive oxygen species,ROS)、线粒体ROS和一些抗氧化基因的表达变化。试验结果表明,TGEV感染后细胞总ROS水平并没有明显升高,而线粒体ROS在感染12 h后有少量增加,在感染24h到48h后出现明显的增加(P0.01);抗氧化基因SCD、Nrf2和eNOS在感染12 h到72 h之间都出现明显上调(P0.01)。另外,用还原剂谷胱甘肽(GSH)降低ROS后,可以抑制TGEV感染(2 h到48 h)引起的LC3-Ⅱ的表达(P0.01)和线粒体数量的减少(P0.01)。为了进一步确定线粒体氧化应激是调控TGEV引起线粒体自噬的主要原因,通过RNA干扰技术降低监控线粒体氧化应激蛋白DJ-1的表达,证明DJ-1干扰能够明显抑制TGEV的复制(P0.01),线粒体数量、异常线粒体比例和细胞凋亡比例在TGEV感染12 h到48 h之间也出现增加(P0.01)。以上结果均显示,线粒体氧化应激在介导TGEV感染引起的线粒体自噬中发挥了重要作用。4、TGEV的N蛋白诱导线粒体自噬的研究某些病毒蛋白可以直接定位到线粒体上,导致线粒体异常和线粒体自噬。为了进一步探究TGEV引起线粒体异常和线粒体自噬的原因,通过构建TGEV的M、N、E、PLP1蛋白的慢病毒表达载体并转染IPEC-J2细胞观察这些蛋白的线粒体定位情况。结果发现,TGEV结构蛋白N有明显的线粒体定位现象。对N蛋白稳定表达细胞的线粒体数量、膜电位、ROS和线粒体ROS的检测发现,N蛋白能促进细胞和线粒体ROS水平升高(P0.01),降低细胞线粒体数量(P0.01),但对膜电位和细胞凋亡没有明显影响。为了进一步确定N蛋白是否是诱导线粒体自噬的原因,通过检测TGEV感染的细胞中N蛋白亚细胞定位情况,证明N蛋白在TGEV感染时也存在明显的线粒体定位现象。以上结果表明,TGEV的N蛋白是诱导IPEC-J2细胞发生线粒体损伤和线粒体自噬的重要原因。本论文以猪小肠上皮细胞为模式细胞证明了细胞线粒体自噬在TGEV的复制中发挥着重要的作用,TGEV的N蛋白是造成线粒体损伤和线粒体自噬的重要原因。本研究为深入研究TGEV感染与线粒体自噬的相互作用机制,揭示TGEV在肠上皮细胞上的致病机理提供了理论基础。
[Abstract]:Porcine transmissible gastroenteritis (TGE) is a high degree of contact caused by swine infectious gastroenteritis (Transmissible gastroenteritis virus, TGEV). The target cell of the acute digestive tract infectious disease.TGEV infection is the small intestinal epithelial cells. The small intestinal epithelium is the main site of the nutrient absorption site. There are a large number of mitochondria. Some viruses specifically target mitochondria to promote their replication and survival in the cells. Some viruses induce autophagy by interacting with autophagic receptors, causing cell stress and other ways to induce autophagy, and some viruses can induce mitochondrial damage by activating mitochondrial autophagy receptors. At present, the study of the interaction between TGEV and mitochondrial autophagy has not been reported. Therefore, in order to explore the role and significance of mitochondrial autophagy in TGEV infection, we used the porcine jejunal epithelial cell IPEC-J2 as an infection model of TGEV. First, the mitochondrial damage was studied by TGEV. And the effect of mitochondrial autophagy; secondly, the effect of mitochondrial autophagy on TGEV replication was studied, and the effect of oxidative stress on TGEV induced mitochondrial autophagy was studied. Finally, through overexpression of TGEV structure protein and unstructured protein, the key proteins to induce mitochondrial autophagy were found. 1, TGEV infected IPEC-J2 Cells induced mitochondrial damage and autophagy in the process of virus infection, some viruses can promote the continuous infection by causing mitochondrial damage and autophagy. First, in order to study whether there is damage and autophagy in the process of mitochondrial TGEV infection IPEC-J2 cells, flow cytometry detected the line particles in the cells after TGEV infection. The changes in the degree of body damage and the number of mitochondria were observed and the mitochondrial structure changes after TGEV infection were observed by transmission electron microscopy. The results showed that the mitochondrial membrane potential in the cells of 12 h and 24 h infected by TGEV was obviously decreased (P 0.01). The mitochondria morphology showed obvious swelling and deformation, the crista disappeared, and the obvious bilayer enveloped lines were observed. Secondly, in order to determine whether TGEV infection activates cell autophagy to remove damaged mitochondria, the changes in autophagy related protein Beclin-1 and LC3- II and the number of mitochondria changes after TGEV infection are detected by Western-blot and flow cytometry. The results show that the content of autophagic protein Beclin-1 and LC3- II is obvious between 24h and 48h in TGEV infection. Increasing (P0.01), after infection of 12 h and 24h, the number of mitochondria decreased significantly (P0.01), but recovered after infection of 48 h. In order to further prove that TGEV infection induced mitochondrial autophagy, the co localization of mitochondria and autophagic in IPEC-J2 cells was observed by confocal microscopy. The results showed that TGEV infection was obvious after 12h. The co localization of mitochondria and autophagosome is more obvious with the prolongation of the infection time. In order to determine whether the mitochondrial autophagy caused by TGEV is complete, the TGEV infection of 24h (P0.05) and 48h (P0.01) can promote the decrease of p62/SQSTM1 protein by detecting the changes of p62/SQSTM1 protein and the fusion of mitochondria and lysosomes. Solution, promote the fusion of mitochondria and lysosomes and induce complete mitochondrial autophagy. The results show that TGEV infected IPEC-J2 cells can cause mitochondrial damage and mitochondrial autophagy.2, mitochondrial autophagy affects TGEV replication and apoptosis. Mitochondrial autophagy can inhibit apoptosis, promote the replication of the virus, and also reduce the replication of the virus. In order to determine whether mitochondrial autophagy affects the replication of TGEV, the autophagy inhibitor 3- methyl adenine (3-Methyladenine, 3-MA), autophagy accelerant rapamycin (Rapamycin) and the mitochondrial autophagy promoter carbonyl cyanide (carbonylcyanide-m-chlorophenylhy) are determined to determine whether mitochondrial autophagy affects replication of the virus. Drazone, CCCP) treated IPEC-J2 cells to change the autophagy level and detected the effect of autophagy inhibition or activation on the titer of TGEV and the expression of N protein. The results showed that the autophagy inhibitor 3-MA could significantly reduce the titer of 24h and 48h (P0.01) of TGEV infection (P0.01), and the cell apoptosis was significantly increased (P0.01); autophagy promoter Rapamycin and mitochondrial autophagy promoted the autophagy. The dose of CCCP could increase the titer of TGEV infection 24 h and 48 h (P 0.01), CCCP significantly promoted the expression of N protein after TGEV infection 24h (P0.05) and 48h (P0.01), and rapamycin had a significant promotion effect on the expression of the protein after the infection 48 (0.05). The expression of phage ATG5 also obtained similar results with 3-MA. The above results show that mitochondrial autophagy promotes the replication of TGEV by inhibiting cell apoptosis and the effect of mitochondrial oxidative stress on the autophagy induced mitochondrial autophagy by TGEV, the oxidative stress of mitochondria is the main pathway to mediate mitochondrial autophagy. In order to determine the line particles caused by TGEV Whether autophagy is associated with mitochondrial oxidative stress, the expression changes of reactive oxygen species (ROS), mitochondrial ROS and some antioxidant genes after TGEV infection were detected by flow cytometry. The results showed that the total ROS level of the cells after TGEV infection was not significantly increased, but the mitochondrial ROS was less after 12 h infection. The amount increased significantly (P0.01) after infection of 24h to 48h; antioxidant genes SCD, Nrf2 and eNOS were significantly up-regulated between 12 h to 72 h (P0.01). Furthermore, the reduction of TGEV infection (2 to 48) and the decrease of mitochondrial number could be inhibited by reducing the reduction of glutathione (GSH) to ROS. It was further determined that mitochondrial oxidative stress was the main reason for the regulation of mitochondrial autophagy by TGEV, and the expression of mitochondrial oxidative stress protein DJ-1 was monitored by RNA interference technique. It was proved that DJ-1 interference could obviously inhibit the replication of TGEV (P0.01). The number of mitochondria, the proportion of abnormal mitochondria and the proportion of apoptosis were 12 h to 48 in TGEV infection. H also showed an increase (P0.01). All the results showed that mitochondrial oxidative stress played an important role in the mitochondrial autophagy mediated by TGEV infection..4, TGEV N protein induced mitochondrial autophagy, some viral proteins can be directly located to mitochondria, resulting in mitochondrial abnormalities and mitochondrial autophagy. Investigate the causes of mitochondrial abnormalities and mitochondrial autophagy. By constructing TGEV M, N, E, PLP1 protein Lentivirus Expression Vector and transfecting IPEC-J2 cells to observe the mitochondrial localization of these proteins, it is found that TGEV structural protein N has a distinct mitochondrial localization phenomenon. The number of mitochondria, membrane electricity and membrane electricity are stable for N protein. The detection of ROS and mitochondrial ROS found that N protein could increase the level of cell and mitochondrial ROS (P0.01), decrease the number of mitochondria (P0.01), but have no significant influence on the membrane potential and apoptosis. In order to further determine whether the N protein is the cause of mitochondrial autophagy, the detection of N protein subcells in TGEV infected cells The results show that the N protein of TGEV is an important reason for inducing mitochondrial damage and mitochondrial autophagy in IPEC-J2 cells. This paper uses porcine small intestinal epithelial cells as model cells to demonstrate that the mitochondrial autophagy of TGEV cells plays a role in the replication of TGEV. The important role of the TGEV N protein is the important cause of mitochondrial damage and mitochondrial autophagy. This study provides a theoretical basis for the in-depth study of the interaction mechanism of TGEV infection and mitochondrial autophagy, and to reveal the pathogenesis of TGEV in intestinal epithelial cells.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651

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