本文選題：TGEV + IPEC-J2細胞��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：豬傳染性胃腸炎(Porcine transmissible gastroenteritis, TGE)是由豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus, TGEV)引起的一種高度接觸性、急性消化道傳染病。TGEV感染的靶細胞是小腸上皮細胞。小腸上皮作為營養(yǎng)吸收的主要位點細胞內(nèi)含有大量的線粒體。有一些病毒專門以線粒體為調(diào)控靶點,促進自身在細胞內(nèi)復(fù)制和存活;有一些病毒通過與自噬受體相互作用、引起細胞應(yīng)激等方式誘導(dǎo)自噬,增加病毒復(fù)制;還有一些病毒通過激活線粒體自噬受體、誘導(dǎo)線粒體損傷等方式引起線粒體自噬,促進病毒的持續(xù)感染。目前,關(guān)于TGEV與線粒體自噬相互作用的研究還未見報道。因此,為了探究線粒體自噬在TGEV感染中的作用和意義,我們應(yīng)用豬空腸上皮細胞IPEC-J2作為TGEV的感染模型,首先研究了 TGEV對線粒體損傷和線粒體自噬的影響;其次研究了線粒體自噬對TGEV復(fù)制的影響;再次研究了細胞氧化應(yīng)激對TGEV誘導(dǎo)線粒體自噬的影響;最后通過對TGEV的結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白的過表達,尋找誘導(dǎo)線粒體自噬的關(guān)鍵蛋白。具體內(nèi)容如下:1、TGEV感染IPEC-J2細胞誘導(dǎo)線粒體損傷和自噬的研究在病毒感染過程中,某些病毒能夠通過引起線粒體損傷和自噬促進自身的持續(xù)感染。首先,為了研究線粒體在TGEV感染IPEC-J2細胞過程中是否存在損傷和自噬,流式細胞術(shù)檢測了 TGEV感染后細胞內(nèi)的線粒體損傷程度和線粒體數(shù)量變化,并通過透射電鏡觀察了 TGEV感染后細胞線粒體的結(jié)構(gòu)變化。結(jié)果發(fā)現(xiàn),TGEV感染12 h和24 h后細胞內(nèi)的線粒體膜電位明顯降低(P 0.01 ),線粒體形態(tài)出現(xiàn)明顯腫脹變形,嵴消失,并觀察到明顯的雙層膜包裹的線粒體。其次,為了確定TGEV感染是否激活細胞自噬清除受損的線粒體,通過Western-blot和流式細胞術(shù)檢測TGEV感染后自噬相關(guān)蛋白Beclin-1和LC3-Ⅱ的變化以及線粒體數(shù)量的變化。結(jié)果證明,自噬蛋白Beclin-1和LC3-Ⅱ在TGEV感染24h到48h之間含量都明顯增加(P0.01),感染12 h和24h后線粒體數(shù)量又明顯減少(P0.01),但在感染48 h后恢復(fù)。為了進一步證明TGEV感染誘導(dǎo)線粒體自噬發(fā)生,通過共聚焦顯微鏡觀察IPEC-J2細胞內(nèi)線粒體和自噬體是否存在共定位現(xiàn)象。試驗結(jié)果表明,TGEV感染12h后可見明顯的線粒體與自噬體的共定位,隨著感染時間延長,共定位現(xiàn)象更加明顯。最后為了確定TGEV引起的線粒體自噬是否完全,通過檢測p62/SQSTM1蛋白變化以及線粒體與溶酶體的融合現(xiàn)象,證明TGEV感染24h (P0.05)和48h (P0.01)能促進p62/SQSTM1蛋白的降解,促進線粒體和溶酶體的融合,誘導(dǎo)產(chǎn)生完全的線粒體自噬。以上結(jié)果顯示TGEV感染IPEC-J2細胞能引起線粒體的損傷和線粒體自噬。2、線粒體自噬對TGEV復(fù)制和細胞凋亡影響的研究線粒體自噬可以抑制細胞凋亡,促進病毒的復(fù)制;也可以降低線粒體數(shù)量,減少能量產(chǎn)生,抑制病毒的復(fù)制。為了確定線粒體自噬是否影響TGEV的復(fù)制,通過自噬抑制劑3-甲基腺嘌呤(3-Methyladenine,3-MA)、自噬促進劑雷帕霉素(Rapamycin)和線粒體自噬促進劑羰基氰化物間氯苯腙(carbonylcyanide-m-chlorophenylhydrazone,CCCP)處理IPEC-J2細胞改變細胞自噬水平,檢測自噬抑制或激活對TGEV的滴度和N蛋白表達的影響。試驗結(jié)果表明,自噬抑制劑3-MA能顯著降低TGEV感染24h和48h的滴度(P0.01),并且細胞凋亡明顯增加(P0.01);自噬促進劑Rapamycin和線粒體自噬促進劑CCCP都能增加TGEV感染24 h和48 h后滴度(P 0.01 ),CCCP對TGEV感染24h (P0.05)和48h(P0.01)后N蛋白的表達有顯著促進作用,雷帕霉素對TGEV感染48 h(P 0.05)后N蛋白的表達有顯著促進作用。為了排除藥物處理的非特異性作用,通過RNA干擾技術(shù)抑制自噬蛋白ATG5的表達,同樣得到了與3-MA相似的結(jié)果。以上結(jié)果顯示線粒體自噬通過抑制細胞凋亡促進TGEV的復(fù)制。3、線粒體氧化應(yīng)激對TGEV誘導(dǎo)線粒體自噬影響的研究線粒體的氧化應(yīng)激是介導(dǎo)線粒體自噬的主要途徑。為了確定TGEV引起的線粒體自噬是否與線粒體氧化應(yīng)激有關(guān),通過流式細胞術(shù)檢測了 TGEV感染后細胞的活性氧(reactive oxygen species,ROS)、線粒體ROS和一些抗氧化基因的表達變化。試驗結(jié)果表明,TGEV感染后細胞總ROS水平并沒有明顯升高,而線粒體ROS在感染12 h后有少量增加,在感染24h到48h后出現(xiàn)明顯的增加(P0.01);抗氧化基因SCD、Nrf2和eNOS在感染12 h到72 h之間都出現(xiàn)明顯上調(diào)(P0.01)。另外,用還原劑谷胱甘肽(GSH)降低ROS后,可以抑制TGEV感染(2 h到48 h)引起的LC3-Ⅱ的表達(P0.01)和線粒體數(shù)量的減少(P0.01)。為了進一步確定線粒體氧化應(yīng)激是調(diào)控TGEV引起線粒體自噬的主要原因,通過RNA干擾技術(shù)降低監(jiān)控線粒體氧化應(yīng)激蛋白DJ-1的表達,證明DJ-1干擾能夠明顯抑制TGEV的復(fù)制(P0.01),線粒體數(shù)量、異常線粒體比例和細胞凋亡比例在TGEV感染12 h到48 h之間也出現(xiàn)增加(P0.01)。以上結(jié)果均顯示,線粒體氧化應(yīng)激在介導(dǎo)TGEV感染引起的線粒體自噬中發(fā)揮了重要作用。4、TGEV的N蛋白誘導(dǎo)線粒體自噬的研究某些病毒蛋白可以直接定位到線粒體上,導(dǎo)致線粒體異常和線粒體自噬。為了進一步探究TGEV引起線粒體異常和線粒體自噬的原因,通過構(gòu)建TGEV的M、N、E、PLP1蛋白的慢病毒表達載體并轉(zhuǎn)染IPEC-J2細胞觀察這些蛋白的線粒體定位情況。結(jié)果發(fā)現(xiàn),TGEV結(jié)構(gòu)蛋白N有明顯的線粒體定位現(xiàn)象。對N蛋白穩(wěn)定表達細胞的線粒體數(shù)量、膜電位、ROS和線粒體ROS的檢測發(fā)現(xiàn),N蛋白能促進細胞和線粒體ROS水平升高(P0.01),降低細胞線粒體數(shù)量(P0.01),但對膜電位和細胞凋亡沒有明顯影響。為了進一步確定N蛋白是否是誘導(dǎo)線粒體自噬的原因,通過檢測TGEV感染的細胞中N蛋白亞細胞定位情況,證明N蛋白在TGEV感染時也存在明顯的線粒體定位現(xiàn)象。以上結(jié)果表明,TGEV的N蛋白是誘導(dǎo)IPEC-J2細胞發(fā)生線粒體損傷和線粒體自噬的重要原因。本論文以豬小腸上皮細胞為模式細胞證明了細胞線粒體自噬在TGEV的復(fù)制中發(fā)揮著重要的作用,TGEV的N蛋白是造成線粒體損傷和線粒體自噬的重要原因。本研究為深入研究TGEV感染與線粒體自噬的相互作用機制,揭示TGEV在腸上皮細胞上的致病機理提供了理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.651

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