本文關(guān)鍵詞：藍(lán)舌病病毒感染誘導(dǎo)細(xì)胞自噬的作用和機(jī)制　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 藍(lán)舌病病毒 細(xì)胞自噬 相互作用 信號(hào)通路

【摘要】：藍(lán)舌病(Bluetongue,BT)是由藍(lán)舌病病毒(Bluetongue virus,BTV)引起的一種重要蟲(chóng)媒傳染病,可引起反芻動(dòng)物特別是綿羊較高的發(fā)病率和致死率,對(duì)畜牧業(yè)的發(fā)展和畜產(chǎn)品的經(jīng)濟(jì)貿(mào)易造成潛在的巨大威脅。BTV屬于呼腸孤病毒科環(huán)狀病毒屬,是一種復(fù)雜的雙鏈分節(jié)段RNA病毒,擁有雙層衣殼,無(wú)囊膜。作為環(huán)狀病毒屬的一個(gè)典型代表,BTV在分子結(jié)構(gòu)、生化特征、流行病學(xué)和診斷方法等分子生物學(xué)領(lǐng)域已取得了重大進(jìn)展,然而,BTV感染與宿主細(xì)胞應(yīng)答之間的相互作用并沒(méi)有得到充分的關(guān)注,因此從宿主的角度去探索BTV的致病機(jī)制顯得尤為重要。病毒作為一種專(zhuān)性細(xì)胞內(nèi)寄生物,其在感染過(guò)程中必定會(huì)與宿主的胞內(nèi)應(yīng)答發(fā)生相互作用。細(xì)胞自噬(Autophagy)作為一種保守的胞內(nèi)適應(yīng)性應(yīng)答,在維持細(xì)胞穩(wěn)態(tài),促進(jìn)新陳代謝方面起著舉足輕重的作用。它可將細(xì)胞內(nèi)的衰老細(xì)胞器、外源微生物等成分包裹在一個(gè)稱(chēng)為自噬體的雙層膜結(jié)構(gòu)中,運(yùn)送至溶酶體進(jìn)行降解回收。自噬是對(duì)抗病原體的天然防御機(jī)制,但最近越來(lái)越多的證據(jù)表明許多病毒已進(jìn)化出多種策略抵抗、逃逸、甚至利用細(xì)胞自噬促進(jìn)自身增殖,如流感病毒、丙型肝炎病毒、登革熱病毒、輪狀病毒等。對(duì)于BTV而言,其復(fù)制感染與細(xì)胞自噬之間的相互作用及其啟動(dòng)機(jī)制便是本研究中要探索的問(wèn)題。首先,采用三種經(jīng)典方法對(duì)BTV1感染BSR細(xì)胞后是否誘導(dǎo)自噬進(jìn)行了檢測(cè),包括透射電鏡觀(guān)察自噬體的形成、激光共聚焦檢測(cè)熒光聚點(diǎn)的變化以及Western blot分析LC3的轉(zhuǎn)化。結(jié)果表明,與陰性對(duì)照相比,BTV1感染后誘導(dǎo)BSR細(xì)胞產(chǎn)生了自噬體樣的雙層膜結(jié)構(gòu),GFP-LC3熒光聚點(diǎn)顯著累積,而且LC3發(fā)生了明顯的類(lèi)別轉(zhuǎn)化。此外,在BTV的自然宿主靶細(xì)胞-原代羊舌上皮細(xì)胞上進(jìn)行了分析,得到了類(lèi)似的結(jié)果。由此證實(shí)BTV1感染可以誘導(dǎo)細(xì)胞自噬的發(fā)生。其次,進(jìn)行了基于自噬性降解的自噬潮分析。自噬潮,即自噬的整個(gè)動(dòng)態(tài)連續(xù)的過(guò)程,可通過(guò)p62的降解、LC3-II的周轉(zhuǎn)、自噬體和溶酶體共定位分析等綜合評(píng)估。研究結(jié)果顯示,BTV1感染明顯促進(jìn)了自噬底物p62/SQSTM1的降解,而且溶酶體抑制劑CQ的配合使用導(dǎo)致了LC3-II和p62的蓄積,BTV1感染后自噬體標(biāo)記GFP-LC3和溶酶體標(biāo)記LysoTracker也出現(xiàn)明顯的共定位,表明BTV1誘導(dǎo)了自噬的起始和自噬性降解過(guò)程,即BTV1感染后觸發(fā)了完整的自噬潮反應(yīng)。再次,判定BTV1誘導(dǎo)自噬是否嚴(yán)格依賴(lài)于病毒的有效復(fù)制。滅活病毒UV-BTV1不能誘導(dǎo)相同的自噬現(xiàn)象證實(shí)了該推論。進(jìn)一步應(yīng)用自噬早期阻斷藥物3-MA和晚期阻斷藥物CQ處理細(xì)胞或者干擾自噬關(guān)鍵基因Beclin 1,可見(jiàn)自噬活性明顯降低,病毒復(fù)制也受到了顯著的抑制;相反,自噬誘導(dǎo)劑Rapamycin處理細(xì)胞后激活了自噬反應(yīng),促進(jìn)了BTV1的復(fù)制。上述結(jié)果證明了BTV1的有效復(fù)制是誘導(dǎo)自噬的關(guān)鍵因素,并可以利用自噬促進(jìn)自身增殖。最后,為了深入探索BTV1感染激活自噬的機(jī)制,對(duì)可能涉及的多條信號(hào)級(jí)聯(lián)反應(yīng)進(jìn)行了系統(tǒng)分析。結(jié)果顯示,BTV1感染后明顯抑制了自噬的中央調(diào)控分子mTOR的磷酸化及其下游底物p70S6K的活性,從而啟動(dòng)了自噬。而后探索mTOR上游可能的調(diào)控信號(hào),首先檢測(cè)發(fā)現(xiàn)BTV1感染激發(fā)細(xì)胞自噬不依賴(lài)于ERK1/2信號(hào)通路。而藥物處理和突變質(zhì)粒轉(zhuǎn)染顯示mTOR活性降低部分取決于PI3K/Akt的失活,相反,激活A(yù)kt后p-mTOR有所恢復(fù),自噬也相應(yīng)被抑制,說(shuō)明Akt信號(hào)通路參與了BTV1誘導(dǎo)的自噬的啟動(dòng)過(guò)程。最為明顯的是AMPK途徑的影響,通過(guò)藥物處理和RNA干擾實(shí)驗(yàn)等聯(lián)合使用,證實(shí)病毒感染顯著提高了胞漿內(nèi)Ca2+的濃度,并介導(dǎo)了Ca MKKβ的激活,作用于下游的AMPK,從而導(dǎo)致mTOR的抑制和自噬的激活。試驗(yàn)進(jìn)一步證實(shí)TSC2是Akt/AMPK及mTOR之間信號(hào)傳導(dǎo)的關(guān)鍵中介分子。結(jié)果表明BTV1可通過(guò)抑制Akt和活化Ca2+介導(dǎo)的AMPK來(lái)共同作用于下游的TSC2-mTOR途徑,從而誘導(dǎo)自噬的激活,促進(jìn)病毒自身的復(fù)制。綜上所述,本研究首次闡明了BTV1感染誘導(dǎo)的細(xì)胞自噬在BTV1的復(fù)制中發(fā)揮著重要作用,而且系統(tǒng)分析了調(diào)控兩者之間相互作用的信號(hào)途徑。這些發(fā)現(xiàn)推動(dòng)了BTV與宿主相互作用領(lǐng)域的研究,成為探索BTV發(fā)病機(jī)制的新視角,為抗病毒藥物研發(fā)奠定了基礎(chǔ)。
[Abstract]:Bluetongue (Bluetongue, BT) by bluetongue virus (Bluetongue virus BTV) is an important infectious disease caused by insect, especially sheep ruminant animal can cause high morbidity and mortality, causing huge potential threat to the development of animal husbandry and animal products trade. BTV belongs to the Reoviridae like virus, is a kind of complex double stranded segments of RNA virus, has a double capsid, no envelope. As a typical representative of the orbivirus genus, BTV in the molecular structure, biochemical characteristics, epidemiology and diagnostic methods of molecular biology have made significant progress, however, the interaction between BTV infection and host cell responses have not been fully concerned, because the host from the point of view to explore the pathogenic mechanism of BTV is is particularly important. The virus, as a specific intracellular parasite, is bound to interact with the intracellular response of the host in the process of infection. Cellular autophagy (Autophagy), as a conserved intracellular adaptive response, plays an important role in maintaining cell homeostasis and promoting metabolism. It can encapsulate cell senescent organelles and exogenous microorganisms in a bilayer membrane structure called autophagosome, transported to lysosomes, and can be degraded and recovered. Autophagy is a natural defense mechanism against pathogens, but recently more and more evidence that many viruses have evolved a variety of strategies of resistance, escape, and even the use of autophagy promote their proliferation, such as influenza virus, hepatitis C virus, dengue virus, rotavirus. For BTV, the interaction between the replicative infection and the autophagy and the mechanism of its initiation are the problems to be explored in this study. First of all, using three classical methods to induce autophagy in BTV1 infected BSR cells were detected, including conversion change formation, transmission electron microscope observation of autophagosomes detected by laser confocal fluorescence technique and Western blot analysis LC3. The results show that, compared with the negative control, the double membrane autophagosomes like BSR cells induced by BTV1 infection, GFP-LC3 fluorescence and LC3 accumulation significantly accumulated obviously category transformation. In addition, the target cells of the natural host of BTV, the primary goat tongue epithelial cells, were analyzed, and similar results were obtained. It is confirmed that BTV1 infection can induce autophagy. Secondly, autophagy analysis based on autophagy was carried out. Autophagy, the whole process of autophagy, can be evaluated through p62 degradation, turnover of LC3-II, CO localization of autophagosomes and lysosomes. The results showed that BTV1 infection could promote the degradation of autophagy substrate p62/SQSTM1, and with the lysosomal inhibitor CQ use has led to the accumulation of LC3-II and p62, BTV1 after infection of autophagosome marker GFP-LC3 and lysosomal marker LysoTracker also appeared significant co localization, suggesting initiation and autophagic degradation of BTV1 induced autophagy, BTV1 infection after the complete reaction of tidal triggering autophagy. Again, it is determined whether the BTV1 induced autophagy is strictly dependent on the effective replication of the virus. Inactivated virus UV-BTV1 can not induce the same autophagy, which confirms this inference. Further application of autophagy early blocking drugs and late 3-MA cells treated with CQ blocking drugs or interfere with the key autophagy gene Beclin 1, visible autophagy activity decreased significantly, virus replication was significantly inhibited; on the contrary, the agent Rapamycin cells after activation of autophagy reaction induced autophagy, promote the replication of BTV1. The above results show that the effective replication of BTV1 is the key factor in inducing autophagy and can use autophagy to promote the proliferation of autophagy. Finally, in order to explore the mechanism of activation of autophagy by BTV1 infection, a systematic analysis of the possible multi signal cascade reaction was carried out. The results showed that BTV1 infection significantly inhibited the phosphorylation of the autophagy, the central regulatory molecule mTOR, and the activity of the downstream substrate p70S6K, thus initiating autophagy. Then we explored the possible regulatory signals in the upstream of mTOR. First, it was found that the autophagy of BTV1 infected cells was not dependent on the ERK1/2 signaling pathway. Drug treatment and mutation plasmid transfection showed that the decrease of mTOR activity was partly determined by the inactivation of PI3K/Akt. On the contrary, after activation of Akt, p-mTOR recovered and autophagy was also inhibited, indicating that the Akt signaling pathway was involved in the initiation process of BTV1 induced autophagy. The most obvious is the influence of AMPK pathway. The combination of drug treatment and RNA interference test confirmed that virus infection significantly increased the concentration of Ca2+ in the cytoplasm, and mediated the activation of Ca MKK beta, which played a role in the downstream AMPK, resulting in the inhibition of mTOR and the activation of autophagy. The test further confirmed that TSC2 is the key mediator of signal transduction between Akt/AMPK and mTOR. The results showed that BTV1 could co activate downstream TSC2-mTOR pathway by inhibiting Akt and activated Ca2+ mediated AMPK, thereby inducing autophagy and promoting the replication of virus itself. To sum up, this study first elucidates that BTV1 induced autophagy plays an important role in the replication of BTV1, and systematically analyzes the signaling pathways regulating the interaction between them. These findings promote the research on the interaction between BTV and host, and become a new perspective to explore the pathogenesis of BTV and lay a foundation for the research and development of antiviral drugs.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S852.65

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