【摘要】：豬瘟(Classical swine fever,CSF)是由豬瘟病毒(Classical swine fever virus,CSFV)引起的一種以高熱、出血和免疫抑制為典型特征的嚴重危害養(yǎng)豬業(yè)的重大傳染病,是世界動物衛(wèi)生組織(OIE)規(guī)定的必須報告的疫病,對我國乃至全球的養(yǎng)豬業(yè)造成巨大的經(jīng)濟損失。CSFV屬于黃病毒科(Flaviviridae)瘟病毒屬(Pestivirus),是一種單股正鏈RNA病毒。目前對CSFV的致病機制雖有一定的研究,但對于CSFV在宿主細胞中感染復(fù)制的機制,以及免疫逃逸的致病機理仍缺乏全面深入的了解。由于病毒要建立持續(xù)性感染,必須利用宿主細胞中可調(diào)控蛋白合成、能量代謝、細胞存活生長的關(guān)鍵信號通路。因此,探討CSFV感染的宿主細胞中參與調(diào)控細胞存活、蛋白合成和細胞自噬等過程的關(guān)鍵信號通路,闡明其參與CSFV復(fù)制增殖的機制,為豬瘟的防治提供新的靶點和理論依據(jù)。哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信號通路連接著從感應(yīng)能量和生長因子到調(diào)控細胞存活生長及大分子合成代謝的過程,除了在腫瘤發(fā)生發(fā)展中發(fā)揮重要的作用,也參與多種DNA、RNA病毒的感染復(fù)制,在病毒復(fù)制中扮演重要的角色。但對于CSFV與mTOR信號通路的關(guān)系,目前尚沒有明確報道。因此,本研究擬闡明CSFV對mTOR信號通路的調(diào)控,以及mTOR信號通路調(diào)控CSFV復(fù)制的分子機制,從mTOR的角度為解釋CSFV的復(fù)制和持續(xù)性感染提供新的理論依據(jù)。本研究首先利用細胞內(nèi)信號通路蛋白芯片技術(shù)以及western-blot等方法證實與未感染CSFV的細胞相比較,CSFV(石門強毒株)感染ST細胞能夠顯著抑制Akt/mTOR信號通路的激活,該抑制在6-24 h最為顯著,隨后逐漸恢復(fù),至48 h恢復(fù)至處理前水平。而CSFV結(jié)構(gòu)蛋白Ems、非結(jié)構(gòu)蛋白NS3、NS5A在抑制Akt/mTOR信號通路的激活中發(fā)揮重要的作用。為了探討Akt/mTOR信號通路對CSFV自身復(fù)制的影響,利用mTOR抑制劑rapamycin和激活劑insulin改變宿主細胞mTOR通路活性后感染CSFV,western-blot和熒光定量PCR等結(jié)果表明在24 h內(nèi),rapamycin雖抑制細胞增殖但顯著促進CSFV的復(fù)制;而insulin促進細胞增殖但顯著抑制CSFV復(fù)制;值得注意的是在48 h時這一調(diào)控趨勢并不存在;我們推測:CSFV感染抑制mTOR/S6K1,導致S6K1通過IRS(胰島素受體)誘導Akt負反饋激活,進而抵消了由mTOR抑制引起的病毒復(fù)制。隨后,我們進一步探討mTOR上游Akt對CSFV復(fù)制的影響;分別應(yīng)用Akt特異性抑制劑(LY294002)抑制Akt活性,以及Akt激活劑(SC79)激活A(yù)kt后感染CSFV,熒光定量PCR結(jié)果表明,與未經(jīng)處理的ST細胞相比較,抑制Akt活性能夠在72h內(nèi)持續(xù)上調(diào)CSFV基因組拷貝數(shù);而激活A(yù)kt則持續(xù)降低CSFV基因組拷貝數(shù)。與單獨改變mTOR活性引起的只在24 h內(nèi)調(diào)控病毒復(fù)制有所不同,這很有可能是由于CSFV感染引起mTOR抑制誘導Akt負反饋激活以及Akt作為mTOR上游引起比mTOR更強的作用導致。上述研究結(jié)果表明Akt/mTOR信號通路可負調(diào)控CSFV的復(fù)制,也提示CSFV感染ST細胞可能通過誘導Akt負反饋激活來調(diào)控病毒復(fù)制和維持細胞穩(wěn)態(tài)。為了深入探究Akt/mTOR信號通路負調(diào)控CSFV復(fù)制的分子機制,本研究以mTOR信號通路下游調(diào)控自噬及蛋白合成的兩個效應(yīng)分子ULK1和S6K1為切入點,應(yīng)用透射電鏡、western-blot、共聚焦顯微鏡、LC3雙熒光自噬慢病毒、IFA檢測病毒滴度以及熒光定量PCR等方法證明CSFV感染能夠增強ST細胞的自噬流,隨后應(yīng)用rapamycin和insulin以及ULK1抑制劑抑制或激活mTOR/ULK1活性,結(jié)果顯示自噬被顯著促進或抑制,而病毒的復(fù)制被相應(yīng)的促進或抑制。證明CSFV通過mTOR/ULK1依賴的信號通路誘導細胞自噬進而促進病毒復(fù)制。另一方面,應(yīng)用雙熒光素酶報告基因系統(tǒng),通過構(gòu)建含CSFV內(nèi)部核糖體進入位點(IRES)的螢火蟲熒光素酶重組載體(CSFV-IRES),應(yīng)用過表達和shRNA干擾等方法證明過表達和干擾表達S6K1能夠抑制和促進CSFV-IRES蛋白驅(qū)動活性,隨后病毒滴度檢測和熒光定量PCR結(jié)果顯示,過表達和干擾表達S6K1能抑制和促進CSFV復(fù)制。接著,利用免疫共沉淀、核糖體分離技術(shù)以及熒光定量PCR等方法證實,CSFV感染ST細胞能抑制mTOR/S6K1磷酸化激活,進而促進S6K1與真核起始因子3(elF3A)的結(jié)合,釋放CSFV-IRES與eIF3競爭結(jié)合40S核糖體,促進病毒mRNA翻譯。Akt/mTOR信號通路抑制能夠?qū)е录毎鲋骋种坪图毎蛲?而在體外,CSFV感染宿主細胞通常不抑制細胞增殖和凋亡。因此結(jié)合以上的研究,我們探討CSFV感染抑制Akt/mTOR信號通路是否通過誘導Akt負反饋維持細胞增殖和抗凋亡,以便于為病毒復(fù)制提供穩(wěn)定的細胞內(nèi)環(huán)境。通過western-blot檢測CSFV感染后Akt磷酸化水平,結(jié)果表明Akt磷酸化水平先降低后恢復(fù),進一步證實CSFV感染ST細胞可誘導Akt負反饋激活。隨后流式細胞檢測發(fā)現(xiàn)CSFV感染初期能夠?qū)⒓毎芷谧铚贕0/G1期,而48 h后沒有阻滯作用;另外使用Akt抑制劑LY294002單獨或與CSFV共同作用ST細胞,SRB實驗檢測細胞增殖,結(jié)果顯示抑制Akt活性同時感染CSFV較單獨抑制Akt或單獨感染CSFV時,對細胞增殖的抑制顯著增強,間接提示CSFV感染誘導宿主細胞Akt負反饋激活,維持宿主細胞增殖,而一旦Akt被抑制劑持續(xù)抑制,感染CSFV誘導的Akt負反饋激活也被相應(yīng)地抑制,因此不能維持細胞增殖。接著,細胞凋亡檢測結(jié)果顯示,抑制Akt活性同時感染CSFV較單獨抑制Akt或單獨感染CSFV時對細胞引起的凋亡率更大,證明CSFV誘導的負反饋激活能夠維持細胞存活。最后,以Akt抑制劑與CSFV共同作用ST細胞至24 h、48 h、72 h,熒光定量PCR檢測結(jié)果表明,與單獨感染CSFV細胞相比較,在72 h內(nèi)持續(xù)阻斷Akt能夠持續(xù)上調(diào)病毒基因組拷貝數(shù),進一步提示CSFV感染引起Akt負反饋激活,進而維持病毒的復(fù)制。以上結(jié)果充分證實CSFV感染ST細胞誘導Akt負反饋激活的存在并且能夠維持細胞存活和病毒自身復(fù)制。綜上,本研究全面證實CSFV感染宿主細胞能夠通過劫持mTOR信號通路以維持病毒復(fù)制和細胞存活,為以Akt/mTOR激活防治豬瘟提供新的靶點和思路。
[Abstract]:Classical swine fever (CSF) is a serious infectious disease caused by classical swine fever virus (CSFV), which is characterized by high fever, hemorrhage and immunosuppression. It is an epidemic disease that must be reported by the World Organization for Animal Health (OIE). It has caused tremendous damage to the pig industry in China and even the world. CSFV belongs to the genus Pestivirus of Flaviviridae. It is a single stranded positive-stranded RNA virus. Although the pathogenesis of CSFV has been studied, the mechanism of infection and replication of CSFV in host cells and the pathogenesis of immune escape are still poorly understood. Continuous infection requires the use of key signaling pathways that regulate protein synthesis, energy metabolism, and cell survival and growth in host cells. The mammalian target of rapamycin (mTOR) signaling pathway links the processes from induction energy and growth factors to regulation of cell survival, growth and macromolecule synthesis and metabolism. Infection replication plays an important role in viral replication. However, the relationship between CSFV and mTOR signaling pathway has not been clearly reported. Therefore, this study aims to elucidate the regulation of CSFV on mTOR signaling pathway and the molecular mechanism of mTOR signaling pathway on CSFV replication, providing an explanation for CSFV replication and persistent infection from the perspective of mTOR. In this study, compared with non-CSFV-infected cells, CSFV-infected ST cells significantly inhibited the activation of Akt/mTOR signaling pathway. The inhibition was most significant at 6-24 h, then gradually recovered until 48 h. In order to investigate the effect of Akt/mTOR signaling pathway on CSFV self-replication, we used mTOR inhibitor rapamycin and activator insulin to change the activity of host cell mTOR pathway and infected CSFV, Western-blot and fluorescence. Quantitative PCR and other results showed that rapamycin inhibited cell proliferation but significantly promoted CSFV replication within 24 hours; insulin promoted cell proliferation but significantly inhibited CSFV replication; it is noteworthy that this regulatory trend did not exist at 48 hours; we speculated that CSFV infection inhibited mTOR/S6K1, resulting in S6K1 inducing Akt negative reaction via IRS (insulin receptor). Subsequently, we further investigated the effect of Akt upstream of mTOR on CSFV replication; Akt specific inhibitor (LY294002) was used to inhibit Akt activity; and Akt activator (SC79) was used to activate Akt to infect CSFV. Fluorescence quantitative PCR results showed that it was inhibited compared with untreated ST cells. Akt-producing activity could continuously up-regulate the copy number of CSFV genome within 72 hours, while activation of Akt could continuously decrease the copy number of CSFV genome. These results suggest that the Akt/mTOR signaling pathway can negatively regulate the replication of CSFV, and that ST cells infected with CSFV may regulate virus replication and maintain cell homeostasis by inducing Akt negative feedback activation. Two effector molecules ULK1 and S6K1, which regulate autophagy and protein synthesis downstream of the pathway, were used as entry points. TEM, Western-blot, confocal microscopy, LC3 double fluorescent autophagy lentivirus, IFA detection of virus titer and fluorescence quantitative PCR proved that CSFV infection could enhance the autophagy of ST cells. K1 inhibitors inhibit or activate mTOR/ULK1 activity, indicating that autophagy is significantly promoted or inhibited, whereas virus replication is correspondingly promoted or inhibited. It is demonstrated that CSFV induces cell autophagy through mTOR/ULK1-dependent signaling pathway and promotes virus replication. On the other hand, double luciferase reporter gene system is used to construct CSFV-containing interior. The fluorescein enzyme recombinant vector (CSFV-IRES) at the ribosomal entry site (IRES) was used to demonstrate that the overexpression and interference of S6K1 could inhibit and promote the drive activity of CSFV-IRES protein. The results of viral titer detection and fluorescence quantitative PCR showed that overexpression and interference of S6K1 could inhibit and promote CSFV-IRES protein. FV replication. Immunocoprecipitation, ribosome isolation and fluorescence quantitative PCR confirmed that CSFV-infected ST cells inhibited the phosphorylation of mTOR/S6K1, thereby facilitating the binding of S6K1 to eukaryotic initiation factor 3 (elF3A), releasing CSFV-IRES to compete with eIF3 to bind to 40S ribosomes and promoting the translation of viral mRNA. Akt/mTOR signaling pathway was inhibited. CSFV infection usually does not inhibit cell proliferation and apoptosis in vitro. Therefore, in combination with the above studies, we explore whether CSFV infection inhibits Akt/mTOR signaling pathway by inducing Akt negative feedback to maintain cell proliferation and anti-apoptosis in order to provide stable details for virus replication. Akt phosphorylation was detected by Western-blot. The results showed that Akt phosphorylation decreased first and then recovered. It was further confirmed that CSFV-infected ST cells could induce Akt negative feedback activation. Using Akt inhibitor LY294002 alone or in conjunction with CSFV, the proliferation of ST cells was detected by SRB assay. The results showed that inhibition of Akt activity and infection with CSFV increased significantly compared with inhibition of Akt alone or infection with CSFV alone. Once Akt was inhibited by the inhibitor, the negative feedback activation of Akt induced by CSFV was inhibited accordingly, so the cell proliferation could not be maintained. Next, the results of cell apoptosis detection showed that the rate of cell apoptosis induced by inhibiting Akt activity and infecting CSFV at the same time was higher than that induced by inhibiting Akt alone or CSFV alone. Finally, the results of fluorescence quantitative PCR assay showed that continuous blockade of Akt for 72 hours could continuously increase the number of viral genomic copies compared with single infection of CSFV cells, suggesting that CSFV infection could cause negative feedback activation of Akt and consequently maintain the disease. These results fully confirm that CSFV-infected ST cells induce Akt negative feedback activation and can maintain cell survival and virus replication. In conclusion, this study fully confirms that CSFV-infected host cells can maintain virus replication and cell survival by hijacking the mTOR signaling pathway, providing a basis for the prevention and treatment of swine fever with Akt/mTOR activation. New targets and ideas.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651

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4 顧兵;腦出血后mTOR信號通路激活及雷帕霉素腦保護作用機制研究[D];蘇州大學;2015年

5 張劍;食管鱗狀細胞癌中mTOR表達及其與癌癥惡性程度和患者機體免疫反應(yīng)水平相關(guān)性研究[D];青島大學;2015年

6 周璇;mTOR信號通路在小鼠B淋巴細胞成熟及抗體產(chǎn)生中的作用及其機制[D];南方醫(yī)科大學;2014年

7 楊雪帆;慈菇多糖對小鼠免疫功能及mTOR信號通路的影響[D];福建醫(yī)科大學;2015年

8 夏傳友;組蛋白甲基化酶SMYD3在前列腺癌中mTOR通路的機制研究[D];山東大學;2016年

9 王嘉禎;食管鱗癌細胞中LSD1與mTOR通路相互調(diào)控作用研究[D];鄭州大學;2016年

10 張海環(huán);豬小腸賴氨酸轉(zhuǎn)運體對氮源的響應(yīng)規(guī)律及mTOR信號通路的調(diào)控研究[D];吉林農(nóng)業(yè)大學;2016年

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