發(fā)布時(shí)間：2018-05-18 04:28

  本文選題：腸道病毒71型 + 柯薩奇病毒A組16型　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：手足口病(hand-foot-and-mouth disease, HFMD)是一種全球性的疾病,自上個(gè)世紀(jì)九十年代以來,在中國(guó)大陸、臺(tái)灣、新加坡和亞洲其它地區(qū)一直保持高發(fā)態(tài)勢(shì),其易感人群主要是5歲以下嬰幼兒,該疾病易致罹患者并發(fā)神經(jīng)系統(tǒng)相關(guān)重癥,甚至可能引發(fā)死亡,已經(jīng)成為嚴(yán)重危害兒童健康和公共衛(wèi)生安全的重要傳染性疾病。研究表明,HFMD的致病原主要是小核糖核酸病毒科的腸道病毒71型(Enterovirus 71, EV71)和柯薩奇病毒A組16型(Coxsackie virus A 16, CA16)。近年來對(duì)于HFMD的研究大多數(shù)集中于EV71的感染,而對(duì)CA16的感染缺乏足夠的重視。臨床上,EV71感染易引發(fā)重癥病例及死亡病例,而CA16感染所致的癥狀普遍較輕,但CA16感染所致HFMD患者一旦發(fā)展到重癥階段,其基本情況和臨床癥狀特征基本與EV71感染病例一致。另外,CA16病毒在人群中的重復(fù)感染的流行病學(xué)現(xiàn)象也越來越引起人們的廣泛關(guān)注。盡管EV71滅活疫苗已經(jīng)上市,但EV71與CA16感染導(dǎo)致的臨床癥狀的差異,以及兩者的感染機(jī)制與免疫機(jī)理仍不十分清楚,這些問題依然需要更深入的研究�；诒緦�(shí)驗(yàn)室在前期研究中建立的CA16嬰猴感染模型,本論文的第一部分首先對(duì)CA16重復(fù)感染恒河猴嬰猴的全基因組表達(dá)譜進(jìn)行了分析,結(jié)果顯示,與恒河猴嬰猴感染CA16前相比,恒河猴嬰猴感染CA16后各個(gè)時(shí)間共出現(xiàn)了948個(gè)共同差異基因；將這些差異基因進(jìn)行基因本體論(Gene Ontology, GO)分析后發(fā)現(xiàn)：這些差異基因主要集中在5個(gè)生物學(xué)過程,包括：細(xì)胞通訊、細(xì)胞周期、免疫系統(tǒng)過程、轉(zhuǎn)錄調(diào)節(jié)、代謝過程,說明在恒河猴嬰猴感染CA16的不同階段中,這5個(gè)生物學(xué)過程參與了其引發(fā)疾病的進(jìn)程；隨后,我們對(duì)各類基因中上調(diào)和下調(diào)最顯著的10個(gè)基因進(jìn)行了共表達(dá)網(wǎng)絡(luò)分析,發(fā)現(xiàn)IL-6、IL-8、IL-18等作為了關(guān)鍵調(diào)控基因,它們是參與炎癥反應(yīng)及免疫反應(yīng)的重要相關(guān)基因；緊接著我們對(duì)免疫相關(guān)的基因進(jìn)行了通路分析,發(fā)現(xiàn)其主要分布在趨化因子和細(xì)胞因子介導(dǎo)的炎癥通路以及白介素信號(hào)通路,進(jìn)一步說明在恒河猴嬰猴感染CA16的不同階段中,炎癥反應(yīng)與免疫反應(yīng)發(fā)揮著主要作用。圍繞著機(jī)體抗病毒的固有免疫反應(yīng)Ⅰ型干擾素(Type Ⅰ Interferon, IFN-I)動(dòng)員的特征,最后我們還對(duì)芯片中與IFN-I產(chǎn)生相關(guān)的基因及與細(xì)胞自噬相關(guān)的基因進(jìn)行了分析,結(jié)果發(fā)現(xiàn)與IFN-I產(chǎn)生相關(guān)的基因和自噬相關(guān)的基因大多發(fā)生了下調(diào)表達(dá),這為后續(xù)的研究提供了線索和依據(jù)。本論文的第二部分根據(jù)第一部分提供的線索,將EV71與CA16直接感染人正常呼吸道上皮細(xì)胞16HBE,并檢測(cè)兩者感染后IFN-I產(chǎn)生相關(guān)基因的改變,結(jié)果發(fā)現(xiàn),與對(duì)照組相比,EV71感染組中TLR3、MAVS、MDA5、MyD88、IRF7、 IFNα和IFNβ的基因表達(dá)量均發(fā)生了顯著性地上調(diào),而在CA16感染組中TLR3和IRF3的基因表達(dá)水平顯著性地下降,TLR7、MAVS、RIG-I、MyD88、IRF7、 IFNα和IFNβ的基因表達(dá)水平均無顯著性地變化。此外,病毒滴度和病毒載量的檢測(cè)結(jié)果顯示,相比于EV71感染組,CA16感染組在16HBE上的復(fù)制效率更高。上述結(jié)果提示：盡管EV71感染16HBE后可能會(huì)顯著性地誘發(fā)IFN-I產(chǎn)生相關(guān)通路的激活,但產(chǎn)生的IFN-I尚不足以抵抗EV71的入侵,故EV71感染16HBE后仍可實(shí)現(xiàn)增殖,而CA16感染16HBE后并不能觸發(fā)IFN-I的產(chǎn)生,因此,推測(cè)CA16感染16HBE后可能誘發(fā)了某種機(jī)制使其逃避了其在16HBE上的天然免疫反應(yīng),并使其更容易在16HBE上實(shí)現(xiàn)快速地增殖,這揭示了EV71與CA16可能通過不同的途徑增強(qiáng)其自身的感染能力。本論文的第三部分在第二部分的基礎(chǔ)上,進(jìn)一步探究引發(fā)EV71和CA16感染所致IFN-I產(chǎn)生差異性的原因,故我們鎖定在EV71和CA16感染16HBE后誘發(fā)的細(xì)胞自噬現(xiàn)象對(duì)IFN-I產(chǎn)生及病毒受體表達(dá)的影響。結(jié)果顯示,無論是16HBE中的內(nèi)源性LC3染色還是將外源性轉(zhuǎn)熒光標(biāo)記的LC3質(zhì)粒導(dǎo)入16HBE,我們均發(fā)現(xiàn)在EV71感染后可誘導(dǎo)16HBE細(xì)胞發(fā)生完全自噬,而CA16感染則誘導(dǎo)16HBE細(xì)胞發(fā)生非完全自噬；在給予自噬抑制劑3MA后,EV71與CA16感染的16HBE的細(xì)胞活性顯著性地下降,病毒空斑形成單位(plaque forming units, PFUs)顯著性地下降,且在自噬現(xiàn)象被抑制后,在基因水平上,TLR7、MyD88、 IRF7介導(dǎo)的IFN-I的產(chǎn)生在EV71與CA16感染的16HBE中顯著性地上升,而在蛋白水平上,TLR7、MyD88、IRF7也在EV71與CA16感染的16HBE中顯著性地上升,另外,免疫熒光的結(jié)果也顯示TLR7與內(nèi)體標(biāo)志物M6PR不僅存在共定位現(xiàn)象,而且在抑制EV71與CA16誘導(dǎo)的自噬后TLR7的表達(dá)強(qiáng)度顯著性增加,這些結(jié)果說明EV71與CA16感染均可以通過誘導(dǎo)細(xì)胞自噬而抑制TLR7、 MyD88、IRF7介導(dǎo)的IFN-I的產(chǎn)生,進(jìn)而促進(jìn)病毒自身的包裝成熟。此外,我們還發(fā)現(xiàn),在抑制EV71與CA16誘導(dǎo)的自噬后,EV71與CA16在16HBE上的病毒入侵受體SCARB2和CD162的陽性細(xì)胞比例也會(huì)顯著性地下降,說明EV71與CA16可能通過誘發(fā)自噬的發(fā)生而促進(jìn)其感染細(xì)胞受體的表達(dá),進(jìn)而增強(qiáng)其自身的入侵。上述結(jié)果提示EV71與CA16感染可能通過抑制IFN-I和促進(jìn)病毒受體的表達(dá)來協(xié)作病毒參與免疫逃逸。綜上所述,本研究首先通過對(duì)CA16感染恒河猴的全基因組表達(dá)譜的分析,首次揭示了CA16感染引起的差異基因表達(dá)譜；隨后通過比較EV71與CA16感染16HBE后IFN-I產(chǎn)生相關(guān)基因的變化,發(fā)現(xiàn)EV71和CA16可以通過不同的模式識(shí)別受體及下游信號(hào)分子而誘發(fā)差異性的IFN-I產(chǎn)生,且IFN-I的產(chǎn)生可能會(huì)直接影響兩者在16HBE上的感染和增殖能力；最后還探討了EV71和CA16誘導(dǎo)的自噬對(duì)WN-I產(chǎn)生和病毒受體表達(dá)的影響,發(fā)現(xiàn)EV71和CA16感染可分別引發(fā)完全自噬與非完全自噬現(xiàn)象,且兩者均可以通過誘發(fā)自噬而抑制TLR7、 MyD88、IRF7的表達(dá),從而來抑制IFN-I的產(chǎn)生水平,另外,EV71和CA16也均可以通過誘發(fā)的自噬來促進(jìn)病毒受體的表達(dá)。本論文的研究結(jié)果為CA16感染和免疫機(jī)理研究提供了線索,初步解釋了CA16重復(fù)感染的機(jī)理,并部分了揭示了EV71和CA16參與免疫逃逸的機(jī)理,為EV71和CA16引起的HFMD的治療提供了新的靶點(diǎn)。
[Abstract]:Hand-foot-and-mouth disease (HFMD) is a global disease. Since the 90s of last century, it has maintained high incidence in mainland China, Taiwan, Singapore and other regions of Asia. Its susceptible population is mainly children under 5 years of age. Death, which has become an important infectious disease that seriously endangers children's health and public health safety, has shown that HFMD's pathogenicity is mainly the enterovirus 71 (Enterovirus 71, EV71) and the coxsackievirus A group 16 (Coxsackie virus A 16, CA16) in the family of small ribonucleic viruses. In recent years, most studies have concentrated on the study of HFMD. The infection of EV71 is not enough to pay attention to the infection of CA16. In clinical, EV71 infection may lead to severe cases and death cases, and the symptoms of CA16 infection are generally mild, but the basic and clinical symptoms of HFMD patients caused by CA16 infection are basically consistent with those of EV71 infection. In addition, CA16 virus is the same. The epidemiological phenomenon of repeated infection in the population has attracted more and more attention. Although the EV71 inactivated vaccine has been listed, the differences in the clinical symptoms caused by EV71 and CA16 infection, and the mechanism and immune mechanism of the two are still not very clear, and these problems still need more in-depth study. The first part of the CA16 infinfancy model was established in the previous study. The first part of this paper was first to analyze the whole genome expression profile of the monkey and baby monkey with repeated CA16 infection in Ganges RIver. The results showed that, compared with the infection of the monkey and monkey of Ganges RIver monkey, the monkey and monkey were infected with 948 common difference genes at each time after infection with the CA16. The analysis of Gene Ontology (GO) found that these differential genes were mainly concentrated in 5 biological processes, including cell communication, cell cycle, immune system process, transcriptional regulation, metabolic process, indicating that the 5 biological processes involved in the development of the disease in the different stages of the infection of the monkey and monkey of the monkey and monkey in Ganges RIver. After that, we carried out a co expression network analysis of the most significant 10 genes up and down in various genes, and found that IL-6, IL-8, IL-18 as the key regulatory genes, they are important related genes involved in the inflammatory response and immune response. The inflammatory pathway and the interleukins signaling pathway, which are distributed in chemokines and cytokine mediated pathways, further demonstrate that the inflammatory response and immune response play a major role in different stages of CA16 infection in monkeys and monkeys in Ganges RIver. The mobilization of Type I Interferon (IFN-I) around the innate immune response to the body's antiviral activity In the end, we also analyzed genes associated with IFN-I and autophagy related genes in the chip. The results showed that most of the genes associated with IFN-I and autophagy related genes were down regulated, which provided clues and basis for subsequent studies. The second part of this paper was provided in the first part. EV71 and CA16 were directly infected with 16HBE in human normal respiratory epithelial cells, and the changes in the related genes produced by IFN-I were detected after the infection. The results showed that the gene expression of TLR3, MAVS, MDA5, MyD88, IRF7, IFN alpha and IFN beta in the EV71 infection group were significantly higher than those of the control group. TLR7, MAVS, RIG-I, MyD88, IRF7, IFN, and IFN beta had no significant changes in gene expression levels. In addition, the viral titer and viral load showed that the replication efficiency of the CA16 infection group was higher on 16HBE than in the EV71 infection group. The results suggested that the EV71 infection after 16HBE was available. It can significantly induce activation of the related pathway of IFN-I production, but the production of IFN-I is not enough to resist the invasion of EV71, so EV71 can still achieve proliferation after 16HBE infection, and CA16 infection 16HBE can not trigger the production of IFN-I. Therefore, it is presumed that CA16 infection 16HBE may induce some mechanism to escape its natural immunity in 16HBE. The third part of this paper, based on the second part, further explores the reasons for the difference between IFN-I and EV71 caused by EV71 and CA16 infection, so we lock up the sense of EV71 and CA16 in the third part of this paper. The effects of autophagy induced by 16HBE on the production of IFN-I and the expression of viral receptor. The results showed that both endogenous LC3 staining in 16HBE or the introduction of exogenous LC3 plasmids into 16HBE was found to induce complete autophagy in 16HBE cells after EV71 infection, and CA16 infection induced 16HBE cells. After the autophagy inhibitor 3MA, the activity of EV71 and CA16 infected 16HBE cells decreased significantly, and the plaque formation unit (plaque forming units, PFUs) descended significantly, and the production of TLR7, MyD88, and IRF7 mediated IFN-I, at the gene level, was at the gene level. At the protein level, TLR7, MyD88, and IRF7 also increased significantly at the protein level of 16HBE infected 16HBE. In addition, the results of immunofluorescence also showed that there was not only a co localization phenomenon between the TLR7 and the internal body marker M6PR, but also the significant increase in the expression of TLR7 after the inhibition of EV71 and CA16 induced autophagy, these results said Both EV71 and CA16 infection can inhibit the production of TLR7, MyD88, IRF7 mediated IFN-I by inducing autophagy and further promoting the packaging maturity of the virus itself. In addition, we also found that the proportion of EV71 and CA16 in 16HBE on 16HBE on the 16HBE and CA16 is also significant after inhibition of the autophagy induced by EV71 and CA16. Down down, indicating that EV71 and CA16 may promote the expression of autophagy by inducing autophagy to promote the expression of its cell receptor and further enhance its own invasion. These results suggest that EV71 and CA16 infection may cooperate with the virus to participate in immune escape by inhibiting the expression of IFN-I and promoting the expression of the virus receptor. To sum up, this study first passed the sense of CA16. The analysis of the whole genome expression profiles of Ganges RIver monkeys revealed the differential gene expression profiles caused by CA16 infection for the first time. Subsequently, by comparing the changes in the related genes produced by EV71 and CA16 infected with 16HBE, it was found that EV71 and CA16 could induce differential IFN-I production by different modes of recognition and downstream signal molecules, and IFN-I, and IFN-I It may directly affect the infection and proliferation ability of both of them on 16HBE. Finally, the effects of autophagy induced by EV71 and CA16 on the production of WN-I and the expression of virus receptors are also discussed. It is found that EV71 and CA16 infection can induce complete autophagy and incomplete autophagy, and both can inhibit TLR7, MyD88, IRF7 by inducing autophagy. In addition, EV71 and CA16 can also promote the expression of the virus receptor by inducing autophagy. The results of this paper provide clues for the study of CA16 infection and immune mechanism, preliminarily explain the mechanism of CA16 repeat infection, and partly reveal the mechanism of EV71 and CA16 in immune escape. Rationale provides a new target for the treatment of HFMD induced by EV71 and CA16.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R512.5

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