CA16感染恒河猴全基因组表达谱分析及其感染诱发的自噬抑制Ⅰ型干扰素产生的研究

发布时间：2018-05-18 04:28

本文选题：肠道病毒71型 + 柯萨奇病毒A组16型　；参考：《北京协和医学院》2016年博士论文

【摘要】：手足口病(hand-foot-and-mouth disease, HFMD)是一种全球性的疾病,自上个世纪九十年代以来,在中国大陆、台湾、新加坡和亚洲其它地区一直保持高发态势,其易感人群主要是5岁以下婴幼儿,该疾病易致罹患者并发神经系统相关重症,甚至可能引发死亡,已经成为严重危害儿童健康和公共卫生安全的重要传染性疾病。研究表明,HFMD的致病原主要是小核糖核酸病毒科的肠道病毒71型(Enterovirus 71, EV71)和柯萨奇病毒A组16型(Coxsackie virus A 16, CA16)。近年来对于HFMD的研究大多数集中于EV71的感染,而对CA16的感染缺乏足够的重视。临床上,EV71感染易引发重症病例及死亡病例,而CA16感染所致的症状普遍较轻,但CA16感染所致HFMD患者一旦发展到重症阶段,其基本情况和临床症状特征基本与EV71感染病例一致。另外,CA16病毒在人群中的重复感染的流行病学现象也越来越引起人们的广泛关注。尽管EV71灭活疫苗已经上市,但EV71与CA16感染导致的临床症状的差异,以及两者的感染机制与免疫机理仍不十分清楚,这些问题依然需要更深入的研究。基于本实验室在前期研究中建立的CA16婴猴感染模型,本论文的第一部分首先对CA16重复感染恒河猴婴猴的全基因组表达谱进行了分析,结果显示,与恒河猴婴猴感染CA16前相比,恒河猴婴猴感染CA16后各个时间共出现了948个共同差异基因；将这些差异基因进行基因本体论(Gene Ontology, GO)分析后发现：这些差异基因主要集中在5个生物学过程,包括：细胞通讯、细胞周期、免疫系统过程、转录调节、代谢过程,说明在恒河猴婴猴感染CA16的不同阶段中,这5个生物学过程参与了其引发疾病的进程；随后,我们对各类基因中上调和下调最显著的10个基因进行了共表达网络分析,发现IL-6、IL-8、IL-18等作为了关键调控基因,它们是参与炎症反应及免疫反应的重要相关基因；紧接着我们对免疫相关的基因进行了通路分析,发现其主要分布在趋化因子和细胞因子介导的炎症通路以及白介素信号通路,进一步说明在恒河猴婴猴感染CA16的不同阶段中,炎症反应与免疫反应发挥着主要作用。围绕着机体抗病毒的固有免疫反应Ⅰ型干扰素(Type Ⅰ Interferon, IFN-I)动员的特征,最后我们还对芯片中与IFN-I产生相关的基因及与细胞自噬相关的基因进行了分析,结果发现与IFN-I产生相关的基因和自噬相关的基因大多发生了下调表达,这为后续的研究提供了线索和依据。本论文的第二部分根据第一部分提供的线索,将EV71与CA16直接感染人正常呼吸道上皮细胞16HBE,并检测两者感染后IFN-I产生相关基因的改变,结果发现,与对照组相比,EV71感染组中TLR3、MAVS、MDA5、MyD88、IRF7、 IFNα和IFNβ的基因表达量均发生了显著性地上调,而在CA16感染组中TLR3和IRF3的基因表达水平显著性地下降,TLR7、MAVS、RIG-I、MyD88、IRF7、 IFNα和IFNβ的基因表达水平均无显著性地变化。此外,病毒滴度和病毒载量的检测结果显示,相比于EV71感染组,CA16感染组在16HBE上的复制效率更高。上述结果提示：尽管EV71感染16HBE后可能会显著性地诱发IFN-I产生相关通路的激活,但产生的IFN-I尚不足以抵抗EV71的入侵,故EV71感染16HBE后仍可实现增殖,而CA16感染16HBE后并不能触发IFN-I的产生,因此,推测CA16感染16HBE后可能诱发了某种机制使其逃避了其在16HBE上的天然免疫反应,并使其更容易在16HBE上实现快速地增殖,这揭示了EV71与CA16可能通过不同的途径增强其自身的感染能力。本论文的第三部分在第二部分的基础上,进一步探究引发EV71和CA16感染所致IFN-I产生差异性的原因,故我们锁定在EV71和CA16感染16HBE后诱发的细胞自噬现象对IFN-I产生及病毒受体表达的影响。结果显示,无论是16HBE中的内源性LC3染色还是将外源性转荧光标记的LC3质粒导入16HBE,我们均发现在EV71感染后可诱导16HBE细胞发生完全自噬,而CA16感染则诱导16HBE细胞发生非完全自噬；在给予自噬抑制剂3MA后,EV71与CA16感染的16HBE的细胞活性显著性地下降,病毒空斑形成单位(plaque forming units, PFUs)显著性地下降,且在自噬现象被抑制后,在基因水平上,TLR7、MyD88、 IRF7介导的IFN-I的产生在EV71与CA16感染的16HBE中显著性地上升,而在蛋白水平上,TLR7、MyD88、IRF7也在EV71与CA16感染的16HBE中显著性地上升,另外,免疫荧光的结果也显示TLR7与内体标志物M6PR不仅存在共定位现象,而且在抑制EV71与CA16诱导的自噬后TLR7的表达强度显著性增加,这些结果说明EV71与CA16感染均可以通过诱导细胞自噬而抑制TLR7、 MyD88、IRF7介导的IFN-I的产生,进而促进病毒自身的包装成熟。此外,我们还发现,在抑制EV71与CA16诱导的自噬后,EV71与CA16在16HBE上的病毒入侵受体SCARB2和CD162的阳性细胞比例也会显著性地下降,说明EV71与CA16可能通过诱发自噬的发生而促进其感染细胞受体的表达,进而增强其自身的入侵。上述结果提示EV71与CA16感染可能通过抑制IFN-I和促进病毒受体的表达来协作病毒参与免疫逃逸。综上所述,本研究首先通过对CA16感染恒河猴的全基因组表达谱的分析,首次揭示了CA16感染引起的差异基因表达谱；随后通过比较EV71与CA16感染16HBE后IFN-I产生相关基因的变化,发现EV71和CA16可以通过不同的模式识别受体及下游信号分子而诱发差异性的IFN-I产生,且IFN-I的产生可能会直接影响两者在16HBE上的感染和增殖能力；最后还探讨了EV71和CA16诱导的自噬对WN-I产生和病毒受体表达的影响,发现EV71和CA16感染可分别引发完全自噬与非完全自噬现象,且两者均可以通过诱发自噬而抑制TLR7、 MyD88、IRF7的表达,从而来抑制IFN-I的产生水平,另外,EV71和CA16也均可以通过诱发的自噬来促进病毒受体的表达。本论文的研究结果为CA16感染和免疫机理研究提供了线索,初步解释了CA16重复感染的机理,并部分了揭示了EV71和CA16参与免疫逃逸的机理,为EV71和CA16引起的HFMD的治疗提供了新的靶点。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.5

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