手足口病病毒EV71感染与细胞表面受体表达以及胞内天然免疫通路的关系

发布时间：2018-07-20 13:25

【摘要】：肠道病毒71型(EV71)为单正链的RNA病毒,小核糖核酸病毒科,肠道病毒属。EV71是引起小儿手足口病的常见病原体,重症感染可引起脑干脑炎、脑脊髓炎及急性迟缓性麻痹等严重并发症。自2008年以来,EV71在我国大陆多省市持续的暴发流行,导致数百婴幼儿死亡,已造成严重的社会危害。病毒感染细胞的首要环节是其表面特定的配体与宿主细胞膜表面特异性受体的识别吸附。此外,先天免疫系统对于识别病毒感染及激活随后的适应性免疫应答有至关重要的作用,目前研究发现的三种模式识别受体：RLRs, TLRs以及NLRs,这些受体能够识别病毒感染后产生的非自身的核酸成分,通过招募下游的接头蛋白,激活Ⅰ型干扰素及细胞因子的产生,发挥抗病毒效应。其中,多种细胞系(非免疫细胞)利用RLRs识别RNA病毒的感染,而pDC利用TLRs识别病毒的入侵,NLRs在识别ssRNA病毒感染方面发挥一定作用。然而,并不是只要有病毒进入细胞就能够引起抗病毒免疫的过程,激发有效抗病毒先天免疫的两个主要的条件是：1)宿主免疫系统具有有效识别入侵病毒的受体；2)触发干扰素级联反应的蛋白系统,这二者缺一不可。 2009年日本学者发现EV71感染上皮细胞的特异性受体为SCARB2,在不易感的细胞中过表达SCARB2可明显促进病毒的感染。在本实验中,我们首先对不同细胞系细胞表面受体高低水平与病毒感染能力的相关性进行分析,结果发现：EV71对于细胞表面受体水平较低的细胞难以感染,但对于受体表达均处于较高水平且表达量基本一致的不同细胞系,EV71对其感染能力却存在巨大的差异。例如对EV71非常易感的RD细胞及较难感染的HeLa细胞,其细胞表面SCARB2表达水平基本一致,但病毒产率却相差2个log以上。究其原因,我们用生物素标记的病毒进行病毒吸附实验,用ELISA方法对吸附病毒进行定量检测,发现EV71对RD及HeLa这两种细胞吸附能力相同,流式细胞术也进一步证实了这一现象,从而排除细胞表面因素的影响,进一步探讨胞内主要发挥抗病毒作用的蛋白——RIG-I、MDA5、NOD2及MAVS等的表达水平,结果显示RD细胞内源性RIG-I表达水平特别低,并且不能有效诱导干扰素相关抗病毒基因ISGs等的表达。为进一步证实RIG-I在EV71感染中的作用,在内源性水平低的RD细胞过表达HA-RIG-I,可明显抑制病毒的感染,反之,在表达水平高的HeLa细胞内用siRNA干扰RIG-I的表达可明显增加病毒的感染,且具有剂量依赖性。综上：病毒对细胞的感染能力受细胞表面特异性受体及胞内天然免疫通路双重调控,具体来讲,对于手足口病病毒EV71,除了SCARB2这一细胞表面特异性受体外,胞内天然免疫通路中主要识别RNA病毒感染的蛋白RIG-I的表达水平也决定了病毒对细胞的感染能力。
[Abstract]:Enterovirus 71 (EV71) is a single-stranded RNA virus, small ribonucleic acid virus family, enterovirus. EV71 is a common pathogen of hand, foot and mouth disease in children. Severe infection can cause brainstem encephalitis. Severe complications such as encephalomyelitis and acute delayed paralysis. Since 2008, the outbreak of EV71 in many provinces and cities of China has caused hundreds of infant deaths and caused serious social harm. The first step of viral infection is the recognition and adsorption of specific ligands on the surface of the cell surface and specific receptors on the surface of the host cell membrane. In addition, the innate immune system plays a crucial role in identifying viral infections and activating subsequent adaptive immune responses. Three types of pattern recognition receptors, namely: RLRRs, TLRs and NLRss, have been found to recognize non-self-contained nucleic acids produced by virus infection and activate the production of interferon type I and cytokines by recruiting downstream junction proteins. Give play to the antiviral effect. Among them, many cell lines (non-immune cells) use RLRs to recognize RNA virus infection, while pDC uses TLRs to recognize the invasion of virus and NLRs play a role in the identification of ssRNA virus infection. However, it is not possible to induce antiviral immunity as long as viruses enter the cells. The two main conditions for triggering effective innate antiviral immunity are: 1) the host immune system has an effective receptor to recognize invading viruses; In 2009, Japanese researchers found that the specific receptor of EV71 infected epithelial cells was SCARB2, and that overexpression of SCARB2 in unsusceptible cells could significantly promote the infection of the virus. 2) the protein system that triggers the cascade reaction of interferon is indispensable. In 2009, Japanese researchers found that the specific receptor of EV71 infected epithelial cells was SCARB2. In this experiment, we first analyzed the relationship between the cell surface receptor level and virus infection ability in different cell lines. The results showed that it was difficult to infect cells with lower cell surface receptor level. However, there were great differences in the infection ability of different cell lines (EV71) with high level of receptor expression and similar expression levels. For example, the expression level of SCARB2 on the surface of Rd cells, which is very susceptible to EV71, and HeLa cells, which are difficult to infect, is basically the same, but the virus yield is more than two log. The reason is that we used biotin labeled virus to carry out virus adsorption test and Elisa method to quantitatively detect the adsorbed virus. We found that EV71 has the same adsorption ability to Rd and HeLa cells, and flow cytometry further confirmed this phenomenon. In order to eliminate the effect of cell surface factors, the expression levels of RIG-Igna MDA5NOD2 and MAVs were further investigated. The results showed that the expression of endogenous RIG-I was very low in Rd cells, and the expression level of RIG-I was very low in Rd cells. Moreover, it can not effectively induce the expression of interferon-associated antiviral genes such as ISGs. In order to further confirm the role of RIG-I in EV71 infection, overexpression of HA-RIG-I in Rd cells with low endogenous level could significantly inhibit virus infection, whereas siRNA interference with RIG-I expression in HeLa cells with high expression level could significantly increase virus infection. And it is dose dependent. In conclusion, the ability of virus to infect cells is regulated by cell surface specific receptor and intracellular innate immune pathway. Specifically, for HFMD virus EV71, in addition to SCARB2, a cell surface specific receptor, The expression level of RIG-I, which mainly recognizes RNA virus infection in intracellular innate immune pathway, also determines the ability of virus to infect cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

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