發(fā)布時(shí)間：2018-09-14 17:13

【摘要】：一、 研究背景和目的腸道病毒71型(Human Enterovirus 71,簡稱EV71)屬于小核糖核酸病毒科、腸道病毒A屬,是引起嬰幼兒手足口病(Hand Foot Mouth Disease, HFMD)的主要病原體之一,其感染常引起兒童手足口病、皰疹性咽峽炎等較輕的臨床癥狀。然而,越來越多的報(bào)道揭示EV71的感染可引起嚴(yán)重的神經(jīng)系統(tǒng)綜合征,包括無菌性腦膜腦炎、腦干腦炎、脊髓灰質(zhì)炎樣麻痹、神經(jīng)源性肺水腫,嚴(yán)重者甚至死亡。自從1969年首次在美國加利福尼亞州分離該病毒,此后在保加利亞、馬來西亞、新加坡、日本、越南、中國均引起過大規(guī)模流行。據(jù)廣東省衛(wèi)計(jì)委5月17日發(fā)布的手足口病預(yù)警信息,截止5月16日,全省今年共報(bào)告手足口病88366例,其中死亡3例,較2015年同期病例數(shù)(51327例)上升72%,以EV71病毒致死率最高。作為手足口病的主要病原之一,EV71已經(jīng)成為目前全球范圍內(nèi)取代脊髓灰質(zhì)炎病毒,對公共衛(wèi)生安全威脅最嚴(yán)重的中樞神經(jīng)毒性病原。因此,探討EV71致神經(jīng)系統(tǒng)感染的影響因素對EV71的防治意義重大。從分子生物學(xué)的角度來看,EV71為RNA病毒,在復(fù)制過程中缺乏核酸外切酶的校對功能而導(dǎo)致較高的出錯(cuò)率,約每復(fù)制一個(gè)基因就有一個(gè)自發(fā)突變。VP1區(qū)由于其高度多樣性和較少參與重組而被認(rèn)為是腸道病毒進(jìn)化研究中最有意義的基因區(qū)域。有研究表明,VP1的變異與神經(jīng)系統(tǒng)的感染可能存在相關(guān)關(guān)系,決定EV71神經(jīng)毒性可能是單一位點(diǎn)的突變,也有可能是多個(gè)位點(diǎn)突變共同作用的結(jié)果。有研究在分析具有神經(jīng)毒性的EV71分離株時(shí)發(fā)現(xiàn),VP1上有一個(gè)重要的氨基酸變異Alal 70Val(丙氨酸170頡氨酸),猜測該變異使得病毒蛋白結(jié)構(gòu)發(fā)生變化,改變了病毒與受體的結(jié)合能力。然而,此研究只是基于少量的樣本發(fā)現(xiàn)的突變位點(diǎn),突變位點(diǎn)少,且突變的位點(diǎn)對人群發(fā)病的影響有多大意義并不清楚。因此,我們旨在通過更大樣本的人群來篩選,以驗(yàn)證VP1蛋白變異是否與EV71神經(jīng)系統(tǒng)感染有關(guān)。此外,一項(xiàng)關(guān)于大腦組織TLR-5和TLR-6受體的表達(dá)與EV71感染致神經(jīng)系統(tǒng)損傷的研究發(fā)現(xiàn),腦組織病毒受體的高表達(dá)與神經(jīng)系統(tǒng)損傷有關(guān)。另有研究也證實(shí)EV71受體對于病毒感染細(xì)胞和組織至關(guān)重要。病毒進(jìn)入細(xì)胞的第一步便是與細(xì)胞表面的病毒受體結(jié)合,病毒進(jìn)入細(xì)胞的主要途徑通過病毒受體介導(dǎo)的RNA釋放或者胞吞作用,因此猜測病人自身的受體數(shù)量、病毒蛋白與受體的相互作用可能也與神經(jīng)系統(tǒng)的感染有關(guān)。目前已知的EV71的受體有：P-selectin glycoprotein ligand-1 (PSGL-1)、 Human scavenger receptor class B2(SCARB2), Vimentin(VIM)。近年來,越來越多研究報(bào)道了VIM在病原體吸附、胞吞、遷移過程中的重要作用。VIM是新近發(fā)現(xiàn)的EV71受體,在人腦微血管細(xì)胞中表達(dá)豐富并介導(dǎo)大腸桿菌細(xì)菌侵襲,EV71感染與相類似。因此猜測EV71通過VIM受體介導(dǎo)進(jìn)入人腦微血管內(nèi)皮細(xì)胞而感染神經(jīng)系統(tǒng)。根據(jù)已有文獻(xiàn)報(bào)道我們提出：EV71病毒的變異和患者自身受體的差異可能是影響EV71感染神經(jīng)系統(tǒng)的因素,二者或獨(dú)立作用或存在交互作用。本文主要從廣東省2008年～2010年EV71的流行病學(xué)分析、VIM在EV71感染HBMEC中的作用兩方面來探究EV71所致神經(jīng)系統(tǒng)的影響因素,以確定EV71病毒VP1蛋白的A289T變異,以及VIM受體與EV71神經(jīng)系統(tǒng)感染的關(guān)系；并明確EV71病毒感染血腦屏障細(xì)胞是神經(jīng)系統(tǒng)感染的一條重要途徑。本研究不但有助于分析EV71的流行病特征、病毒變異情況；也將為EV71病毒的神經(jīng)系統(tǒng)感染建立有意義的預(yù)警信號,及早發(fā)現(xiàn)易感人群,及時(shí)采取有效的防范措施,對于減少神經(jīng)系統(tǒng)感染的發(fā)病率具有重要的意義。二、 研究方法1.廣東省2008～2010年EV71的流行病學(xué)分析收集2008～2010年廣東省多個(gè)市區(qū)送至廣東省疾病預(yù)防控制中心檢驗(yàn)的樣本(包括糞便、咽拭子、腦脊液等),采用熒光定量PCR技術(shù)和病毒分離培養(yǎng)技術(shù)確診為EV71感染的共194例樣本。根據(jù)癥狀的差異分成EV71神經(jīng)系統(tǒng)感染組和非神經(jīng)系統(tǒng)感染兩組。分別提取病人的RNA,逆轉(zhuǎn)錄合成cDNA,并擴(kuò)增全長的病毒VP1序列。通過比對病毒VP1序列和構(gòu)建系統(tǒng)進(jìn)化樹確定病毒的分型；采用Logistic回歸分析影響患者致神經(jīng)系統(tǒng)感染的因素；病毒的VP1基因序列用DNAman軟件翻譯成蛋白質(zhì)序列,定義在同一個(gè)位點(diǎn)上,出現(xiàn)兩種以上氨基酸為一個(gè)變異。2.人腦微血管內(nèi)皮細(xì)胞VIM敲除細(xì)胞系的建立在美國國立生物技術(shù)信息中心(NCBI)的數(shù)據(jù)庫中找到VIM的基因序列,找到VIM基因CDS區(qū),分析其基因結(jié)構(gòu),明確CDS的外顯子部分。按照基因本身的性質(zhì),選擇候選的待敲除位點(diǎn)后設(shè)計(jì)并合成識別靶位點(diǎn)的一對DNAOligos。把sgRNA連接到Lenti CRISPRv2載體中并構(gòu)建Cas9質(zhì)粒。將構(gòu)建好的目的質(zhì)粒及空白對照分別包裝成慢病毒再感染HBMEC,篩選出穩(wěn)定表達(dá)VIM-KO的細(xì)胞株以及正常對照株。3.VIM在EV71感染HBMEC中的作用分析在RD細(xì)胞中擴(kuò)增并滴定的病毒分別感染正常和VIM-KO的HBMEC,觀察HBMEC的病變效應(yīng)。采用熒光定量PCR技術(shù)檢測EV71病毒在正常和VIM-KO的細(xì)胞中吸附能力、復(fù)制能力和病毒核酸釋放水平的差異。同時(shí)檢測在HBMEC正常和VIM-KO細(xì)胞中病毒VPl蛋白合成的差異。三、 結(jié)果1.廣東省2008～2010年EV71的主要型別為C4a型在2008～2010年期間,廣東省各個(gè)地區(qū)流行的EV71病毒株為C4a型,也是目前我國主要的流行株。同時(shí)發(fā)現(xiàn)此型在廣東省流行過程中已經(jīng)開始演化,增加了三個(gè)族,即C4a2-C4a4族。2.EV71病毒株型別的時(shí)間、地區(qū)分布從各病毒株的分布時(shí)間來看：2008年四種均有,但主要集中在C4a1型；2009年的流行株為C4a1和C4a2型；而在2010年,除湛江的一株屬于C4a4型,其余均為C4a1型。從地區(qū)分布來看：大部分地區(qū)的病毒株型別為C4a1和C4a2型；只有2008年東莞分株009-08-M單獨(dú)構(gòu)成了C4a3型；C4a4也僅分別出現(xiàn)在佛山和湛江。綜合來看各病毒株的流行并沒有地區(qū)和時(shí)間集中趨勢。3.病毒A289T變異與神經(jīng)系統(tǒng)感染有關(guān)隨著年齡的增加,神經(jīng)系統(tǒng)感染患者的發(fā)病率明顯下降(P0.05)。對患者進(jìn)一步用Logistic回歸法分析其性別、年齡、病毒型別、VP1變異對患者臨床癥狀輕重有無神經(jīng)系統(tǒng)癥狀的影響。結(jié)果表明EV71患者的臨床癥狀與性別、病毒的型別與有無神經(jīng)系統(tǒng)癥狀沒有無統(tǒng)計(jì)學(xué)相關(guān)性：年齡和病毒A289T變異與患者的臨床表現(xiàn)相關(guān),病毒289A(289丙氨酸)容易致EV71神經(jīng)系統(tǒng)感染(P0.05,OR=2.360,95% CI為1.163-4.659)。4.人腦微血管內(nèi)皮細(xì)胞VIM敲除細(xì)胞系的成功建立采用CRISPR-Cas9技術(shù)及慢病毒載體包裝系統(tǒng)包裝出VIM-KO和正常對照的病毒后,感染HBMEC 48 h后觀察到GFP的表達(dá)接近70%-80%。用嘌呤霉素篩選出穩(wěn)定的HBMEC VIM-KO和對照細(xì)胞株后,用VIM的特異性抗體分別檢測HBMEC對照和VIM-KO中的VIM表達(dá)量。結(jié)果發(fā)現(xiàn)VIM-KO的細(xì)胞中VIM的表達(dá)量下降了90%以上,說明此次VIM敲除效果較好。5.VIM在EV71感染HBMEC中的作用分析EV71進(jìn)入細(xì)胞后,由于其對細(xì)胞的損傷,會(huì)導(dǎo)致細(xì)胞的形態(tài)發(fā)生改變。分別用病毒感染對照以及VIM-KO的HBMEC,發(fā)現(xiàn)病毒感染HBMEC正常對照細(xì)胞24 h后,細(xì)胞出現(xiàn)了皺縮,變圓甚至破碎；隨著感染時(shí)間的延長,到了48h后,部分細(xì)胞已經(jīng)脫落而飄浮在培養(yǎng)中。相比之下,EV71同樣的MOI感染VIM-KO,24 h后細(xì)胞的形態(tài)幾乎不變,即使到了48 h,細(xì)胞的形態(tài)改變很小,只有很小一部分細(xì)胞開始變圓。說明EV71感染能引起HBMEC病變效應(yīng),但在VIM敲除的細(xì)胞中,細(xì)胞狀態(tài)變化不大,病變效應(yīng)很弱。EV71侵襲細(xì)胞的第一步是吸附于細(xì)胞表面。用熒光定量PCR檢測HBMEC對照和VIM-KO表面的病毒核酸量的差異,結(jié)果表明與對照相比,VIM敲除后EV71在細(xì)胞表面的吸附下降了40%。EV71感染細(xì)胞24 h和48h后,正常的HBMEC中病毒量分別同比增加到3倍和8倍,而VIM-KO細(xì)胞復(fù)制的非常少,24 h后僅為0 h的1.3倍,48h后病毒量也僅為0 h的2倍左右。EV71感染HBMEC 48 h后,VIM敲除細(xì)胞EV71病毒核酸量是對照細(xì)胞的1/12。細(xì)胞培養(yǎng)液上清EV71病毒核酸量檢測結(jié)果表明,VIM敲除的細(xì)胞比對照細(xì)胞少1.4倍。EV71病毒在細(xì)胞內(nèi)的核酸復(fù)制和合成蛋白是基本同步的,通過檢測EV71在HBMEC對照和敲除的細(xì)胞內(nèi)合成蛋白的差異,發(fā)現(xiàn)VIM敲除后病毒VP1蛋白的合成也比HBMEC對照細(xì)胞少很多。四、 結(jié)論1、在2008～2010年期間,廣東省各個(gè)地區(qū)流行的EV71病毒株為C4a型,也是目前我國主要的流行株。同時(shí)發(fā)現(xiàn)此型在廣東省流行過程中已經(jīng)開始演化,增加了三個(gè)族,即C4a2-C4a4族。EV71病毒亞型在時(shí)間、地區(qū)上的分布無明顯趨勢。2、病毒的A289T變異與與EV71神經(jīng)系統(tǒng)感染有關(guān),病毒289A(289丙氨酸)容易致EV71神經(jīng)系統(tǒng)感染。本次所獲得的A289T變異位點(diǎn)是廣東省194例EV71感染人群的分子流行病學(xué)的研究結(jié)果,此位點(diǎn)的進(jìn)一步研究,對于廣東省乃至全國的EV71感染的防治具有實(shí)際的指導(dǎo)意義。3、VIM是HBMEC表面EV71的受體,EV71通過與受體VIM的結(jié)合而侵襲HBMEC,并且進(jìn)一步影響EV71在細(xì)胞中的復(fù)制甚至釋放。本課題提出EV71感染神經(jīng)系統(tǒng)的通路假說：EV71通過VIM受體介導(dǎo)的RNA釋放,進(jìn)入HBMEC細(xì)胞,穿越血腦屏障,最終感染神經(jīng)系統(tǒng)。此項(xiàng)研究將有助于揭示EV71的神經(jīng)系統(tǒng)感染機(jī)制。4、本文將病毒變異研究與受體研究相結(jié)合,認(rèn)為病毒的變異與自身的受體同時(shí)影響EV71致神經(jīng)系統(tǒng)感染。這為EV71致神經(jīng)系統(tǒng)感染的分析提供一條有意義的線索。
[Abstract]:Background and Objectives: Human Enterovirus 71 (EV71) belongs to the family of small ribonucleoviruses. Enterovirus A (EV71) is one of the main pathogens causing Hand Foot Mouth Disease (HFMD) in infants and young children. Its infection often causes minor clinical symptoms such as hand-foot-mouth disease (HFMD) and herpes angina. A growing number of reports have revealed that EV71 infection can cause severe neurological syndrome, including aseptic meningoencephalitis, brainstem encephalitis, poliomyelitis-like paralysis, neurogenic pulmonary edema, and even death. The virus was first isolated in California, USA, in 1969, and has since been found in Bulgaria, Malaysia, Singapore, etc. As of May 16, 88 366 cases of hand-foot-mouth disease (HFMD) have been reported in Guangdong Province, including 3 deaths, 72% higher than the corresponding number of cases (51 327 cases) in 2015. EV71 is the leading cause of HFMD. First, EV71 has become the most serious central neurotoxic pathogen in the world, replacing poliovirus and threatening public health. Therefore, it is of great significance to explore the influencing factors of nervous system infection caused by EV71 for the prevention and treatment of EV71. VP1 region is considered to be the most significant gene region in the evolution of enterovirus because of its high diversity and less recombination. Studies have shown that VP1 mutation may be associated with nervous system infection. Neurotoxicity of EV71 may be a single site mutation or a combination of multiple site mutations. In the analysis of neurotoxic EV71 isolates, an important amino acid variant Alal 70Val (alanine 170 pi) was found in VP1. It was speculated that the mutation caused structural changes in the protein of the virus. However, this study is based on a small number of mutation sites, and the significance of the mutation site on the pathogenesis of the population is not clear. Therefore, we aim to screen a larger sample of people to verify whether VP1 protein mutation is associated with EV71 infection of the nervous system. In addition, a study on the expression of TLR-5 and TLR-6 receptors in brain tissue and neurological damage caused by EV71 infection found that the high expression of virus receptors in brain tissue was associated with neurological damage. Other studies have also confirmed that EV71 receptors are essential for viral infection of cells and tissues. The first step for viruses to enter cells is with the cell surface. Viral receptors bind to the surface of the virus, and the main way the virus enters the cell is through RNA release or cytophagy mediated by the virus receptor. Therefore, it is speculated that the number of receptors in the patient and the interaction between the virus protein and the receptor may also be related to the infection of the nervous system. The known receptors for EV71 are P-selectin glycoprotein ligand-1 (PS-71). GL-1, Human scavenger receptor class B2 (SCARB2), Vimentin (VIM). In recent years, more and more studies have reported that VIM plays an important role in pathogen adsorption, endocytosis and migration. We have reported that the mutation of EV71 virus and the difference of the patient's own receptor may be the factors influencing the infection of EV71 in the nervous system. The two factors either act independently or interact with each other. This paper mainly from 2008 to 2010 in Guangdong Province. Epidemiological analysis of EV71 in 1997 showed that the role of VIM in EV71 infection with HBMEC was related to the factors affecting the nervous system caused by EV71 in order to determine the A289T variation of VP1 protein of EV71 and the relationship between VIM receptor and the infection of EV71 nervous system. This study will not only help to analyze the epidemiological characteristics of EV71 and the variation of EV71 virus, but also establish a meaningful early warning signal for the infection of the nervous system of EV71 virus, early detection of susceptible groups, and timely and effective preventive measures. It is of great significance to reduce the incidence of infection of the nervous system. 2. Research methods 1. Guangdong 200 Epidemiological analysis of EV71 from August to 2010 collected 194 samples (including feces, pharyngeal swabs, cerebrospinal fluid, etc.) from several urban districts of Guangdong Province sent to the Guangdong Center for Disease Control and Prevention for examination (including feces, swabs, cerebrospinal fluid) from 2008 to 2010. Systemic infection group and non-nervous system infection group. RNA was extracted from patients, reverse transcription was used to synthesize cDNA, and full-length VP1 sequence was amplified. Viral typing was determined by comparing VP1 sequence and constructing phylogenetic tree. Logistic regression analysis was used to analyze the influencing factors of nervous system infection. NAman software translation into protein sequence, defined at the same site, there are more than two amino acids for a mutation. 2. Human brain microvascular endothelial cell VIM knockout cell line established in the United States National Center for Biotechnology Information (NCBI) database to find the VIM gene sequence, find the VIM gene CDS region, analyze its gene structure, clear C According to the nature of the gene itself, a pair of DNA Oligos was designed and synthesized to identify the target site. The sgRNA was linked to the Lenti CRISPRv2 vector and the Cas9 plasmid was constructed. The role of VIM in the infection of HBMEC by EV71 was analyzed. The viruses amplified and titrated in RD cells were infected with normal and VIM-KO HBMEC respectively, and the pathological changes of HBMEC were observed. The adsorption, replication and nucleic acid release of EV71 in normal and VIM-KO cells were detected by fluorescence quantitative PCR. At the same time, the difference of VPl protein synthesis between normal HBMEC and VIM-KO cells was detected. 3. Results 1. The main type of EV71 in Guangdong Province from 2008 to 2010 was C4a. During 2008 to 2010, the EV71 strain prevalent in Guangdong Province was C4a, which was also the main prevalent strain in China. In the course of evolution, three families, C4a2-C4a4.2.EV71 strain, were added, and the distribution time of each strain was as follows: all the four strains in 2008, but mainly concentrated in C4a1; the epidemic strains in 2009 were C4a1 and C4a2; in 2010, except one strain in Zhanjiang, which belonged to C4a4, the others were C4a1. From the regional distribution point of view: most of the virus strains are C4a1 and C4a2; only in 2008 Dongguan ramet 009-08-M constituted C4a3 alone; C4a4 only appeared in Foshan and Zhanjiang. In a comprehensive view, the epidemic of each virus strain has no regional and temporal concentration trend. With age increasing, the incidence of neurological infection decreased significantly (P 0.05). Logistic regression was used to analyze the influence of gender, age, viral type and VP1 mutation on the severity of clinical symptoms of patients with neurological symptoms. There was no statistically significant correlation between age and viral A289T variation. Viral 289A (289 alanine) was associated with EV71 nervous system infection (P 0.05, OR = 2.360, 95% CI was 1.163-4.659). 4. The successful establishment of VIM knockout cell lines in human brain microvascular endothelial cells using CRISPR-Cas9 technology and lentiviral vector packaging. After 48 hours of infection with HBMEC, the expression of GFP was nearly 70%-80%. After screening stable HBMEC VIM-KO and control cell lines with purinomycin, the expression of VIM in HBMEC control and VIM-KO was detected with specific antibody of VIM. The results showed that the expression of VIM in VIM-KO cells decreased. More than 90% of the cells were knocked out by VIM. 5. Analysis of the role of VIM in EV71 infection of HBMEC. When EV71 enters the cell, the cell morphology will change due to its damage to the cell. By contrast, the morphology of the cells infected with VIM-KO by the same MOI of EV71 remained almost unchanged 24 hours after infection. Even at 48 hours, the morphological changes of the cells were very small, and only a small number of cells began to become round. The first step of EV71 invasion was to adsorb on the cell surface. The difference in the amount of viral nucleic acid between HBMEC control and VIM-KO surface was detected by fluorescence quantitative PCR. The results showed that the adsorption of EV71 on the cell surface decreased by 40% after VIM knockout. After 24 hours and 48 hours of infection, the viral quantity in normal HBMEC increased to 3 times and 8 times respectively, while the viral quantity in VIM-KO cells was only 1.3 times of that in 0 hours after 24 hours. After 48 hours of infection with HBMEC, the nucleic acid content of EV71 virus in VIM knockout cells was 1/12 of that in control cells. The results of acid assay showed that the number of cells knocked out by VIM was 1.4 times less than that of the control cells. The replication of nucleic acid and the synthesis of protein of EV71 virus in the cells were basically synchronous. During 2008-2010, the EV71 virus strain in Guangdong Province was C4a, which was also the main epidemic strain in China. It was found that the EV71 virus strain had evolved during the epidemic process in Guangdong Province, and three families, C4a2-C4a4, were added. The EV71 virus subtype had no obvious trend in time and region. 2. The A289T variation and distribution of the virus were not significant. Virus 289A (289 alanine) is easy to cause the infection of EV71 nervous system. The A289T mutation locus is the result of molecular epidemiology of 194 EV71 infected people in Guangdong Province. Further study on this locus is of practical significance for the prevention and treatment of EV71 infection in Guangdong Province and even in the whole country. 3. VIM is the receptor of EV71 on the surface of HBMEC. EV71 invades HBMEC by binding to the receptor VIM, and further affects the replication and even release of EV71 in cells. The hypothesis that EV71 infects the nervous system is put forward: EV71 enters HBMEC cells through the release of RNA mediated by VIM receptors, crosses the blood-brain barrier, and eventually infects the nervous system. The study will be helpful to reveal the mechanism of EV71 infection in the nervous system. 4. In this paper, we combine the study of virus mutation with the study of receptors, and conclude that the virus mutation and its receptors affect both the nervous system infection caused by EV71.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R512.5;R742.9

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