本文關(guān)鍵詞： 手足口病 腸道病毒71型 SCARB2受體 miR-127-5p 病毒感染　出處：《南京大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：手足口病是由多種腸道病毒感染引起的一個(gè)高度傳染性疾病,主要在春季到秋季期間影響著嬰幼兒的健康,它的臨床表現(xiàn)范圍廣泛,從輕度和自限性到無(wú)菌性腦膜炎,脊髓灰質(zhì)炎樣急性松弛性麻痹,腦干腦炎等死亡率高的罕見(jiàn)致命表現(xiàn)和嚴(yán)重后遺癥。手足口病最常見(jiàn)的是由柯薩奇病毒A16感染引起的,其次由腸道病毒71(EV71)感染引起。重要的是,EV71造成的手足口病往往與致命的并發(fā)癥有關(guān)。EV71屬于小核糖核酸病毒科家族的單股正鏈RNA病毒,首先從患有神經(jīng)系統(tǒng)疾病的感染患者中被分離出來(lái)。EV71感染開(kāi)始于與宿主細(xì)胞受體的結(jié)合,人類(lèi)清道夫受體B類(lèi)成員2(SCARB2)被證明是組織中廣泛表達(dá)的EV71的受體。作為EV71所有病毒株的受體,SCARB2能夠介導(dǎo)病毒結(jié)合,內(nèi)化和脫殼,并且在EV71感染的早期步驟中起到關(guān)鍵作用。MicroRNA(miRNA)是一類(lèi)在動(dòng)物,植物和病毒中發(fā)現(xiàn)的19至25nt的高度保守的非編碼RNA寡核苷酸。這些小RNA主要通過(guò)結(jié)合靶mRNA上的互補(bǔ)序列來(lái)調(diào)節(jié)特異性基因的表達(dá),以調(diào)控其翻譯或穩(wěn)定性。在人類(lèi)中有數(shù)百個(gè)miRNA編碼基因,其調(diào)節(jié)蛋白質(zhì)編碼基因,越來(lái)越多的證據(jù)表明miRNA可以調(diào)節(jié)廣泛的生物過(guò)程,包括發(fā)育,分化,細(xì)胞增殖,細(xì)胞凋亡和免疫應(yīng)答。正鏈RNA病毒在復(fù)制過(guò)程的各個(gè)步驟中需要招募特定的宿主因子,這些宿主因子有助于病毒基因組復(fù)制,病毒蛋白質(zhì)合成和防御宿主免疫反應(yīng)。然而,最近有研究證實(shí)病毒感染可以改變其宿主細(xì)胞中miRNA的表達(dá),并且miRNA可以通過(guò)靶向細(xì)胞因子來(lái)間接調(diào)節(jié)病毒的復(fù)制或通過(guò)直接靶向病毒mRNA來(lái)影響病毒復(fù)制,miRNA被認(rèn)為在病毒和宿主之間的復(fù)雜相互作用的網(wǎng)絡(luò)中作為一個(gè)關(guān)鍵的效應(yīng)分子。然而,miRNA在EV71復(fù)制和發(fā)病機(jī)制中的作用尚不是很清楚,有研究報(bào)道m(xù)iR-127-5p可以抑制SCARB2的表達(dá),于是我們首先通過(guò)生物信息學(xué)分析,發(fā)現(xiàn)miR-127-5p在SCARB2 mRNA的3'UTR區(qū)域存在兩個(gè)靶結(jié)合位點(diǎn),然后,我們利用熒光素酶報(bào)告基因?qū)嶒?yàn)證實(shí)了 miR-127-5p可以與SCARB2 3'UTR上的兩個(gè)靶位點(diǎn)結(jié)合來(lái)影響SCARB2的表達(dá)。SCARB2作為EV71的重要受體,于是我們想進(jìn)一步探究,miR-127-5p在EV71感染過(guò)程是否會(huì)發(fā)揮作用。首先我們發(fā)現(xiàn)EV71感染細(xì)胞后,胞內(nèi)miR-127-5p的水平發(fā)生了上升,值得注意的是,此時(shí)SCARB2總的蛋白水平相應(yīng)的發(fā)生了微小的下調(diào)。另外,我們將細(xì)胞轉(zhuǎn)染miR-127-5p模擬物后再感染EV71,發(fā)現(xiàn)EV71在宿主細(xì)胞內(nèi)的復(fù)制和翻譯均受到明顯的抑制,同時(shí)我們用miR-127-5p inhibitor來(lái)抑制內(nèi)源性的miR-127-5p的表達(dá),發(fā)現(xiàn)這可以明顯的促進(jìn)接下來(lái)病毒的感染。此外,我們也探究了 miR-127-5p是否會(huì)與病毒的基因組直接結(jié)合而影響了病毒復(fù)制,結(jié)果表明miR-127-5p不會(huì)與病毒基因組結(jié)合,并不影響進(jìn)入后病毒的復(fù)制�？傊�,我們的實(shí)驗(yàn)結(jié)果表明miR-127-5p通過(guò)直接靶向SCARB2 mRNA,介導(dǎo)SCARB2的表達(dá),從而抑制EV71黏附和感染。
[Abstract]:Foot and mouth disease is a highly contagious disease caused by a variety of intestinal virus infection, mainly during the spring to fall affects the health of infants and young children, its clinical manifestations range from mild and self limiting to aseptic meningitis, poliomyelitis like acute flaccid paralysis, a rare fatal manifestation of brainstem encephalitis such a high mortality rate and serious sequelae. HFMD is most commonly caused by coxsackievirus A16 infection, followed by enterovirus 71 (EV71) infection. It is important that EV71 caused HFMD and often fatal complications related to.EV71 belongs to the Picornaviridae family of positive strand RNA virus. First of all from the infected patients suffering from neurological disorders have been separated with.EV71 infection began in the host cell receptor, human scavenger receptor class B member 2 (SCARB2) was shown to be widely in table As the EV71 receptor. All strains as EV71 receptor, SCARB2 can mediate virus binding, internalization and shelling, and in the early steps of EV71 infection plays a key role in the.MicroRNA (miRNA) is a kind of animal, plant and found 19 to 25nt virus in the highly conserved non RNA oligonucleotide encoding. These small RNA mainly through the combination of complementary sequences on target mRNA expression to regulate specific gene regulation, to the translation or stability. There are hundreds of miRNA encoding gene in humans, the regulation of protein encoding genes, there is growing evidence that miRNA can regulate the biological process, including extensive development, differentiation, cell proliferation, apoptosis and immune response. Positive strand RNA virus replication process needs at every step in the recruitment of host specific factors, these host factors contribute to the replication of the virus genome, protein synthesis and anti virus The host immune response. However, recent studies have confirmed that the virus infection can change the expression of miRNA in the host cell, and miRNA by targeting cytokines to indirectly regulate the replication of the virus or by directly targeting mRNA virus to affect viral replication, miRNA is considered as a key effector molecule of complex interactions between the virus and the host in the network. However, the role of miRNA in EV71 replication and pathogenesis are still not very clear, some studies reported that miR-127-5p can inhibit the expression of SCARB2, so we first through bioinformatics analysis, it is found that there are two target binding sites in the 3'UTR region, miR-127-5p SCARB2 mRNA and then we used luciferase reporter gene assays confirmed that miR-127-5p can be combined with the two target sites of SCARB2 3'UTR to SCARB2 as an important effect on the expression of.SCARB2 by EV71 The body, so we want to further explore the miR-127-5p in the course of EV71 infection might play a role. First, we found that EV71 infected cells, intracellular miR-127-5p levels rise, it is worth noting that the total SCARB2 protein level corresponding to the occurrence of tiny fall. In addition, we will re infection of cells transfected with EV71 analogue of miR-127-5p, found that EV71 replication in host cells and translation was inhibited, the expression at the same time we use miR-127-5p inhibitor to inhibit endogenous miR-127-5p, found that it could promote the virus infection. In addition, we also explored whether miR-127-5p directly binds to the viral genome and affect the virus the results showed that miR-127-5p replication does not bind with the viral genome does not affect after entering the replication of the virus. In conclusion, our results suggest that miR-127- 5p mediates the expression of SCARB2 by targeting SCARB2 mRNA directly, thus inhibiting the adhesion and infection of EV71.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R512.5
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本文編號(hào)：1497242