本文關(guān)鍵詞： 白紋伊蚊 中腸 microRNAs(miRNA) 高通量測序 登革病毒(DENV) C6/36細(xì)胞　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文　論文類型：學(xué)位論文

【摘要】：登革熱、登革出血熱（Dengue hemorrhagic fever，DHF）是重要的蟲媒傳染病之一，廣泛流行于熱帶和亞熱帶地區(qū)，埃及伊蚊和白紋伊蚊是其主要傳播媒介。由于尚無有效的疫苗和治療藥物，登革熱的防控仍然依賴于媒介蚊蟲的防治。近二十年來，由于氣候變暖、城市化進(jìn)程加快以及國際間交流增加，其感染范圍不斷擴(kuò)大，并時有暴發(fā)，因此，有必要發(fā)展新的媒介控制方法。而通過基因調(diào)控改變蚊蟲媒介效能，研究新型媒介控制措施成為諸多學(xué)者關(guān)注的焦點(diǎn)。媒介效能的關(guān)鍵指標(biāo)是媒介蚊蟲對病毒的易感性，蚊蟲體內(nèi)存在多個阻止病原感染、擴(kuò)散和傳播的天然免疫防御屏障，其中，中腸是防止病原體入侵、建立體內(nèi)病毒感染的第一個靶器官，病原體能否感染中腸上皮細(xì)胞，是影響蚊蟲對蟲媒病毒易感性的重要因素。目前，對于中腸感染屏障的分子機(jī)制尚不明確，因此也成為媒介蚊蟲對登革病毒易感性調(diào)控機(jī)制研究的瓶頸所在。闡明中腸與病原體相互作用的分子機(jī)制，將使通過基因操縱來調(diào)節(jié)媒介效能，進(jìn)而阻斷病原傳播成為可能，對制定新的媒介控制措施具有重要的理論意義。 MicroRNA（miRNA）作為一類新型的基因調(diào)控因子，通過轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)，在調(diào)控病毒感染和內(nèi)在免疫調(diào)節(jié)中發(fā)揮重要作用。近年來，蚊類miRNA研究中，用RNA干擾（RNAi）的方法去除岡比亞按蚊Dcr1或Ago1（miRNA生成過程中必不可少的兩種酶）會導(dǎo)致中腸瘧原蟲感染增加，而在埃及伊蚊中腸上皮細(xì)胞整合入穩(wěn)定表達(dá)具有RNAi作用的反向重復(fù)序列后，其中腸登革病毒復(fù)制得到明顯抑制，證明miRNA在蚊類中腸病原感染中發(fā)揮了重要作用，為研究蚊蟲抗感染機(jī)制提供了新的思路。目前，在埃及伊蚊的研究表明登革病毒感染會引起埃及伊蚊身體及中腸組織miRNA的上調(diào)或下調(diào)，其中的一些miRNA在體外細(xì)胞實驗中表現(xiàn)出對病毒復(fù)制的抑制或增強(qiáng)作用。但與白紋伊蚊中腸感染登革病毒相關(guān)的miRNA研究尚未見報道，而諸多研究證實，白紋伊蚊比埃及伊蚊中腸上皮細(xì)胞更容易感染登革病毒，但登革病毒從白紋伊蚊中腸擴(kuò)散到其它組織的能力低于埃及伊蚊，說明兩者中腸屏障的機(jī)制可能存在差異。雖有研究證實不同發(fā)育階段白紋伊蚊存在不同種類miRNA,但miRNA的表達(dá)具有組織特異性，對于白紋伊蚊中腸內(nèi)特異表達(dá)的miRNA種類及其與登革病毒感染的相關(guān)性研究尚無報道。本研究針對白紋伊蚊中腸特異性miRNA及其在登革病毒感染過程中的變化規(guī)律進(jìn)行分析，并篩選部分miRNA進(jìn)行體外細(xì)胞轉(zhuǎn)染實驗，試圖通過研究探討影響白紋伊蚊中腸細(xì)胞感染登革病毒的miRNA分子，為闡明中腸感染屏障機(jī)制奠定基礎(chǔ)，同時也為設(shè)計新的方法來干擾或阻斷蚊蟲傳播病原體的過程提供思路。 本研究以野外采集的廣州株白紋伊蚊為研究對象，在實驗室條件下，用含有登革2型病毒（DENV-2）標(biāo)準(zhǔn)株（NGC）的人工血餐飼喂受試蚊蟲，同時分別設(shè)立未吸血蚊和吸食不含病毒血餐蚊為空白對照和血餐對照，在飼喂后不同時間點(diǎn)獲取中腸組織，以傳統(tǒng)膠切割回收方法構(gòu)建小RNA庫并進(jìn)行高通量測序及生物信息學(xué)分析，獲得保守miRNA表達(dá)譜并預(yù)測新miRNA。進(jìn)一步通過研究中腸miRNA感染后變化特點(diǎn)，分析感染與未感染中腸miRNA表達(dá)差異，尋找可能與登革病毒感染有關(guān)的miRNA分子。選擇差異表達(dá)miRNA，用人工合成的miRNA類似物(mimic)和反義抑制物（inhibitor）轉(zhuǎn)染C6/36細(xì)胞，研究目的miRNA對C6/36細(xì)胞內(nèi)DENV-2復(fù)制的影響作用。 結(jié)果： 1．白紋伊蚊對DENV-2敏感性較高，中腸感染率高于身體其它部分感染率。 本實驗中白紋伊蚊對DENV-2的感染率在5-7天迅速上升至63.3%，之后平穩(wěn)緩慢上升。中腸感染率高于身體其它部分感染率，兩組均數(shù)配對t檢驗結(jié)果表明差異具有統(tǒng)計學(xué)意義，t=3.872，P=0.018。 2．白紋伊蚊中腸miRNA表達(dá)種類豐富，并具有組織特異性。 吸血和未吸血中腸樣本中，通過高通量測序和生物信息學(xué)分析共鑒定112種保守miRNA并預(yù)測了43種新miRNA，莖環(huán)引物qRT-PCR驗證了之前在該蚊種未曾發(fā)現(xiàn)的aal-miR-1174、aal-miR-2951、aal-miR-956-3p和aal-miR-956-5p的表達(dá)。 3．白紋伊蚊吸血后中腸miRNA變化具有多種模式。 吸血后1、2、5、7天，與未吸血蚊相比，miRNA變化特征被分為7大類，分別是先降再升、降-升-降、速升速降、慢升略降、持續(xù)升高、持續(xù)降低、基本不變，其中，先降再升、降-升-降是主要模式，構(gòu)成比分別為20%和32%，幾個高水平表達(dá)的miRNA，如aal-miR-184、aal-miR-956-3p、aal-let-7、aal-miR-281和aal-miR-34均屬于此類。變化不顯著的類型中，多數(shù)miRNA表達(dá)水平非常低。吸食含DENV-2血餐后中腸miRNA變化模式種類未變，但有些miRNA所屬類型發(fā)生改變，如aal-miR-276-3p和aal-miR-275-3p由“先降再升”改變?yōu)椤跋壬俳怠薄?4．感染與未感染蚊中腸miRNA表達(dá)差異顯著，上調(diào)種類多于下調(diào)種類。 通過相同時間點(diǎn)配對比較感染與未感染蚊中腸miRNA表達(dá)差異，共篩選到74個差異表達(dá)顯著的miRNA。按fold-change2、歸一化表達(dá)量至少在一組中不低于30的標(biāo)準(zhǔn)，進(jìn)一步篩選出22個差異表達(dá)miRNA，其中16個呈上調(diào)表達(dá)，包括aal-miR-1767、aal-miR-276-3p、aal-miR-193-5p、aal-miR-1951、aal-miR-4728-5p、aal-miR-6134、aal-miR-622、aal-miR-1420b-5p、aal-miR-998-5p等，4個呈下調(diào)表達(dá)，包括aal-miR-1273f、aal-miR-4448、 aal-miR-6666-3p和aal-miR-15-3p。而aal-miR-989和aal-miR-2941在不同的對比方式中分別表現(xiàn)出相反的變化趨勢。 5．差異表達(dá)miRNA在埃及伊蚊預(yù)測的靶基因數(shù)量多，具有多種功能，43種miRNA也在DENV-2預(yù)測到靶基因。 用RNAhybrid軟件預(yù)測差異表達(dá)的miRNA在埃及伊蚊基因組的靶基因，根據(jù)自由能高低篩選出靶基因數(shù)量為3608，結(jié)合位點(diǎn)有63386個，GO富集及代謝通路分析表明這些靶基因與多種生理功能有關(guān)。共有43種差異表達(dá)miRNA在DENV-2基因組預(yù)測到靶基因，分別位于DENV-2基因組非結(jié)構(gòu)蛋白NS1、NS2A、NS3、NS4A、NS5和E蛋白等。 6．C6/36細(xì)胞轉(zhuǎn)染實驗表明4個miRNA對細(xì)胞內(nèi)DENV-2復(fù)制有影響作用。 對5個隨機(jī)選取的miRNA人工合成mimic和inhibtor，體外C6/36細(xì)胞轉(zhuǎn)染實驗表明， aal-miR-1767、aal-miR-4728-5p和aal-miR-276-3p能夠增強(qiáng)C6/36細(xì)胞內(nèi)DENV-2復(fù)制，aal-miR-4448能夠抑制C6/36細(xì)胞內(nèi)DENV-2復(fù)制。 結(jié)論： 1．首次對白紋伊蚊中腸特異表達(dá)的miRNA進(jìn)行了鑒定分析，發(fā)現(xiàn)了新的在白紋伊蚊體內(nèi)表達(dá)的保守miRNA，預(yù)測了新miRNA，豐富了蚊蟲miRNA的種類。 2．白紋伊蚊吸血后，中腸miRNA的變化復(fù)雜多樣，，但具有一定的規(guī)律性，隨時間變化主要表現(xiàn)為7大類型，分別是先降再升、降-升-降、速升速降、慢升略降、持續(xù)升高、持續(xù)降低、基本不變。其中，先降再升、降-升-降是主要模式，分別占構(gòu)成比的20%和32%。吸食含DENV-2血餐后，miRNA變化模式的類型未發(fā)生變，但有少數(shù)miRNA所屬模式發(fā)生了改變。表達(dá)模式發(fā)生改變的miRNA可能在中腸感染DENV-2過程中發(fā)揮調(diào)控作用。 3．感染與未感染蚊配對差異分析結(jié)果表明，經(jīng)口感染DENV-2后中腸miRNA變化顯著，且不同時期表現(xiàn)不同。感染后24h，多數(shù)miRNA表達(dá)上調(diào)，感染后48h，多數(shù)miRNA表達(dá)下調(diào)；攝入DENV-2后第7天，感染與未感染蚊中腸差異表達(dá)miRNA的種類最多，變化幅度最大。不同時間點(diǎn)差異表達(dá)的miRNA種類變化較大，表明白紋伊蚊中腸miRNA調(diào)控DENV-2感染的作用機(jī)制非常復(fù)雜，miRNA的變化呈現(xiàn)出瞬時性、復(fù)雜性和多變性的特點(diǎn)。 4．體外細(xì)胞轉(zhuǎn)染實驗表明，aal-miR-1767、aal-miR-4728-5p和aal-miR-276-3p能夠促進(jìn)C6/36細(xì)胞內(nèi)DENV-2復(fù)制，aal-miR-4448能夠抑制C6/36細(xì)胞內(nèi)DENV-2復(fù)制，這些miRNA可能在白紋伊蚊中腸感染DENV-2過程中也發(fā)揮重要調(diào)節(jié)作用。
[Abstract]:Dengue fever, dengue hemorrhagic fever (Dengue hemorrhagic, fever, DHF) is one of the important infectious disease, widely spread in tropical and subtropical regions, Aedes aegypti and Aedes albopictus is the main medium of communication. Because there is no effective vaccine and drug prevention, prevention and control of dengue fever still relies on mosquito vectors in recent twenty years. Come, due to climate warming, city urbanization and international exchanges increased, expanding the scope of the infection, and when the outbreak, therefore, it is necessary to develop new media control method. Through the change of gene regulation in mosquito control measures of effectiveness, the new media has become the focus of attention of many scholars. The key indicators of the effectiveness of media is the mosquito susceptibility to the virus in mosquitoes, there are multiple stop infection, spread the innate immune defense barrier, which is to prevent the intestinal pathogens into the Invasion, the establishment of the first target organ in virus infection, the pathogen can infect midgut epithelial cells, is an important factor affecting the susceptibility of mosquito arbovirus. At present, the molecular mechanism of mesenteronal infection barrier is not clear, so it has become a bottleneck of mosquito vectors of dengue virus disease and regulation mechanism of susceptibility to elucidate the molecular mechanism of midgut. Interaction with pathogens, will make through genetic manipulation to regulate the media efficiency, thereby blocking the spread of the pathogen as possible, has important theoretical significance to develop new media control measures.
MicroRNA (miRNA) as a new type of gene transcription factor through post transcriptional regulation of gene expression and play an important role in the regulation of viral infection and innate immune regulation. In recent years, the research of miRNA mosquitoes, RNA interference (RNAi) method for the removal of Dcr1 or Ago1 (according to the Gambia mosquito two key enzymes in the formation process of miRNA) will lead to increased infection in the midgut of Plasmodium, Aedes aegypti midgut epithelial cells integrated into the stable expression of inverted repeat sequences with RNAi function, including intestinal dengue virus replication was suppressed obviously, indicated that miRNA played an important role in the mosquito midgut infection, provides a new idea for study on the mosquito anti infection mechanism. At present, the research shows that the Aedes aegypti dengue virus infection can cause the body of Aedes aegypti and midgut tissue miRNA up or down, some fine miRNA in vitro Showed enhancement effect on virus replication inhibition or cell experiment. But with the Aedes albopictus midgut infection on miRNA of dengue virus related research has not been reported, and many studies have confirmed that the ratio of Aedes albopictus Aedes aegypti midgut epithelial cells more susceptible to dengue virus, and dengue virus from Aedes albopictus midgut spread to the ability of other tissues is lower than that of Aedes aegypti, indicating a possible mechanism of both intestinal barrier are different. Although research has shown that different developmental stages of Aedes albopictus have different kinds of miRNA, but miRNA expression is tissue specific, for white grain Aedes mosquitoes in the midgut specific expression of miRNA and its correlation with dengue virus infection is this study reports. According to the analysis of Aedes albopictus midgut specific miRNA and dengue virus infection in changes in the process of screening, and part of miRNA transfection in vitro The purpose of this study is to explore the miRNA molecules affecting the dengue virus infected by Aedes albopictus, and to lay a foundation for elucidating the barrier mechanism of midgut infection, and to provide ideas for designing new ways to interfere or block the transmission of pathogens by mosquitoes.
In this study, the field collection of Guangzhou strains of Aedes albopictus as the research object, under laboratory conditions, with dengue virus type 2 (DENV-2) standard strain (NGC) artificial blood meal fed by mosquitoes, and were set up without blood sucking mosquitoes and smoking without virus blood meal mosquitoes as blank control and blood food control, feeding in different time points after obtaining the midgut tissue, in the traditional rubber recycling method to construct small cutting RNA library and high throughput sequencing and bioinformatics analysis, obtain conserved miRNA expression profile and prediction of new miRNA. to further study the characteristic changes in midgut after miRNA infection, analysis of infected and uninfected midgut miRNA expression differences. Looking for a possible and dengue virus infection related miRNA molecules. Selection of differentially expressed miRNA, with a synthetic analogue of miRNA (mimic) and antisense inhibitor (inhibitor) was transfected into C6/36 cells. The research purpose of miRNA cells in C6/36 DENV-2 The impact of replication.
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本文編號：1484448