登革病毒易感性相关的白纹伊蚊中肠特异性microRNA的鉴定、筛选与功能研究

发布时间：2018-02-02 11:55

  本文关键词： 白纹伊蚊 中肠 microRNAs(miRNA) 高通量测序 登革病毒(DENV) C6/36细胞　出处：《中国人民解放军军事医学科学院》2014年博士论文　论文类型：学位论文

【摘要】：登革热、登革出血热（Dengue hemorrhagic fever，DHF）是重要的虫媒传染病之一，广泛流行于热带和亚热带地区，埃及伊蚊和白纹伊蚊是其主要传播媒介。由于尚无有效的疫苗和治疗药物，登革热的防控仍然依赖于媒介蚊虫的防治。近二十年来，由于气候变暖、城市化进程加快以及国际间交流增加，其感染范围不断扩大，并时有暴发，因此，有必要发展新的媒介控制方法。而通过基因调控改变蚊虫媒介效能，研究新型媒介控制措施成为诸多学者关注的焦点。媒介效能的关键指标是媒介蚊虫对病毒的易感性，蚊虫体内存在多个阻止病原感染、扩散和传播的天然免疫防御屏障，其中，中肠是防止病原体入侵、建立体内病毒感染的第一个靶器官，病原体能否感染中肠上皮细胞，是影响蚊虫对虫媒病毒易感性的重要因素。目前，对于中肠感染屏障的分子机制尚不明确，因此也成为媒介蚊虫对登革病毒易感性调控机制研究的瓶颈所在。阐明中肠与病原体相互作用的分子机制，将使通过基因操纵来调节媒介效能，进而阻断病原传播成为可能，对制定新的媒介控制措施具有重要的理论意义。 MicroRNA（miRNA）作为一类新型的基因调控因子，通过转录后水平调控基因的表达，在调控病毒感染和内在免疫调节中发挥重要作用。近年来，蚊类miRNA研究中，用RNA干扰（RNAi）的方法去除冈比亚按蚊Dcr1或Ago1（miRNA生成过程中必不可少的两种酶）会导致中肠疟原虫感染增加，而在埃及伊蚊中肠上皮细胞整合入稳定表达具有RNAi作用的反向重复序列后，其中肠登革病毒复制得到明显抑制，证明miRNA在蚊类中肠病原感染中发挥了重要作用，为研究蚊虫抗感染机制提供了新的思路。目前，在埃及伊蚊的研究表明登革病毒感染会引起埃及伊蚊身体及中肠组织miRNA的上调或下调，其中的一些miRNA在体外细胞实验中表现出对病毒复制的抑制或增强作用。但与白纹伊蚊中肠感染登革病毒相关的miRNA研究尚未见报道，而诸多研究证实，白纹伊蚊比埃及伊蚊中肠上皮细胞更容易感染登革病毒，但登革病毒从白纹伊蚊中肠扩散到其它组织的能力低于埃及伊蚊，说明两者中肠屏障的机制可能存在差异。虽有研究证实不同发育阶段白纹伊蚊存在不同种类miRNA,但miRNA的表达具有组织特异性，对于白纹伊蚊中肠内特异表达的miRNA种类及其与登革病毒感染的相关性研究尚无报道。本研究针对白纹伊蚊中肠特异性miRNA及其在登革病毒感染过程中的变化规律进行分析，并筛选部分miRNA进行体外细胞转染实验，试图通过研究探讨影响白纹伊蚊中肠细胞感染登革病毒的miRNA分子，为阐明中肠感染屏障机制奠定基础，同时也为设计新的方法来干扰或阻断蚊虫传播病原体的过程提供思路。 本研究以野外采集的广州株白纹伊蚊为研究对象，在实验室条件下，用含有登革2型病毒（DENV-2）标准株（NGC）的人工血餐饲喂受试蚊虫，同时分别设立未吸血蚊和吸食不含病毒血餐蚊为空白对照和血餐对照，在饲喂后不同时间点获取中肠组织，以传统胶切割回收方法构建小RNA库并进行高通量测序及生物信息学分析，获得保守miRNA表达谱并预测新miRNA。进一步通过研究中肠miRNA感染后变化特点，分析感染与未感染中肠miRNA表达差异，寻找可能与登革病毒感染有关的miRNA分子。选择差异表达miRNA，用人工合成的miRNA类似物(mimic)和反义抑制物（inhibitor）转染C6/36细胞，研究目的miRNA对C6/36细胞内DENV-2复制的影响作用。 结果： 1．白纹伊蚊对DENV-2敏感性较高，中肠感染率高于身体其它部分感染率。 本实验中白纹伊蚊对DENV-2的感染率在5-7天迅速上升至63.3%，之后平稳缓慢上升。中肠感染率高于身体其它部分感染率，两组均数配对t检验结果表明差异具有统计学意义，t=3.872，P=0.018。 2．白纹伊蚊中肠miRNA表达种类丰富，并具有组织特异性。 吸血和未吸血中肠样本中，通过高通量测序和生物信息学分析共鉴定112种保守miRNA并预测了43种新miRNA，茎环引物qRT-PCR验证了之前在该蚊种未曾发现的aal-miR-1174、aal-miR-2951、aal-miR-956-3p和aal-miR-956-5p的表达。 3．白纹伊蚊吸血后中肠miRNA变化具有多种模式。 吸血后1、2、5、7天，与未吸血蚊相比，miRNA变化特征被分为7大类，分别是先降再升、降-升-降、速升速降、慢升略降、持续升高、持续降低、基本不变，其中，先降再升、降-升-降是主要模式，构成比分别为20%和32%，几个高水平表达的miRNA，如aal-miR-184、aal-miR-956-3p、aal-let-7、aal-miR-281和aal-miR-34均属于此类。变化不显著的类型中，多数miRNA表达水平非常低。吸食含DENV-2血餐后中肠miRNA变化模式种类未变，但有些miRNA所属类型发生改变，如aal-miR-276-3p和aal-miR-275-3p由“先降再升”改变为“先升再降”。 4．感染与未感染蚊中肠miRNA表达差异显著，上调种类多于下调种类。 通过相同时间点配对比较感染与未感染蚊中肠miRNA表达差异，共筛选到74个差异表达显著的miRNA。按fold-change2、归一化表达量至少在一组中不低于30的标准，进一步筛选出22个差异表达miRNA，其中16个呈上调表达，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个呈下调表达，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在不同的对比方式中分别表现出相反的变化趋势。 5．差异表达miRNA在埃及伊蚊预测的靶基因数量多，具有多种功能，43种miRNA也在DENV-2预测到靶基因。 用RNAhybrid软件预测差异表达的miRNA在埃及伊蚊基因组的靶基因，根据自由能高低筛选出靶基因数量为3608，结合位点有63386个，GO富集及代谢通路分析表明这些靶基因与多种生理功能有关。共有43种差异表达miRNA在DENV-2基因组预测到靶基因，分别位于DENV-2基因组非结构蛋白NS1、NS2A、NS3、NS4A、NS5和E蛋白等。 6．C6/36细胞转染实验表明4个miRNA对细胞内DENV-2复制有影响作用。 对5个随机选取的miRNA人工合成mimic和inhibtor，体外C6/36细胞转染实验表明， aal-miR-1767、aal-miR-4728-5p和aal-miR-276-3p能够增强C6/36细胞内DENV-2复制，aal-miR-4448能够抑制C6/36细胞内DENV-2复制。 结论： 1．首次对白纹伊蚊中肠特异表达的miRNA进行了鉴定分析，发现了新的在白纹伊蚊体内表达的保守miRNA，预测了新miRNA，丰富了蚊虫miRNA的种类。 2．白纹伊蚊吸血后，中肠miRNA的变化复杂多样，，但具有一定的规律性，随时间变化主要表现为7大类型，分别是先降再升、降-升-降、速升速降、慢升略降、持续升高、持续降低、基本不变。其中，先降再升、降-升-降是主要模式，分别占构成比的20%和32%。吸食含DENV-2血餐后，miRNA变化模式的类型未发生变，但有少数miRNA所属模式发生了改变。表达模式发生改变的miRNA可能在中肠感染DENV-2过程中发挥调控作用。 3．感染与未感染蚊配对差异分析结果表明，经口感染DENV-2后中肠miRNA变化显著，且不同时期表现不同。感染后24h，多数miRNA表达上调，感染后48h，多数miRNA表达下调；摄入DENV-2后第7天，感染与未感染蚊中肠差异表达miRNA的种类最多，变化幅度最大。不同时间点差异表达的miRNA种类变化较大，表明白纹伊蚊中肠miRNA调控DENV-2感染的作用机制非常复杂，miRNA的变化呈现出瞬时性、复杂性和多变性的特点。 4．体外细胞转染实验表明，aal-miR-1767、aal-miR-4728-5p和aal-miR-276-3p能够促进C6/36细胞内DENV-2复制，aal-miR-4448能够抑制C6/36细胞内DENV-2复制，这些miRNA可能在白纹伊蚊中肠感染DENV-2过程中也发挥重要调节作用。
[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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