长链双链RNA调控内源性免疫和慢性炎症的分子机制研究

发布时间：2018-05-28 03:01

本文选题：双链RNA + 抗病毒　；参考：《第四军医大学》2016年博士论文

【摘要】：双链RNA(dsRNA)是具有两条互补链的RNA,依据其碱基对长度,将长于30bp的dsRNA称为长链dsRNA。近二十年来研究发现短链dsRNA(30bp)在生命活动中起着重要作用,在真核细胞中主要以microRNA的形式调控基因的表达,对其调控机制的研究已经较为完整。microRNA的前体pre-microRNA是真核细胞中目前发现的一类长链dsRNA,而在病理过程中,细胞中也会出现大量长链dsRNA,目前对于这些长链dsRNA的作用机制尚不清楚。本课题分别针对外源性和内源性dsRNA及其生物学功能,通过建立相关的疾病模型,探讨了dsRNA是否以及如何参与疾病的病理过程,揭示了外源性和内源性dsRNA分别在抗病毒免疫应答和氧化应激引起的代谢紊乱中的调控机制。第一部分:靶向外源性dsRNA激活抗病毒固有免疫的分子机制研究病毒感染会导致宿主细胞内dsRNA大量产生。在哺乳动物细胞中,这些dsRNA会被一套特异的dsRNA受体识别成危险信号,其中主要的胞内受体是RLR家族受体。rlr受体可以识别并结合外源性的长链dsrna,并将其作为病原信号(pamps)通过死亡结构域传递给下游,激活干扰素介导的抗病毒信号通路,在严重感染时甚至激活细胞凋亡。本部分研究中,我们提出了一种新的抗病毒策略,即通过识别dsrna、并以类似于rlr受体的方式激活下游信号,诱导程序性坏死以快速清除被感染的细胞,从而达到广谱高效抗病毒的目的。我们首先运用分子生物学方法改构了rlr受体并将其命名为dscare,证明了被腺病毒(adv)和呼吸道合胞病毒(rsv)感染的a549细胞能被dscare快速杀伤。运用实时定量pcr、elisa和westernblotting等方法,我们发现虽然dscare没有激活细胞内ifn-β通路,但是促进了炎性因子il-1β的表达和分泌。通过经典的细胞死亡标志的分析,如细胞形态学检测、磷脂酰丝氨酸外翻、caspase活化、化学抑制剂阻断特定通路等,我们发现dscare主要通过激活细胞程序性坏死来杀死被感染的细胞而没有激活细胞凋亡。通过基因沉默和化学阻断的方式,发现dscare通过溶酶体蛋白组织蛋白酶d介导了necrosome复合物形成,继而激活程序性坏死通路,而通过rna干涉的方法证明了在这个通路中rip1和rip3都是dscare发挥抗病毒活性的重要分子。化学阻断细胞凋亡和程序性坏死的对照实验进一步表明,抑制剂nec-1通过抑制程序性坏死而阻断了dscare的抗病毒活性,抑制剂zvad阻断细胞凋亡则对dscare的抗病毒活性没有影响。研究结果表明,重组rlr受体dscare绕过了通常被激活的干扰素抗病毒途径,直接激活了程序性坏死通路,从而达到了快速杀伤被感染细胞的目的。这个机制的发现和提出为发展新的高效抗病毒药物提供了理论依据。第二部分:内源性dsrna调控血管内皮功能紊乱的分子机制研究在高血糖引起的血管病变过程中,氧化应激和炎症导致的血管内皮功能紊乱是主要发病诱因。虽然ambati等报道了在氧化应激和炎性状态下内源性dsrna会大量累积并对细胞产生毒性,但是dsrna在高血糖诱导的血管内皮病变过程中是否参与以及其生物学功能尚不清楚。本部分研究旨在探索内源性dsrna是否参与血管内皮细胞的氧化应激及功能紊乱,揭示内源性dsrna参与调控的信号通路。我们运用dsrna特异性抗体j2通过免疫组织化学的方法来检测可能在高血糖小鼠和高糖刺激的人脐静脉内皮细胞(huvec)细胞中产生的dsrna,结果显示在高血糖小鼠的胸主动脉血管内皮中和高糖刺激的HUVEC细胞中均有大量的dsRNA产生。通过免疫沉淀的方法,我们捕获了在高糖刺激的HUVEC细胞中产生的dsRNA,并通过构建文库和DNA测序的方法鉴定捕获到的dsRNA,发现这些dsRNA与Alu RNA Sc亚家族高度同源。我们通过基因克隆的方法构建了真核表达Alu RNA的重组质粒。运用实时定量PCR、ELISA、Western Blotting等方法,通过比较高糖刺激的HUVEC细胞与Alu RNA转染的HUVEC细胞的抗氧化酶系统和氧化应激标志物,我们在HUVEC细胞中检查了捕获到的dsRNA对内皮细胞功能的影响。结果显示,与高糖刺激的促氧化应激和促炎作用类似,Alu RNA过表达促进了活性氧簇(ROS)的产生,并且上调了IL-1β的表达和分泌,而且Alu RNA抑制了内皮细胞一氧化氮合酶eNOS和超氧化物歧化酶SOD2的表达。通过化学清除ROS和化学抑制NFκB的活化,我们发现Alu RNA通过促进ROS产生和激活NFκB来抑制抗氧化酶的表达;而通过构建Alu RNA的功能缺失突变体,我们发现Alu RNA对多种酶的表达抑制是依赖于Alu RNA在转录和翻译水平的负调控机制。我们的研究揭示了内源性dsRNA——Alu RNA在高糖刺激下在血管内皮细胞中大量积累的现象,阐述了内源性Alu RNA激活NFκB通路促进氧化应激和炎症,并通过NFκB通路、转录和(或)翻译水平两种方式负调控抗氧化酶eNOS和SOD2表达,从而进一步加重细胞内氧化应激这一分子机制。这些发现更新了对内皮细胞功能紊乱的调控机制的理解,提出了内源性dsRNA调控代谢紊乱类疾病的发生发展的新理论。
[Abstract]:Double stranded RNA (dsRNA) is a RNA with two complementary chains. According to its base pair length and 30bp dsRNA called long chain dsRNA. for nearly twenty years, it has been found that short chain dsRNA (30bp) plays an important role in life activities. In eukaryotic cells, the expression of the gene is regulated mainly in the form of microRNA, and the study of its regulation mechanism has been comparatively studied. The precursor pre-microRNA of the complete.MicroRNA is a class of long chain dsRNA found in eukaryotic cells, and a large number of long chain dsRNA will appear in the cells in the pathological process. The mechanism of the action of these long chain dsRNA is still unclear. This topic is aimed at exogenous and endogenous dsRNA and its biological functions, by establishing related diseases. The disease model, the study of whether and how dsRNA participates in the pathological process of the disease, reveals the regulatory mechanism of exogenous and endogenous dsRNA in the metabolic disorder caused by antiviral immune response and oxidative stress. Part 1: the molecular mechanism of targeting exogenous dsRNA to activate antiviral disease The intracellular dsRNA is produced in large quantities. In mammalian cells, these dsRNA can be identified as a dangerous signal by a set of specific dsRNA receptors, in which the main intracellular receptor is the RLR family receptor.Rlr receptor that recognizes and combines exogenous long chain dsRNA, and passes it as a pathogen signal (PAMPs) through the death domain to the downstream and activates the stem. In this part, we propose a new antiviral strategy for activating downstream signals by identifying dsRNA and activating downstream signals in a manner similar to the RLR receptor, inducing programmed necrosis to quickly remove infected cells and thus achieve broad-spectrum and efficient resistance. The purpose of the virus. We first modified the RLR receptor by molecular biology and named it dscare, proving that A549 cells infected by adenovirus (ADV) and respiratory syncytial virus (RSV) can be quickly killed by dscare. Using real-time quantitative PCR, ELISA, and westernblotting methods, we found that dscare did not activate intracellular if. N- beta pathway, but promotes the expression and secretion of the inflammatory factor IL-1 beta. Through the analysis of the classic cell death markers, such as cell morphological detection, phosphatidyl serine exotropion, caspase activation, chemical inhibitors block specific pathways, we found that dscare mainly killed the infected cells by activating cell programmed necrosis. There was activation of apoptosis. Through gene silencing and chemical blocking, dscare was found to mediate the formation of necrosome complexes through lysosomal protein cathepsin D and then activate the programmed necrosis pathway, and the RNA interference method proved that rip1 and RIP3 are important molecules of dscare to play antiviral activity in this pathway. A control experiment that blocked apoptosis and programmed necrosis further indicated that inhibitor nec-1 blocked the antiviral activity of dscare by inhibiting procedural necrosis. Inhibitor zVAD blocked apoptosis and had no effect on the antiviral activity of dscare. The results showed that the recombinant RLR receptor dscare bypassed the normally activated interferon. The antiviral pathway directly activates the programmed necrosis pathway to achieve the purpose of rapid killing of infected cells. The discovery of this mechanism provides a theoretical basis for the development of new highly effective antiviral drugs. The second part: the molecular mechanism of endogenous dsRNA regulation of vascular endothelial dysfunction in blood vessels caused by hyperglycemia Vascular endothelial dysfunction caused by oxidative stress and inflammation is the main cause of disease during the process of disease. Although ambati and other reports have reported the accumulation and toxicity of endogenous dsRNA in oxidative stress and inflammatory conditions, dsRNA is involved in the process of hyperglycemia induced vascular endothelial disease and its biological function. The purpose of this study is to explore whether endogenous dsRNA is involved in oxidative stress and dysfunction in vascular endothelial cells and to reveal the signaling pathways involved in the regulation of endogenous dsRNA. We use dsRNA specific antibody J2 to detect human umbilical vein endothelial cells in hyperglycemic mice and high glucose stimulated human umbilical veins by immunohistochemical method. The dsRNA produced in the cell (HUVEC) cells showed a large number of dsRNA production in the thoracic aorta endothelium of the hyperglycemic mice and in the high glucose stimulated HUVEC cells. By immunoprecipitation, we captured the dsRNA produced in the HUVEC cells stimulated by high glucose and identified by the construction of Library and DNA sequencing. The obtained dsRNA was found to be highly homologous to the Alu RNA Sc subfamily. We constructed a recombinant plasmid for the eukaryotic expression of Alu RNA by gene cloning. We used real-time quantitative PCR, ELISA, Western Blotting and other methods to compare the antioxidant enzyme system and oxidation response of the cells with high sugar stimulated HUVEC cells and transfected cells. We examined the effects of the captured dsRNA on endothelial cell function in HUVEC cells. The results showed that the overexpression of Alu RNA promoted the production of reactive oxygen clusters (ROS), increased the expression and secretion of IL-1 beta, and the Alu RNA inhibited nitric oxide in endothelial cells. The expression of synthase eNOS and superoxide dismutase SOD2. Through chemical removal of ROS and chemical inhibition of the activation of NF kappa B, we found that Alu RNA inhibits the expression of antioxidant enzymes by promoting ROS production and activating NF kappa B. The negative regulation mechanism of transcriptional and translation levels. Our study revealed the accumulation of endogenous dsRNA - Alu RNA in vascular endothelial cells stimulated by high glucose, and explained that endogenous Alu RNA activates NF kappa B pathway to promote oxidative stress and inflammation, and negatively regulates oxygen resistance through NF kappa B pathway, transtranslation and / or translation levels. The expression of enzyme eNOS and SOD2 further aggravates the molecular mechanism of intracellular oxidative stress. These discoveries update the understanding of the regulatory mechanism of endothelial dysfunction and suggest a new theory that endogenous dsRNA regulates the development of metabolic disorders.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R3416

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