本文選題：雙鏈RNA + 抗病毒�。� 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：雙鏈RNA(dsRNA)是具有兩條互補(bǔ)鏈的RNA,依據(jù)其堿基對(duì)長(zhǎng)度,將長(zhǎng)于30bp的dsRNA稱為長(zhǎng)鏈dsRNA。近二十年來(lái)研究發(fā)現(xiàn)短鏈dsRNA(30bp)在生命活動(dòng)中起著重要作用,在真核細(xì)胞中主要以microRNA的形式調(diào)控基因的表達(dá),對(duì)其調(diào)控機(jī)制的研究已經(jīng)較為完整。microRNA的前體pre-microRNA是真核細(xì)胞中目前發(fā)現(xiàn)的一類長(zhǎng)鏈dsRNA,而在病理過(guò)程中,細(xì)胞中也會(huì)出現(xiàn)大量長(zhǎng)鏈dsRNA,目前對(duì)于這些長(zhǎng)鏈dsRNA的作用機(jī)制尚不清楚。本課題分別針對(duì)外源性和內(nèi)源性dsRNA及其生物學(xué)功能,通過(guò)建立相關(guān)的疾病模型,探討了dsRNA是否以及如何參與疾病的病理過(guò)程,揭示了外源性和內(nèi)源性dsRNA分別在抗病毒免疫應(yīng)答和氧化應(yīng)激引起的代謝紊亂中的調(diào)控機(jī)制。第一部分:靶向外源性dsRNA激活抗病毒固有免疫的分子機(jī)制研究病毒感染會(huì)導(dǎo)致宿主細(xì)胞內(nèi)dsRNA大量產(chǎn)生。在哺乳動(dòng)物細(xì)胞中,這些dsRNA會(huì)被一套特異的dsRNA受體識(shí)別成危險(xiǎn)信號(hào),其中主要的胞內(nèi)受體是RLR家族受體。rlr受體可以識(shí)別并結(jié)合外源性的長(zhǎng)鏈dsrna,并將其作為病原信號(hào)(pamps)通過(guò)死亡結(jié)構(gòu)域傳遞給下游,激活干擾素介導(dǎo)的抗病毒信號(hào)通路,在嚴(yán)重感染時(shí)甚至激活細(xì)胞凋亡。本部分研究中,我們提出了一種新的抗病毒策略,即通過(guò)識(shí)別dsrna、并以類似于rlr受體的方式激活下游信號(hào),誘導(dǎo)程序性壞死以快速清除被感染的細(xì)胞,從而達(dá)到廣譜高效抗病毒的目的。我們首先運(yùn)用分子生物學(xué)方法改構(gòu)了rlr受體并將其命名為dscare,證明了被腺病毒(adv)和呼吸道合胞病毒(rsv)感染的a549細(xì)胞能被dscare快速殺傷。運(yùn)用實(shí)時(shí)定量pcr、elisa和westernblotting等方法,我們發(fā)現(xiàn)雖然dscare沒(méi)有激活細(xì)胞內(nèi)ifn-β通路,但是促進(jìn)了炎性因子il-1β的表達(dá)和分泌。通過(guò)經(jīng)典的細(xì)胞死亡標(biāo)志的分析,如細(xì)胞形態(tài)學(xué)檢測(cè)、磷脂酰絲氨酸外翻、caspase活化、化學(xué)抑制劑阻斷特定通路等,我們發(fā)現(xiàn)dscare主要通過(guò)激活細(xì)胞程序性壞死來(lái)殺死被感染的細(xì)胞而沒(méi)有激活細(xì)胞凋亡。通過(guò)基因沉默和化學(xué)阻斷的方式,發(fā)現(xiàn)dscare通過(guò)溶酶體蛋白組織蛋白酶d介導(dǎo)了necrosome復(fù)合物形成,繼而激活程序性壞死通路,而通過(guò)rna干涉的方法證明了在這個(gè)通路中rip1和rip3都是dscare發(fā)揮抗病毒活性的重要分子�；瘜W(xué)阻斷細(xì)胞凋亡和程序性壞死的對(duì)照實(shí)驗(yàn)進(jìn)一步表明,抑制劑nec-1通過(guò)抑制程序性壞死而阻斷了dscare的抗病毒活性,抑制劑zvad阻斷細(xì)胞凋亡則對(duì)dscare的抗病毒活性沒(méi)有影響。研究結(jié)果表明,重組rlr受體dscare繞過(guò)了通常被激活的干擾素抗病毒途徑,直接激活了程序性壞死通路,從而達(dá)到了快速殺傷被感染細(xì)胞的目的。這個(gè)機(jī)制的發(fā)現(xiàn)和提出為發(fā)展新的高效抗病毒藥物提供了理論依據(jù)。第二部分:內(nèi)源性dsrna調(diào)控血管內(nèi)皮功能紊亂的分子機(jī)制研究在高血糖引起的血管病變過(guò)程中,氧化應(yīng)激和炎癥導(dǎo)致的血管內(nèi)皮功能紊亂是主要發(fā)病誘因。雖然ambati等報(bào)道了在氧化應(yīng)激和炎性狀態(tài)下內(nèi)源性dsrna會(huì)大量累積并對(duì)細(xì)胞產(chǎn)生毒性,但是dsrna在高血糖誘導(dǎo)的血管內(nèi)皮病變過(guò)程中是否參與以及其生物學(xué)功能尚不清楚。本部分研究旨在探索內(nèi)源性dsrna是否參與血管內(nèi)皮細(xì)胞的氧化應(yīng)激及功能紊亂,揭示內(nèi)源性dsrna參與調(diào)控的信號(hào)通路。我們運(yùn)用dsrna特異性抗體j2通過(guò)免疫組織化學(xué)的方法來(lái)檢測(cè)可能在高血糖小鼠和高糖刺激的人臍靜脈內(nèi)皮細(xì)胞(huvec)細(xì)胞中產(chǎn)生的dsrna,結(jié)果顯示在高血糖小鼠的胸主動(dòng)脈血管內(nèi)皮中和高糖刺激的HUVEC細(xì)胞中均有大量的dsRNA產(chǎn)生。通過(guò)免疫沉淀的方法,我們捕獲了在高糖刺激的HUVEC細(xì)胞中產(chǎn)生的dsRNA,并通過(guò)構(gòu)建文庫(kù)和DNA測(cè)序的方法鑒定捕獲到的dsRNA,發(fā)現(xiàn)這些dsRNA與Alu RNA Sc亞家族高度同源。我們通過(guò)基因克隆的方法構(gòu)建了真核表達(dá)Alu RNA的重組質(zhì)粒。運(yùn)用實(shí)時(shí)定量PCR、ELISA、Western Blotting等方法,通過(guò)比較高糖刺激的HUVEC細(xì)胞與Alu RNA轉(zhuǎn)染的HUVEC細(xì)胞的抗氧化酶系統(tǒng)和氧化應(yīng)激標(biāo)志物,我們?cè)贖UVEC細(xì)胞中檢查了捕獲到的dsRNA對(duì)內(nèi)皮細(xì)胞功能的影響。結(jié)果顯示,與高糖刺激的促氧化應(yīng)激和促炎作用類似,Alu RNA過(guò)表達(dá)促進(jìn)了活性氧簇(ROS)的產(chǎn)生,并且上調(diào)了IL-1β的表達(dá)和分泌,而且Alu RNA抑制了內(nèi)皮細(xì)胞一氧化氮合酶eNOS和超氧化物歧化酶SOD2的表達(dá)。通過(guò)化學(xué)清除ROS和化學(xué)抑制NFκB的活化,我們發(fā)現(xiàn)Alu RNA通過(guò)促進(jìn)ROS產(chǎn)生和激活NFκB來(lái)抑制抗氧化酶的表達(dá);而通過(guò)構(gòu)建Alu RNA的功能缺失突變體,我們發(fā)現(xiàn)Alu RNA對(duì)多種酶的表達(dá)抑制是依賴于Alu RNA在轉(zhuǎn)錄和翻譯水平的負(fù)調(diào)控機(jī)制。我們的研究揭示了內(nèi)源性dsRNA——Alu RNA在高糖刺激下在血管內(nèi)皮細(xì)胞中大量積累的現(xiàn)象,闡述了內(nèi)源性Alu RNA激活NFκB通路促進(jìn)氧化應(yīng)激和炎癥,并通過(guò)NFκB通路、轉(zhuǎn)錄和(或)翻譯水平兩種方式負(fù)調(diào)控抗氧化酶eNOS和SOD2表達(dá),從而進(jìn)一步加重細(xì)胞內(nèi)氧化應(yīng)激這一分子機(jī)制。這些發(fā)現(xiàn)更新了對(duì)內(nèi)皮細(xì)胞功能紊亂的調(diào)控機(jī)制的理解,提出了內(nèi)源性dsRNA調(diào)控代謝紊亂類疾病的發(fā)生發(fā)展的新理論。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R3416

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