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模式识别受体在Behcet病和Vogt-小柳原田综合征发病中作用的研究

发布时间:2018-06-22 17:40

  本文选题:Behcet病 + Vogt-小柳原田综合征 ; 参考:《重庆医科大学》2013年博士论文


【摘要】:背景 葡萄膜炎是指累及葡萄膜、视网膜、视网膜血管和玻璃体的一大类炎症性疾病的总称。根据葡萄膜炎病因及临床表现不同,可将其分为100余种。葡萄膜炎多发于青壮年,表现为慢性或反复发作的眼部炎症,且其造成的眼组织损害是不可逆的,可导致不可逆性盲目。Behcet病和Vogt-小柳原田综合征是我国各种葡萄膜炎中最为常见也是致盲率最高的葡萄膜炎类型。目前认为,Behcet病为一种自身炎症性疾病,而Vogt-小柳原田综合征为一种自身免疫性疾病。两者的病因目前还不清楚,但感染和免疫反应被认为均参与了两种疾病的发生。模式识别受体(pattern-recognition receptors,PRR)是固有免疫的重要组成部分,在机体抵抗外来微生物感染中发挥了重要的作用。模式识别受体在接触到外来微生物后可以将其转化为适应性免疫,激活免疫细胞,促进炎症因子释放,进而引发炎症反应消除外来微生物。但是,模式识别受体表达及功能的异常可以导致免疫系统紊乱,从而导致疾病的发生。 本研究正是基于上述背景,进行了以下两大方面的实验:1、探讨模式识别受体在Behcet病和Vogt-小柳原田综合征患者中的表达,以期寻找到差异表达的模式识别受体。2、进一步探讨差异表达的模式识别受体在Behcet病和Vogt-小柳原田综合征发病中所起的作用。通过上述研究,以期能对以上两种疾病的发病机制有一个深入的了解,同时为其预防和治疗寻找到新的靶点和突破口。 第一部分模式识别受体在Behcet病发病中作用的研究 目的: 探讨模式识别受体在Behcet病发病中的作用。 方法: 分别抽取活动期Behcet病患者、静止期的Behcet病患者、急性前葡萄膜炎患者(acute anterior uveitis, AAU)和正常人外周静脉血,分离单核细胞并诱导为外周血单核细胞诱导的巨噬细胞(monocyte-derived macrophages,MDMs)。使用荧光定量PCR技术及流式细胞技术检测TLR2/4的表达;使用ELISA方法检测IL-1β表达水平;使用流式细胞技术检测线粒体受损及活性氧簇(reactive oxygenspecies,ROS)产生水平;使用基因沉默技术使NLRP3低表达;使用流式细胞技术评价MAPK信号通路磷酸化水平。 结果: 1.活动期Behcet病患者MDMs细胞TLR2和TLR4表达明显高于静止期Behcet病患者、AAU患者和正常人。 2.活动期Behcet病患者MDMs产生IL-1β水平明显高于静止期Behcet病患者、AAU患者和正常人。PGN和LPS刺激MDMs后,,可以明显升高各组IL-1β产生水平。但是,活动期Behcet病患者IL-1β升高水平明显高于其它各实验组,其它各实验组之间无明显差异。 3.活动期Behcet病患者体内MDMs细胞线粒体受损及ROS产生水平与静止期Behcet病患者、AAU患者和正常人相比明显升高;PGN和LPS可以使各组MDMs细胞线粒体受损及ROS产生水平明显增高,并且活动期Behcet病患者升高水平明显高于其它组;anti-TLR2/anti-TLR4可以明显降低PGN/LPS对MDMs细胞线粒体损伤和ROS产生作用;rotenone可以明显升高MDMs细胞ROS产生水平及IL-1β分泌水平,而DPI可以明显降低MDMs细胞ROS产生水平及IL-1β分泌水平。 4.基因沉默炎症小体NLRP3后,各组IL-1β产生显著降低。 5. ROS通过激活p38和ERK1/2,进而激活炎症小体NLRP3,从而使IL-1β产生增多。 结论: 我们的结果显示:TLR2/4在活动期Behcet病患者MDMs细胞中表达明显升高。TLR2/4与其配体PGN/LPS相互作用可以使线粒体产生ROS增多,进而激活炎症小体NLRP3,从而使IL-1β产生增多引起炎症反应。 第二部分模式识别受体在Vogt-小柳原田综合征发病中作用的研究 目的: 探讨模式识别受体在Vogt-小柳原田综合征发病中所起的作用。 方法: 分别抽取活动期Vogt-小柳原田综合征患者、静止期Vogt-小柳原田综合征患者、AAU患者和正常人外周静脉血,分离单核细胞并诱导为巨噬细胞。使用荧光定量PCR技术及流式细胞技术检测TLR2/3/4的表达;使用ELISA方法检测IL-1β表达水平;使用流式细胞技术检测线粒体受损及活性氧簇(ROS)产生水平;使用基因沉默技术使NLRP3低表达;使用流式细胞技术评价MAPK信号通路磷酸化水平。 结果: 1.活动期Vogt-小柳原田综合征患者MDMs细胞TLR3和TLR4表达明显高于静止期Vogt-小柳原田综合征患者、AAU患者和正常人;TLR2表达在各组之间无明显差异。 2.活动期Vogt-小柳原田综合征患者MDMs产生IL-1β水平明显高于静止期Vogt-小柳原田综合征患者、AAU患者和正常人。Poly I:C和LPS刺激MDMs后,可以明显升高各组IL-1β产生水平。但是,活动期Vogt-小柳原田综合征患者IL-1β升高水平明显高于其它各实验组,其它各实验组之间无明显差异。 3.活动期Vogt-小柳原田综合征患者体内MDMs细胞线粒体受损及ROS产生水平与静止期Vogt-小柳原田综合征患者、AAU患者和正常人相比明显升高;poly I:C和LPS可以使各组MDMs细胞线粒体受损及ROS产生水平明显增高,并且活动期Vogt-小柳原田综合征患者升高水平明显高于其它组;anti-TLR3/anti-TLR4可以明显降低poly I:C/LPS对MDMs细胞线粒体损伤和ROS产生的作用;rotenone可以明显升高MDMs细胞ROS产生水平及IL-1β产生水平,而DPI可以明显降低MDMs细胞ROS产生水平及IL-1β产生水平。 4.基因沉默炎症小体NLRP3后,各组IL-1β产生水平显著降低。 5. ROS通过激活p38和ERK1/2信号通路进而激活炎症小体NLRP3,从使IL-1β产生增多。 结论: 我们的结果显示:TLR3/4在活动期Vogt-小柳原田综合征患者MDMs细胞中表达明显升高。TLR3/4与其配体poly I:C/LPS相互作用可以通过使线粒体产生ROS增多,激活炎症小体NLRP3,进而使IL-1β产生增多引起炎症反应。
[Abstract]:background
Uveitis is the general name of a large type of inflammatory disease involving the Vineum, retina, retinal vessels and vitreous body. According to the etiology and clinical manifestations of uveitis, it can be divided into more than 100 species. Uveitis is more likely to occur in young and young adults, characterized by chronic or recurrent ocular inflammation, and its ocular tissue damage is irreversible. It can lead to irreversible blindness.Behcet's disease and Vogt- small Liu Yuantian syndrome, the most common type of uveitis in various uveitis and the highest blinding rate in our country. At present, Behcet's disease is a self inflammatory disease, and Vogt- small Liu Yuantian syndrome is a self immune disease. The cause of the two is not yet clear. Chu, but infection and immune response are considered to be involved in the occurrence of two diseases. Pattern-recognition receptors (PRR) is an important part of the inherent immunity and plays an important role in the body's resistance to external microbial infections. Sexual immunity, activating immune cells, promoting the release of inflammatory factors and causing inflammatory reactions to eliminate foreign microorganisms. However, abnormal expression and function of pattern recognition receptors can lead to the disorder of the immune system, which leads to the occurrence of disease.
This study is based on the above background and carries out the following two major experiments: 1, to explore the expression of pattern recognition receptors in patients with Behcet's disease and Vogt- small Liu Yuantian's syndrome, in order to find the differentially expressed pattern recognition receptor.2 and further explore the differential expression pattern recognition receptors in Behcet's disease and Vogt- small Liu Yuantian syndrome The role of the disease, through the above study, is expected to have an in-depth understanding of the pathogenesis of the above two diseases, and to find new targets and breakthroughs for its prevention and treatment.
Part I the role of pattern recognition receptors in the pathogenesis of Behcet's disease
Objective:
Objective to investigate the role of pattern recognition receptors in the pathogenesis of Behcet disease.
Method:
The patients with active Behcet disease, Behcet disease at rest, acute anterior uveitis (acute anterior uveitis, AAU) and normal human peripheral venous blood were isolated and isolated from mononuclear cells (monocyte-derived macrophages, MDMs) induced by peripheral blood mononuclear cells (monocyte-derived macrophages, MDMs). Fluorescence quantitative PCR technique and flow cytometry were used. The expression of TLR2/4 was detected by technique; the expression level of IL-1 beta was detected by ELISA method; the level of mitochondrial damage and reactive oxygen cluster (reactive OXYGENSPECIES, ROS) production was detected by flow cytometry; the expression of NLRP3 was reduced by gene silencing, and the level of phosphorylation of MAPK signaling pathway was evaluated by flow cytometry.
Result锛

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