摘要
背景
葡萄膜炎是一类由感染因素、免疫反应等引起的眼内脉络膜组织的炎症，好发生于青壮年，易反复发作，是常见的致盲眼病之一,也被称为眼内炎症。目前葡萄膜炎的临床类型和病因多达100余种，而其中，Behcet病、Vogt-小柳原田综合征这两类疾病为我国致盲率最高的葡萄膜炎类型，而Fuchs综合征这类疾病则是一种致盲率低的葡萄膜炎类型。Behcet病、Vogt-小柳原田综合征目前普遍被认为主要是由自身免疫反应所介导的疾病，而Fuchs综合征目前报道显示与病毒感染相关。
microRNA（微小RNA，miRNA）是一类保守的小分子非编码RNA。miRNA能与特异性的信使RNAs （mRNAs）的3’端非翻译区（untranslated region，UTR）互补结合，从而在转录后水平调控靶基因的表达，降解靶基因或者抑制转录后的翻译。目前研究显示miRNAs在免疫功能和自身免疫的调节以及病毒感染中发挥着关键的作用。
本研究正是基于上述背景，进行了以下两大方面的实验：1.探讨五种免疫相关的microRNAs在Behcet病和Vogt-小柳原田综合征这两类自身免疫性疾病中的作用机制；2.探讨miR-146a和转录因子Ets-1基因多态是否与中国汉族人群Behcet病（BD）、Vogt-小柳原田综合征（VKH）和Fuchs’综合征（FUS）相关。通过上述研究，以期能对葡萄膜炎的发病机制有一个更深入的认识，同时为其防治寻找新的靶点。
第一部分MiRNAs在Behcet病发病中作用
目的：
microRNAs（miRNAs）在免疫调节和哺乳动物免疫应答反应中发挥着重要调控作用。在本文中，我们研究了microRNAs在Behcet病和Vogt-小柳原田综合征这两类自身免疫性疾病中的作用机制。
方法：
1.分别抽取活动期、静止期的Behcet病、VKH综合征患者和正常人外周静脉血，并分离这些患者和正常人外周血单个核细胞（PBMC）、CD4+T细胞和树突状细胞（DC）。
2.抽提细胞总RNA，运用TaqMan定量PCR的方法检测外周血单个核细胞（PBMC）、CD4+T细胞和DC中五种与免疫相关的microRNAs的表达水平。
3.体外用miRNA-155mimics和miRNA-155inhibitor分别转染DC，运用流式细胞仪检测转染后的DC与成熟度相关的表面分子标记物（CD80, CD40, CD83, CD86和HLA-DR）的表达水平。
4.采用ELISA方法检测转染后的DC分泌的细胞因子IL-6、IL-10、IFN-γ和IL-1β的表达水平。
5.体外共培养转染后的DC和CD4+T细胞，运用流式细胞仪检测CD4+T细胞内的细胞因子IL-17的表达水平
6.运用miRNA数据库预测miRNA-155的靶基因，再运用双荧光素酶报告基因的检测方法验证miRNA-155对预测的靶基因的3’UTR区域的荧光素酶抑制作用，最后用Western Blot的方法进一步确认miRNA-155对靶基因的调控作用。
结果：
1.活动期Behcet病患者的PBMC和DC中miRNA-155的表达水平显著低于正常人，而活动期VKH患者的PBMC、活动期Behcet病患者的CD4+T细胞以及静止期Behcet病患者的PBMC和DC中这五种miRNAs水平与正常人相比均没有统计学差异。
2.体外能成功将miRNA-155mimic及miRNA-155inhibitor转染入DC中，并且转染了miRNA-155mimic及miRNA-155inhibitor的DC能分别高表达和低表达miRNA-155。
3.转染miRNA-155mimic及miRNA-155inhibitor后的DC，其成熟度没有统计学差异。
4.转染miRNA-155mimics后的DC分泌的促炎因子IL-6呈现显著低表达，而抑炎因子IL-10、IL-1β则呈现显著高表达；而转染miRNA-155inhibitor后的DC分泌的促炎因子IL-6呈现显著高表达，而抑炎因子IL-10、IL-1β则呈现显著低表达。
5.转染了miRNA-155mimics的DC共培养的CD4+T细胞中，炎症因子IL-17显著低表达；而在与转染了miRNA-155inhibitor的DC共培养的CD4+T细胞中，炎症因子IL-17显著高表达。
6.通过综合多个生物信息学预测网站和软件的预测结果，我们根据miRNA-155保守的核苷酸序列预测到了其在人TAB2基因的3’UTR区域有一个可能的靶位点。miR-155能够结合到TAB23’UTR区域而抑制荧光素酶的表达。
7.转染了miR-155mimics的DC中TAB2蛋白的表达量显著下降，而转染了miR-155inhibitor的DC中TAB2蛋白的表达量明显上调，两者均以GAPDH作为内参来均衡蛋白的上样量。该结果表明miR-155能够直接调控靶基因TAB2的蛋白水平表达。
结论：
我们的研究发现在活动期Behcet病患者中miR-155显著低表达。而在这类疾病中这种下调的miR-155水平是通过调节DC和DC4+T细胞分泌的细胞因子而发挥作用的，并且这个调节作用是通过miR-155调控了靶基因TAB2而实现的。
第二部分MiRNAs与Behcet病、Vogt-小柳原田综合征和Fuchs综合征遗传易感性研究
目的：
本实验旨在探讨miR-146a和转录因子Ets-1基因多态是否与中国汉族人群Behcet病（BD）、Vogt-小柳原田综合征（VKH）和Fuchs’综合征（FUS）相关。
方法：
1.运用PCR-RFLP和测序的方法对669例BD患者的miR-146a和Ets-1基因的三个多态位点（miR-146a/rs2910164, ets-1/rs1128334和rs10893872）进行了基因分型。
2.运用PCR-RFLP和测序的方法分别对613例VKH患者的miR-146a和Ets-1基因的三个多态位点（miR-146a/rs2910164,ets-1/rs1128334和rs10893872）进行了基因分型。
3.运用PCR-RFLP和测序的方法分别对219例FUS患者的miR-146a和Ets-1基因的三个多态位点（miR-146a/rs2910164,ets-1/rs1128334和rs10893872）进行了基因分型。
4.运用PCR-RFLP和测序的方法分别对1132例正常人的miR-146a和Ets-1基因的三个多态位点（miR-146a/rs2910164,ets-1/rs1128334和rs10893872）进行了基因分型。
5.运用定量PCR的方法检测miR-146a/rs2910164位点三种基因型的正常人PBMCs细胞中miR-146a表达水平。
6.运用ELISA的方法检测miR-146a/rs2910164位点三种基因型的正常人PBMCs培养上清中细胞因子（IL-17、IL-1β、IL-6、IL-8和MCP-1）的表达水平。
结果：
1.正常人的miR-146a和Ets-1基因的3个SNP的分型结果都符合哈迪温伯格平衡（p>0.05）。BD患者的miR-146a基因的rs2910164CC基因型和显著低于正常对照组(pc=2.19×10~-5, OR0.61)，而GC基因型频率则显著高于正常对照组（pc=0.009, OR1.38)；rs2910164C等位基因的频率也显著低于正常对照组（pc=9.3×10~-5, OR0.75）。
2.定量PCR结果显示：rs2910164基因GG基因型的正常人PBMC细胞中miR-146a基因的表达水平是CC型中miR-146a表达的2.45倍，是GC型中miR-146a表达的1.99倍。
3. ELISA结果显示：IL-17和IL-1β表达在rs2910164基因CC基因型的正常人PBMC培养上清中显著低于GG基因型。IL-6和MCP-1的表达水平在三个基因型中没有统计学差异。而IL-8在rs2910164基因CC基因型的正常人PBMC培养上清中则显示出轻微上调的表达。
4. Ets-1基因的2个SNP rs1128334和rs10893872，各位点基因型和等位基因的频率分布在BD病患者和正常对照组之间均统计学差异。miR-146a和Ets-1基因3个SNP的等位基因与BD患者主要临床症状也没有统计学差异。
5.正常人的miR-146a和Ets-1基因的3个SNP的分型结果都符合哈迪温伯格平衡（p>0.05）。miR-146a和Ets-1基因3个SNP的等位基因与VKH患者主要临床症状没有统计学差异。VKH患者的miR-146a和Ets-1基因3个SNP的等位基因、基因型频率与正常对照组之间均无统计学差异。
6.正常人的miR-146a和Ets-1基因的3个SNP的分型结果都符合哈迪温伯格平衡（p>0.05）。FUS患者的miR-146a和Ets-1基因3个SNP的等位基因、基因型频率与正常对照组之间均无统计学差异。结论：
miR-146a基因的rs2910164与中国汉族人群中BD疾病发病有关。rs2910164CC基因型和C等位基因可能是BD疾病的保护基因。miR-146a和Ets-1基因多态与VKH和FUS无相关性。
Background
Uveitis, a common cause of blindness in the world, can be due toinfectious or noninfectious mechanism. It has various clinical patterns andcharacteristics in different countries. Behcet’s disease (BD) andVogt-Koyanagi-Harada (VKH) syndrome are the two most common uveitisentities in China. It is generally recognized that both diseases are inducedby a complicated interaction, such as environmental factors, autoimmuneresponse and other exogenous elements. Although the etiology of FUS hasnot yet been fully understood, several studies have revealed that both viralinfection and genetic risk factors may be involved in its pathogenesis.
Mammalian microRNAs (miRNAs) are small (18-25nt long),endogenous, noncoding RNA oligonucleotides. They are highly conservedduring evolution and have recently emerged as potent regulators of geneexpression linked to most biological function. miRNAsposttranscriptionally regulate gene expression by binding with imperfectcomplementarity to the sequences in the3’ untranslated region (3’ UTR) of target mRNAs.
Based on the background, the present study was designed to explainthe questions as follows:1. Whether these five known immunologicallyrelevant miRNAs (miR-155, miR-146a, miR-326, miR-181a, miR-17)involved in the development of Behcet’s disease or VKH syndrome?2.Whether microRNA-146a and Ets-1gene polymorphisms are associatedwith ocular Behcet’s disease, Vogt-Koyanagi-Harada syndrome and FuchUveitis syndrome?
PartⅠ Effect of microRNA-155on cytokine production bydendritic cells is involved in the pathogenesis of Behcet’sdisease
Purpose
MicroRNA (miRNAs) have emerged as a class of gene expressionregulators in the regulation of immunity and mammalian inflammatoryresponse. In the present study, we investigated the role of miRNA inBehcet’ disease (BD), an autoinflammatory disease.
Methods
MiRNAs expression in peripheral blood mononuclear cells (PBMCs),dendritic cells (DCs) and CD4+T cell were examined using TaqMan real-time PCR. MiR-155mimics and inhibitor were transfected to DCs toevaluate the effects of miR-155on the DC mature and cytokine productionby these cells, and the influence of miR-155-overexpressed DCs on thecytokine production of CD4+T cells. Luciferase reporter assays andWestern blotting were performed to identify the target gene of miR-155.
Results
BD patients with active uveitis showed a significantly decreasedexpression of miR-155in PBMCs and DCs, but not in CD4+T cells, ascompared with normal individuals. Overexpression of miR-155couldinhibit the production of IL-6and IL-1β and promote the expression ofIL-10in DCs. MiR-155transfected DC could significantly inhibitintracellular IL-17expression of allogeneic CD4+T cells when culturedtogether. However, it did not influence the phenotypic DC maturation.Luciferase reporter assays revealed that TAB2was the target gene ofmiR-155. Western blotting results also confirmed this result.
Conclusions
The present results suggest that miR-155expression is decreased inactive BD patients. Downregulated miR-155could be involved in thisdisease through modulating cytokine production of DC and CD4+T cellsby targeting TAB2.
Part Ⅱ MicroRNA-146a and Ets-1gene polymorphisms inocular Behcet’s disease, Vogt-Koyanagi-Harada syndromeand Fuch Uveitis syndrome
Purpose
MicroRNA-146a (miR-146a) is involved in certainimmune-mediated diseases. Transcription factor Ets-1strongly affectsmiR-146a promoter activity and directly regulates miR-146a expression.This study was performed to investigate the association of miR-146a andEts-1gene polymorphisms with Behcet’s disease (BD),Vogt-Koyanagi-Harada (VKH) disease and Fuch Uveitis syndrome (FUS)in a Chinese Han population.
Methods
A total of669BD patients,613VKH patients,219FUS patients and1132normal controls were genotyped for miR-146a/rs2910164,ets-1/rs1128334and rs10893872using a PCR restriction fragment lengthpolymorphism assay. miR-146a expression was examined in PBMCs byreal-time PCR. Cytokine production by PBMCs were measured by ELISA.
Results
A significantly decreased frequency of the homozygous rs2910164CC genotype and C allele was observed in BD patients compared with controls(pc=2.19×10~-5, odds ratio (OR)0.61; pc=9.3×10~-5, OR0.75, respectively).MiR-146a expression in GG cases was2.45-fold and1.99-fold respectivelyhigher than that in CC cases and GC cases. There was no association ofrs1128334or rs10893872with BD. There was also no association of thesethree SNPs with its main clinical features. No associations were found withthe three SNPs tested and with its clinical manifestations in VKH disease.IL-17and IL-1β production from rs2910164CC cases was markedly lowerthan that in GG cases. No effect of genotype was observed on the IL-6andMCP-1production and IL-8expression was slightly higher in CC cases.There was no association of these three SNPs with FUS.
Conclusions
Our study identified a strong association of rs2910164of miR-146awith BD in a Chinese population and an decreased expression of miR-146aand certain proinflammatory cytokines in individuals carrying the CCgenotype.
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