【作者】 王颖；

【导师】 彭聪； 李捷；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2022， 硕士

【摘要】 背景:慢性自发性荨麻疹(Chronic spontaneous urticaria,CSU)是皮肤的一种常见疾病,主要由肥大细胞驱动,是临床关注的热点。经典的肥大细胞活化是由IgE介导其表面FcεRI受体聚集交联,触发一系列级联放大反应,导致脱颗粒和炎症因子的释放。目前,CSU治疗的一线药物是第二代非镇静H1抗组胺药,但三分之一的患者仍会出现抗组胺药治疗无效的情况。DNA甲基化是表观遗传调控机制之一,影响疾病的发生发展。有研究表明DNA甲基化是肥大细胞活化的关键机制之一,IgE受体交联可降低肥大细胞整体DNA甲基化水平。最近有文献提示DNA甲基化位点在CSU中有改变,但没有深入研究。目的:本研究旨在探索DNA甲基化在CSU发病及CSU抗组胺药抵抗中的作用及其机制。方法:(1)提取健康对照者和CSU患者外周血DNA,进行亚硫酸氢盐处理,使用850k芯片对转化后的DNA进行测序。(2)利用测序得到的p值和beta值,筛选出健康对照者和CSU患者的差异甲基化位点。(3)对差异甲基化位点进行GO和KEGG分析(4)焦磷酸测序验证差异甲基化位点。(5)RT-PCR技术验证其注释到的基因是否有差异。(6)ELISA检测CSU患者血浆TSP-1水平。(7)β-HEX释放实验检测肥大细胞脱颗粒效率,RT-PCR检测细胞因子mRNA表达水平。结果:(1)健康对照组与CSU疾病组间有2142个差异甲基化位点,和对照组相比,疾病组中有1804个位点甲基化水平降低,338个位点甲基化水平升高。(2)焦磷酸测序发现疾病组中CD47甲基化水平降低,mRNA表达上调。(3)TSP-1在CSU疾病组血浆、外周血中表达上调,通过生物信息分析发现TSP-1在CSU疾病组组织中表达上调。(4)TSP-1可以促进肥大细胞脱颗粒,细胞因子mRNA水平上调。(5)抗组胺药无效组与有效组间筛选得到2778个差异甲基化位点,和无效组相比,有效组有1872个位点甲基化水平降低,906个位点甲基化水平升高。(6)焦磷酸测序验证有效组中LCLAT1、MICA甲基化水平降低,而mRNA表达上调。结论:(1)与对照组相比,CSU疾病组有1804个位点甲基化水平降低,338个位点甲基化水平升高。与抗组胺药无效组相比,有效组1872个位点甲基化水平降低,906个位点甲基化水平升高。(2)焦磷酸测序验证差异位点cg09643587在CSU疾病组中甲基化水平下降,注释到CD47,差异位点cg17749961、cg12182649在有效组中下降,注释到LCLAT1、MICA。(3)CD47与TSP-1在CSU患者中表达上调。TSP-1与UAS7评分呈正相关,可以促进肥大细胞脱颗粒,促进细胞因子mRNA水平的表达。图21幅,表16个,参考文献99篇更多还原

【Abstract】 Background: Chronic spontaneous urticaria(CSU)is a common skin disease,which is mainly driven by mast cells and is a hot spot of clinical attention.The classic activation of mast cells is mediated by IgE leading to the aggregation and cross-linking of FcεRI receptor,resulting in a series of cascade amplification reactions,leading to degranulation and the release of inflammatory factors.At present,the first-line drug for CSU treatment is the second-generation non sedative H1 antihistamines,but one third of patients will still have ineffective antihistamines.DNA methylation is one of the epigenetic regulation mechanisms,which affects the occurrence and development of diseases.Studies have shown that DNA methylation is one of the key mechanisms of mast cell activation.IgE receptor crosslinking decreased the overall DNA methylation level of mast cells.Recent literature suggests that DNA methylation sites are changed in CSU,but there is no in-depth study.Objective: This study aims to explore the role and mechanism of DNA methylation in CSU and antihistamine resistance.Methods:(1)The peripheral blood DNA of healthy controls and CSU patients was extracted,treated with bisulfite,and the transformed DNA was sequenced by 850 k chip.(2)Using p value and the beta value obtained by sequencing and statistical analysis,the differential methylation sites of healthy controls and CSU patients were calculated.(3)Go and KEGG analysis of differential methylation sites.(4)Pyrophosphate assay to verify differential methylation position.(5)RT-PCR was used to detect whether the annotated genes were different.(6)The level of plasma TSP-1 in CSU patients was detected by ELISA.(7)The degranulation efficiency of mast cells was detected by β-hex release test.Results:(1)There were 2142 differential methylation sites between the healthy control group and the CSU disease group.Compared with the control group,the methylation level of 1804 sites in the disease group decreased and 338 sites increased.(2)Pyrosequencing showed that the methylation level of CD47 decreased and the mRNA expression was up-regulated in the disease group.(3)The expression of TSP-1 was up-regulated in plasma and peripheral blood of CSU patients.Through biological information analysis,it was found that the expression of TSP-1 was up-regulated in CSU patients.(4)TSP-1 can promote degranulation of mast cells and up regulate cytokine mRNA levels.(5)2778 differential methylation sites were screened between the effective group and the ineffective group.Compared with the ineffective group,the methylation level of 1872 sites in the effective group decreased and 906 sites increased.(6)Pyrosequencing confirmed that the methylation levels of LCLAT1 and MICA in the effective group decreased,while the mRNA expression was up-regulated.Conclusion:(1)Compared with the control group,the methylation levels of 1804 sites decreased and 338 sites increased in the CSU disease group.Compared with the antihistamine ineffective group,the methylation level of 1872 sites decreased and 906 sites increased in the effective group.(2)Pyrosequencing verified that the methylation level of differential site cg09643587 decreased in CSU disease group,annotated to CD47,and differential sites cg17749961 and cg12182649 decreased in effective group,annotated to lclat1 and mica.(3)CD47 and TSP-1 were up-regulated in CSU patients.TSP-1 is positively correlated with UAS7 score,which can promote mast cell degranulation and cytokine mRNA expression.更多还原