DNA甲基化对人单核细胞FcεRIγ亚基基因表达的调节及其在特应性皮炎发病中的作用

发布时间：2018-05-06 20:16

本文选题：5-azaC + FcεRI　；参考：《中南大学》2010年硕士论文

【摘要】： 特应性皮炎是一种遗传、免疫和环境等因素相互作用的炎症性皮肤病,其确切的发病机制尚未完全明了,现有研究证实树突状细胞和单核细胞表面IgE高亲和力受体(FcεRI)异常表达与特应性皮炎发病密切相关,而FcεRIγ亚基的表达在树突状细胞FcεRI表达中起关键作用。研究发现FcεRIγ亚基基因转录启动子中存在两处Alu重复序列,而Alu序列是DNA甲基化的高发区,那么DNA甲基化是否参与FcεRIγ亚基基因转录表达的调控?病理性DNA低甲基化对FcεRIγ亚基基因的调控是否参与特应性皮炎的发病?本课题以DNA甲基化对FcεRIγ亚基基因转录表达的调控作用为着眼点,研究树突状细胞的前提细胞即单核细胞通过DNA甲基化转移酶抑制剂处理后FcεRIγ亚基基因的表达,特应性皮炎患者和正常人单核细胞FcεRIγ亚基基因表达及DNA甲基化状态的差异；从而探讨病理性DNA低甲基化对人单核细胞FcεRIγ亚基表达的调控在特应性皮炎发病中的作用。目的研究DNA甲基化转移酶抑制剂5-氮杂胞苷(5-azaC)干预对正常单核细胞FcεRIγ亚基基因表达的影响,从而探讨DNA甲基化对单核细胞FcεRIγ亚基基因表达的调控。方法密度梯度离心分离3名健康志愿者的外周血单个核细胞,磁珠分选单核细胞,脂多糖(LPS)刺激培养,分别用或不用DNA甲基化转移酶抑制剂(5-azaC)处理3天；而后收集细胞分别作流式细胞检测,提取全基因组DNA、mRNA及蛋白；然后用亚硫酸盐氢钠基因组测序法检测药物处理与未处理单核细胞FcεRIγ亚基启动子DNA甲基化调控序列DNA甲基化水平；western-bolt法、实时定量聚合酶链反应(real-time RT-PCR)分别检测FcεRIγ亚基的蛋白与mRNA的表达水平。结果与未处理组相比,(1)5-azaC处理组单核细胞FcεRIγ亚基mRNA水平明显升高(P=0.008)；(2)5-azaC处理组单核细胞FcεRIγ亚基蛋白水平明显升高(P=0.007)；(3)5-azaC处理组单核细胞表面FcεRI表达水平明显升高(细胞百分率P=0.001,荧光平均强度P=0.005)；(4)5-azaC处理组单核细胞FcεRIγ亚基基因调控序列DNA甲基化水平明显降低(P=0.003)；结论单核细胞FcεRIγ亚基基因表达受DNA甲基化调控,并影响单核细胞FcεRI表面的表达。目的研究AD患者外周血单核细胞是否存在FcεRIγ亚基及FcεRI表达异常,并是否受DNA甲基化调控。方法密度梯度离心分离10例AD患者和10例正常人的外周血单个核细胞,磁珠分选单核细胞,用流式细胞仪检测FcεRI蛋白在单核细胞表面的表达水平；实时定量聚合酶链反应(real-time RT-PCR)检测FcεRIγ亚基的mRNA表达水平；western-blot检测FcεRIγ亚基的蛋白表达水平；亚硫酸氢钠测序检测FcεRIγ亚基启动子DNA甲基化调控序列的甲基化状态。结果与正常对照组相比,(1)AD组单核细胞FcεRIγ亚基mRNA水平明显升高(P=0.01)；(2)AD组单核细胞FcεRIγ亚基蛋白水平明显升高(P=0.000)；(3)AD组单核细胞表面FcεRI表达水平明显升高(细胞百分率P=0.045,荧光平均强度P=0.000)；(4)AD组单核细胞FcεRIγ亚基基因调控序列DNA甲基化水平明显降低(P=0.001)；(5)我们还发现AD患者FcεRIγ亚基启动子DNA甲基化水平与FcεRIγ亚基蛋白表达呈负相关性(R=-0.711,P=0.021)。结论AD患者单核细胞FcεRIγ亚基基因调节序列甲基化水平低下,导致FcεRIγ亚基与FcεRI在细胞表面过度表达,从而参与疾病的发病过程。
[Abstract]:The expression of Fc.epsilon . RI gamma subunit gene is closely related to the pathogenesis of atopic dermatitis , and the expression of Fc.epsilon . RI gamma subunit plays a key role in the expression of Fc.epsilon . RI 纬 subunit gene .
Objective To investigate the role of low methylation of pathological DNA in the pathogenesis of atopic dermatitis .

Objective To investigate the effect of 5 - azaC intervention on the expression of Fc.epsilon . RI 纬 subunit gene in normal monocytes .

Methods Peripheral blood mononuclear cells ( PBMC ) , magnetic beads ( mononuclear cells ) and lipopolysaccharide ( LPS ) were isolated from three healthy volunteers by density gradient centrifugation , and treated with or without DNA methylation - transferase inhibitor ( 5 - azaC ) for 3 days .
then collecting the cells for flow cytometry respectively to extract the whole genome DNA , the mRNA and the protein ;
then detecting the DNA methylation level of the DNA methylation regulatory sequence of the drug treatment and the untreated Monocyte Fc.epsilon . RI gamma subunit promoter by using a sodium bisulfite genome sequencing method ;
Real - time RT - PCR ( real - time RT - PCR ) was used to detect the protein and mRNA expression levels of Fc.epsilon . RI .

Results Compared with untreated group , ( 1 ) 5 - azaC treated group had higher expression of Fc.epsilon . RI 纬 subunit mRNA ( P = 0.008 ) .
( 2 ) The level of Fc.epsilon . RI 纬 subunit protein increased significantly in 5 - azaC treated group ( P = 0.007 ) .
( 3 ) The expression level of Fc 蔚RI in the monocyte surface of 5 - azaC treated group was significantly increased ( P = 0.001 , P = 0.005 ) .
( 4 ) DNA methylation level was significantly lower in 5 - azaC treated group ( P = 0.003 ) .

Conclusion The expression of Fc.epsilon . RI 纬 subunit gene in monocytes is regulated by DNA methylation .

Objective To investigate the presence of Fc.epsilon . RI 纬 subunit and Fc.epsilon RI in peripheral blood mononuclear cells of AD patients .

Methods Peripheral blood mononuclear cells and magnetic beads were isolated from 10 AD patients and 10 normal controls by density gradient centrifugation , and the level of Fc 蔚RI protein expression on monocytes was detected by flow cytometry .
Real - time RT - PCR was used to detect the mRNA expression level of Fc.epsilon . RI 纬 subunit .
Western - blot was used to detect the protein expression level of Fc.epsilon . RI 纬 subunit .
The methylation status of DNA methylation regulatory sequences of Fc.epsilon . RI . gamma . subunit promoter was detected by sodium bisulfite sequencing .

Results Compared with the control group , ( 1 ) the mRNA level of Fc.epsilon . RI in AD group increased significantly ( P = 0.01 ) ;
( 2 ) The level of Fc 蔚RI 纬 subunit protein in AD group increased significantly ( P = 0.000 ) .
( 3 ) The expression level of Fc 蔚RI in the monocytes of AD group was significantly increased ( P = 0 . 45 , P = 0.000 ) .
( 4 ) DNA methylation level was significantly lower in AD group ( P = 0.001 ) .
( 5 ) We also found that the DNA methylation level of Fc.epsilon . RI . gamma . subunit promoter in AD patients was negatively correlated with the expression of Fc.epsilon . RI . gamma . subunit protein ( R = - 0.711 , P = 0.021 ) .

Conclusion The methylation level of Fc.epsilon . RI 纬 subunit gene in AD patients is low , resulting in overexpression of Fc.epsilon . RI gamma subunit and Fc.epsilon . RI in the surface of the cell , thereby participating in the pathogenesis of the disease .

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R758.2

【参考文献】