【作者】 王萍；

【导师】 文海泉；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2010， 硕士

【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。更多还原

【Abstract】 Background Environmental factors influencing the manifestation of atopic diseases, which imply epigenetic modification of key genes may contribute to the pathogenesis of atopic disease. The high-affinity receptor for IgE (FcεRI) is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases,FcεRI gamma subunit(FcεRIγ) is key modulator for the maturation and surface expression of FcεRI. In this context, Structural factors influencing the expression of FcεRIγhave gained considerable interest. Recent research revealed that there are two Alu repeats commonly regulated by DNA methylation in the FcεRIγsubunit transcription pomotor, so we investigated the putative role of DNA methylation in the regulation of FcεRIγgene expression in atopic monocytes.Objective To study the putative role of DNA methylation in the regulation of FcεRIγgene and FcεRI expression in human monocytes.Methods PBMCs (peripheral blood mononuclear cells) from 3 healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation, MOs were isolated from the PBMC using magnetic cell separation technique. MOs were cultured in lipopolysaccharide(LPS), then cells were harvested at 72h after addition of 5-azaC. Bisulfite sequencing was used to determine the methylation status of the FcεRIγpromoter region. FcεRIγwas detected by real-time RT-PCR and Western blotting and FcεRI surface expression was detected by flow cytometry. Results Compared with the untreated MOs, treating healthy monocytes with the demethylating agent 5-azacytidine decreased promoter methylation level (P=0.003), and increased FcεRIγand surface FcεRI expression (FcεRIγprotein level:P=0.007, mRNA level:P=0.008, FcεRI surface (%):P=0.001, MFI:P=0.005).Conclusion DNA methylation regulated FcεRIγsubunit gene expression and contributed to the expression of FcεRI on MOs.Objective To investigate the expression levels and methylation patterns of FcεRIγin MOs from AD patients and healthy controls.Methods PBMCs were isolated from the peripheral venous blood of 10 AD patients and 10 healthy donors by density gradient centrifugation, MOs were isolated from the PBMC using CD 14 beads. Bisulfite sequencing was used to determine the methylation status of the FcεRIγpromoter region. Levels of FcεRIγwere measured by real-time RT-PCR and Western blotting, and using the flowcytometry to detect the expression levels of FcεRI in MOs.Results Compared with the healthy controls, the levels of FcεRIγand surface FcεRI expression in monocytes are increased in AD patients (FcεRIγprotein level:P=0.000, mRNA level:P=0.01, FcεRI surface(%): P=0.045, MFI:P=0.000). The DNA methylation levels within FcεRIγregulatory domains are reduced in atopic monocytes relative to healthy controls (P=0.001) and negatively correlate with FcεRIγexpression (R=-0.711,P=0.021).Conclusion DNA hypomethylation leads to FcεRIγsubunit and surface FcεRI overexpression in atopic dermatitis.更多还原