本文選題：5-azaC + FcεRI��； 參考：《中南大學(xué)》2010年碩士論文

【摘要】： 特應(yīng)性皮炎是一種遺傳、免疫和環(huán)境等因素相互作用的炎癥性皮膚病,其確切的發(fā)病機(jī)制尚未完全明了,現(xiàn)有研究證實(shí)樹(shù)突狀細(xì)胞和單核細(xì)胞表面IgE高親和力受體(FcεRI)異常表達(dá)與特應(yīng)性皮炎發(fā)病密切相關(guān),而FcεRIγ亞基的表達(dá)在樹(shù)突狀細(xì)胞FcεRI表達(dá)中起關(guān)鍵作用。研究發(fā)現(xiàn)FcεRIγ亞基基因轉(zhuǎn)錄啟動(dòng)子中存在兩處Alu重復(fù)序列,而Alu序列是DNA甲基化的高發(fā)區(qū),那么DNA甲基化是否參與FcεRIγ亞基基因轉(zhuǎn)錄表達(dá)的調(diào)控?病理性DNA低甲基化對(duì)FcεRIγ亞基基因的調(diào)控是否參與特應(yīng)性皮炎的發(fā)病?本課題以DNA甲基化對(duì)FcεRIγ亞基基因轉(zhuǎn)錄表達(dá)的調(diào)控作用為著眼點(diǎn),研究樹(shù)突狀細(xì)胞的前提細(xì)胞即單核細(xì)胞通過(guò)DNA甲基化轉(zhuǎn)移酶抑制劑處理后FcεRIγ亞基基因的表達(dá),特應(yīng)性皮炎患者和正常人單核細(xì)胞FcεRIγ亞基基因表達(dá)及DNA甲基化狀態(tài)的差異；從而探討病理性DNA低甲基化對(duì)人單核細(xì)胞FcεRIγ亞基表達(dá)的調(diào)控在特應(yīng)性皮炎發(fā)病中的作用。 目的研究DNA甲基化轉(zhuǎn)移酶抑制劑5-氮雜胞苷(5-azaC)干預(yù)對(duì)正常單核細(xì)胞FcεRIγ亞基基因表達(dá)的影響,從而探討DNA甲基化對(duì)單核細(xì)胞FcεRIγ亞基基因表達(dá)的調(diào)控。 方法密度梯度離心分離3名健康志愿者的外周血單個(gè)核細(xì)胞,磁珠分選單核細(xì)胞,脂多糖(LPS)刺激培養(yǎng),分別用或不用DNA甲基化轉(zhuǎn)移酶抑制劑(5-azaC)處理3天；而后收集細(xì)胞分別作流式細(xì)胞檢測(cè),提取全基因組DNA、mRNA及蛋白；然后用亞硫酸鹽氫鈉基因組測(cè)序法檢測(cè)藥物處理與未處理單核細(xì)胞FcεRIγ亞基啟動(dòng)子DNA甲基化調(diào)控序列DNA甲基化水平；western-bolt法、實(shí)時(shí)定量聚合酶鏈反應(yīng)(real-time RT-PCR)分別檢測(cè)FcεRIγ亞基的蛋白與mRNA的表達(dá)水平。 結(jié)果與未處理組相比,(1)5-azaC處理組單核細(xì)胞FcεRIγ亞基mRNA水平明顯升高(P=0.008)；(2)5-azaC處理組單核細(xì)胞FcεRIγ亞基蛋白水平明顯升高(P=0.007)；(3)5-azaC處理組單核細(xì)胞表面FcεRI表達(dá)水平明顯升高(細(xì)胞百分率P=0.001,熒光平均強(qiáng)度P=0.005)；(4)5-azaC處理組單核細(xì)胞FcεRIγ亞基基因調(diào)控序列DNA甲基化水平明顯降低(P=0.003)； 結(jié)論單核細(xì)胞FcεRIγ亞基基因表達(dá)受DNA甲基化調(diào)控,并影響單核細(xì)胞FcεRI表面的表達(dá)。 目的研究AD患者外周血單核細(xì)胞是否存在FcεRIγ亞基及FcεRI表達(dá)異常,并是否受DNA甲基化調(diào)控。 方法密度梯度離心分離10例AD患者和10例正常人的外周血單個(gè)核細(xì)胞,磁珠分選單核細(xì)胞,用流式細(xì)胞儀檢測(cè)FcεRI蛋白在單核細(xì)胞表面的表達(dá)水平；實(shí)時(shí)定量聚合酶鏈反應(yīng)(real-time RT-PCR)檢測(cè)FcεRIγ亞基的mRNA表達(dá)水平；western-blot檢測(cè)FcεRIγ亞基的蛋白表達(dá)水平；亞硫酸氫鈉測(cè)序檢測(cè)FcεRIγ亞基啟動(dòng)子DNA甲基化調(diào)控序列的甲基化狀態(tài)。 結(jié)果與正常對(duì)照組相比,(1)AD組單核細(xì)胞FcεRIγ亞基mRNA水平明顯升高(P=0.01)；(2)AD組單核細(xì)胞FcεRIγ亞基蛋白水平明顯升高(P=0.000)；(3)AD組單核細(xì)胞表面FcεRI表達(dá)水平明顯升高(細(xì)胞百分率P=0.045,熒光平均強(qiáng)度P=0.000)；(4)AD組單核細(xì)胞FcεRIγ亞基基因調(diào)控序列DNA甲基化水平明顯降低(P=0.001)；(5)我們還發(fā)現(xiàn)AD患者FcεRIγ亞基啟動(dòng)子DNA甲基化水平與FcεRIγ亞基蛋白表達(dá)呈負(fù)相關(guān)性(R=-0.711,P=0.021)。 結(jié)論AD患者單核細(xì)胞FcεRIγ亞基基因調(diào)節(jié)序列甲基化水平低下,導(dǎo)致FcεRIγ亞基與FcεRI在細(xì)胞表面過(guò)度表達(dá),從而參與疾病的發(fā)病過(guò)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 緯 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 緯 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 緯 subunit mRNA ( P = 0.008 ) .
( 2 ) The level of Fc.epsilon . RI 緯 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 緯 subunit gene in monocytes is regulated by DNA methylation .



Objective To investigate the presence of Fc.epsilon . RI 緯 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 緯 subunit .
Western - blot was used to detect the protein expression level of Fc.epsilon . RI 緯 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 緯 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 緯 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 .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R758.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 楊丹榕,修清玉,韓煥興;人FcεRIα亞基細(xì)胞外區(qū)的原核表達(dá)及其和IgE結(jié)合的機(jī)制[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年11期

2 畢媛;張宏斌;王捷;;IgE高親和力受體FcεRⅠ與過(guò)敏性疾病的治療[J];廣東醫(yī)學(xué);2009年10期

3 唐昊,修清玉,韓煥興;IgE與其高親和力受體FcεRⅠ的相互作用[J];免疫學(xué)雜志;2005年S1期

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本文編號(hào)：1853724