胆道闭锁中ITGAL基因启动子区甲基化状态改变及其意义  

DNA methylation of ITGAL promoter in T cells from the peripheral blood and hepatic porta lymph node in biliary atresia

作　　者：赵瑞[1] 董瑞[1] 郑珊[1] 

机构地区：[1]复旦大学附属儿科医院外科,上海201102

出　　处：《中华小儿外科杂志》2011年第3期174-178,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金（30973139）和上海市科委基础科学重点项目基金资助（09JC1402800）

摘　　要：目的 研究胆道闭锁外周血及肝门淋巴结中T淋巴细胞ITGAL基因的表达，并分析其启动子区甲基化情况及对基因表达的影响。方法收集7例胆道闭锁（胆道闭锁组）及7例非肝脏病变（对照组）患儿外周血2ml，CD2磁珠分离T淋巴细胞，以BSP法对ITGAL基因启动子区甲基化状态进行检测，Real-timePCR法对ITGAL基因mRNA表达情况进行检测；同时收集7例胆道闭锁及10例胆总管囊肿患儿肝门淋巴结，CD2磁珠分离T淋巴细胞，以BSP法和Real-timePCR法分别对ITGAL基因启动子区甲基化状态及mRNA表达情况进行检测。5-azaC处理Jurkat细胞分析IT-GAL基因启动子区低甲基化对该基因表达的影响。结果 ITGAL基因-250bp到250bp在各组外周血T细胞和肝门淋巴结T细胞中均处于持续的非甲基化状态，甲基化率均为0%（P〉0．05）。胆道闭锁组外周血T淋巴细胞与对照组相比ITGAL基因启动子区-1450bp到-950bp甲基化率分别为60．2％和54．4％（P=0．196），而ITGAL基因mRNA表达差异亦无统计学意义（P=0．0735）。胆道闭锁肝门淋巴结与胆总管囊肿肝门淋巴结T淋巴细胞ITGAL基因启动子区-1450bp到-950bp甲基化率分别为43．6％和65．6％（P=0．000），且ITGAL基因mRNA表达明显升高（P=0．0234）。5-azaC处理后Jurkat细胞ITGAL启动子区甲基化程度分别为0．46±0．08和0．21±0．04（P=0．038），处理后ITGAI。基因mRNA表达较之前升高2．3倍。结论胆道闭锁肝门淋巴结中T淋巴细胞存在ITGAL基因启动子区低甲基化改变，同时ITGAL基因高表达，提示甲基化异常是胆道闭锁中T淋巴细胞功能异常的潜在原因。Objective The present research examines ITGAL promoter methylation that may affect LFA-1 expression in T cells from the peripheral blood and hepatic porta lymph node in biliary atresia （BA）. Methods We collected blood samples from 7 BA patients and 7 healthy controls. Meanwhile, hepatic porta lymph nodes were collected from 7 BA patients and 10 congenital biliary dilation （CBD） patients. T cells were isolated using CD2 microbeads. Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter in T cells. Real-time RT-PCR was performed to quantify the expression of LFA-1 mRNA. T-cell lines have been treated with 5-azaC to analysis the effects of hypomethylation on LFA-lgene expression. Results The -250 to 250 bp near the transcription start site, recognized by transcription factors, is constitutively demethylated in the T cells isolated from both peripheral and hepatic porta lymph node in BA and control（P〉0. 05）. The methylation index of ITGAL promoter （-1450 to -950bp） and mRNA expression in the T cell from BA and healthy control peripheral blood was not significantly different（P〉0. 05）. The methylation index of ITGAL promoter （-1450 to -950bp） in the T cells isolated from hepatic porta lymph node in BA and CBD patients were 43.6% and 65.6%（P = 0. 000）. The mRNA expressions were elevated significantly in the T ceils isolated from hepatic porta lymph node in BA than CBD patients（P = 0. 0234）. 5-azaC inhibited the methylation in ITGAL promoter and enhanced the expression of ITGAL mRNA（P〈0. 05）. Conclusions ITGAL promoter was hypomethylated and the hypomethylation caused overexpression of LFA-1 in the hepatic porta lymph node in BA, which might resulte in the autoimmunity of T cells.

关 键 词：胆道闭锁 甲基化 T淋巴细胞 

分 类 号：R726.5[医药卫生—儿科]