摘要
目的探讨内皮素受体B（EDNRB）基因启动子上游CpG岛甲基化在先天性巨结肠（Hirschsprung disease，HD）发病机制中的作用。方法采用甲基化特异性PCR（MSP）技术检测2007年9月至2009年3月收集的经病检等证实的25例HD患儿病变组织标本EDNRB基因CpG岛甲基化状态．以同期收集的16例非HD患儿正常结肠组织标本作为阴性对照，采用荧光定量PCR（SYBR-Green法）和Western-blot技术检测这15例HD患儿病变组织标本中EDNRB基因mRNA和蛋白的相对表达量。结果15例标本MSP检测．有2例发现甲基化，甲基化率为2／25（8％）；16例阴性对照均未发现甲基化。荧光定量PCR发现：甲基化组织EDNRB基因Ct值（扩张段0．13±0．08，移行段0．10±0．06．挛缩段（1．09±0．05）均较非甲基化组织（扩张段0．59±0．24．移行段0．57±0．32，挛缩段0．48±0．30）有明显下凋．差异具有统计学意义（P〈0．05）。Western-blot发现：甲基化组织EDNRB基因蛋白相对灰度值（扩张段1．56±0．43，移行段1.32±0．32．挛缩段1．27±0．21）较非甲基化组织（扩张段1．72±0．36．移行段1．52±0．62，挛缩段1．48±0．49）有所下降，但差异无统计学意义（P〉0．05）。结论内皮素受体B（EDNRB）基因启动子上游CpG岛甲基化可能通过影响其mRNA的表达水平导致其表观遗传学的改变．进而导致HD的发生。
Objective To investigate the pathogenesis effect of hypermethylation of CpG sites at upstream of transcription start site of Endothelin receptor B gene （EDNRB） on Hirschsprung disease （HD） . Methods Methylation specific polymerase chain reaction （MSP） for the CpG sites of EDNRB was performed in samples collected from 25 patients diagnosed with Hirschsprung Disease between September 2007 and March 2009 in Wuhan Union Hospital; and the colonic samples of 16 non Hir schsprung Disease patients was selected as controls. Fluorescence quantitative polymerase chain reaction （SYBR Green method） and western blot were performed to detect the gene and protein expression of EDNRB. Results EDNRB methylation was found in 2 of the 25 HD patients （8%）. No EDNRB methylation was found in the 16 controls. In HD patients, Ct of EDNRB in methylated tissues （enlarged segment 0. 13 ± 0. 08, transitional segment 0. 10 ± 0. 06, contracted segment 0. 09 ± 0. 05） were significantly decreased than that of non methylated tissues （enlarged segment 0. 59 ± 0. 24, transitional segment 0.57 ± 11. 32, contracted segment 0. 48 ± 0. 30） （P〈0. 05）. There is no difference of EDNRB protein expression between methylated tissues and non methylated tissues （P〉0. 05）. Conclusions EDNRB hypermethylation at CpG sites down regulates the transcriptional expression of EDNRB, which may play a role in the pathogenesis of HD.
出处
《中华小儿外科杂志》
CSCD
北大核心
2009年第12期849-853,共5页
Chinese Journal of Pediatric Surgery
基金
基金项目：国家自然科学基金面上项目（30872473）
关键词
甲基化
遗传学研究
Methylation
Genetic researeh
