内皮素受体B基因启动子上游CpG岛甲基化对神经嵴细胞迁移的影响

Effect of Methylation at CpG Sites Upstream of Transcription Start Site of Endothelin Receptor B Gene on Migration of Neural Crest Cells

目的探讨内皮素受体B(EDNRB)基因启动子上游CpG岛甲基化对肠神经系统(ENS)发育中神经嵴细胞迁移的影响。方法采用甲基化特异性PCR(MSP)技术检测2007年9月-2008年12月华中科技大学同济医学院附属协和医院经病理检查等证实的16例先天性巨结肠症(HD)患儿挛缩段组织标本中EDNRB基因启动子上游CpG岛甲基化状态;采用荧光定量PCR(SYBRGreen法)检测HD患儿病变组织标本(包括扩张段、移行段和挛缩段3段组织)中EDNRB基因mRNA的相对表达量。并收集同期16例非HD患儿的正常结肠组织标本作为对照。结果HD患儿挛缩段标本行MSP检测,甲基化率为12.5%(2/16例);非HD患儿正常组织均未发现甲基化。16例HD患儿病变组织标本(包括扩张段、移行段、挛缩段)荧光定量PCR检测发现2例甲基化病变组织标本(包括扩张段、移行段、挛缩段)中EDNRB基因表达水平较非甲基化组织明显下调,差异有统计学意义(P<0.05)。结论在HD发病机制中,EDNRB基因启动子上游CpG岛甲基化可能对ENS发育中神经嵴细胞的迁移有一定影响。

Objective To investigate the effect of methylation at CpG sites upstream of the transcription start site of endothelin receptor B （EDNRB） gene on migration of neural crest cells in the development of enteric nervous system （ENS）. Methods Methylation specific polymerase chain reaction （MSP） for the CpG sites of EDNRB gene was performed on postoperative collected contracture sections of colonic tissue samples from 16 children harbouring clinically localised Hirschsprung disease（HD） in Union Hospital,Tongji Medical College,Huazhong University of Science and Technology from Sep. 2007 to Dec. 2008 ,and in a control group of 16 children with normal tissue samples of non HD. Fluorescence quantitative polymerase chain reaction（ SYBR Green method） was used to detect the mRNA expression of EDNRB gene on the postoperative collected colonic tissue samples （included expansion, transitional and contracture sections）from 16 children of HD. Results EDNRB gene methylation was found in 2 out of 16 contracture section samples of the HD,the rate of EDNRB gene methylation was 12.5%. And no EDNRB gene methylation was found in the paired normal tissue samples of control group. The expressing level of mRNA in 2 tissues of CpG sites methylation of EDNRB gene were declined heavily by fluorescence quantitative polymerase chain reaction, contrasting with the normal tissues of non HD（P 〈 0. 05 ）. Conclusions Methylation at CpG sites upstream of the transcription start site of EDNRB gene may have an effect on the migration of neural crest cells in the development of ENS in the pathogenesis of HD.