摘要
目的通过对先天性肛门直肠畸形(anorectal malformation,ARM)患儿末端直肠的组织病理学研究,探索Shh/Bmp4信号通路基因甲基化修饰对ARM末端直肠神经系统(enteric nervous system,ENS)的调控作用。方法收集复旦大学附属儿科医院收治的20例ARM及8例非胃肠道疾病死亡患儿直肠末端组织标本。通过PCR、免疫组织化学和Western blot等方法检测末端直肠组织中DNA甲基转移酶(DNMT1、DNMT3a、DNMT3b)含量及活性、Shh基因启动子区甲基化状态、Shh/Bmp4信号通路关键组分的表达及ENS发育情况。结果ARM组患儿直肠组织中DNMTs(DNMT1、DNMT3a、DNMT3b)的表达在mRNA和蛋白水平上分别为3.54±1.08、2.37±0.75和2.00±0.14,均较对照组的1.00±0.17、1.00±0.30和1.00±0.44明显升高,且差异有统计学意义(P=0.0141、0.0413、0.0120)。ARM组Shh基因启动子区甲基化水平为3.31±0.53,明显高于对照组的5.47±2.20;蛋白表达水平为27655.03±9032.63,较对照组的73738.36±22061.03明显降低,组间比较,差异均有统计学意义(P=0.0435和0.0052)。ARM组Shh/Bmp4信号通路关键组分Bmp4表达水平为1.00±0.16,较对照组3.84±0.71明显降低,组间比较,差异有统计学意义(P=0.0022)。ARM组直肠组织中S-100标记的神经丛分布稀疏分散,其在黏膜下层和肌间神经丛的阳性神经丛染色密度分别为1.23±0.24和1.31±0.11,与对照组相同位置的值(4.21±0.36和4.36±0.65)比较,表达均明显减少,差异均有统计学意义(P<0.0001)。ARM组直肠中c-kit标记的cajal间质细胞和NSE标记的神经节细胞未见明显表达。结论先天性ARM末端直肠组织整体处于高甲基化水平,且Shh基因启动子区处于高甲基化状态,Shh/Bmp4关键组分在ARM组织中表达下降,提示Shh高甲基化状态可能抑制Shh/Bmp4信号通路的表达。
Objective To explore the effects of gene methylation on the development of enteric nervous system (ENS) in rectal end of pediatric congenital anorectal malformation (ARM) by regulating Shh/Bmp4 signaling pathway. Methods Tissue samples from terminal rectum of pediatric ARM patients (n=20) and patients dead from non-gastrointestinal tract malformation (n=8) were collected. The contents of DNA methyltransferases (DNMT1, DNMT3a & DNMT3b), the methylation status of Shh gene promoter region, the expression of key components in Shh/Bmp4 signaling pathway and the development of ENS in rectal end were detected by polymerase chain reaction (PCR), immunohistochemistry and Western blot. Results The expressions of DNMTs (DNMT1, DNMT3a & DNMT3b) were significantly higher in rectal tissue of ARM group than that of control group at the levels of mRNA and protein (DNMT1: 3.54±1.08 vs 1.00±0.17, P=0.014 1;DNMT3a: 2.37±0.75 vs 1.00±0.30, P=0.041 3;DNMT3b: 2.00±0.14 vs 1.00±0.44, P=0.012 0). The methylation level of Shh gene promoter was significantly higher in ARM group than that in control group (3.31±0.53 vs 5.47±2.20, P=0.043 5) and the protein expression level significantly decreased in ARM group (27 655.03±9 032.63 vs 73 738.36±22 061.03, P=0.005 2). The Bmp4 expression level of Shh/Bmp4 signaling pathway was significantly lower in ARM group than that of control group (1.00±0.16 vs 3.84±0.71, P=0.002 2). The distribution of S-100 labeled nerve plexus was sparse and scattered in rectal tissue of ARM group and submucosal (1.23±0.24 vs 4.21±0.36, P<0.000 1) and intermuscular (1.31±0.11 vs 4.36±0.65, P<0.000 1) nerve plexus significantly decreased compared with that of normal group. And c-kit-labeled Cajal stromal and NSE-labeled ganglion cells were not obviously expressed. Conclusions Overall ARM rectal end is at a high methylation level and Shh gene promoter region stays in a high methylation state. The key component of Shh/Bmp4 signaling pathway decreases in ARM rectal tissue and there is a sparse distribution
作者
周莹
杨一凡
董瑞
黄焱磊
Zhou Ying;Yang Yifan;Dong Rui;Huang Yanlei(Department of Pediatric Surgery, Children’s Hospital of Fudan University, Children’s National Medical Center (Shanghai), Shanghai 201102, China)
出处
《中华小儿外科杂志》
CSCD
北大核心
2018年第12期914-917,共4页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金青年项目(81401243)
上海复旦大学"卓学计划"(2015-2018).
关键词
先天性肛门直肠畸形
DNA甲基化
肠神经系统
Congenital ano-rectal malformation
DNA methylation
Enteric nervous system
