DNA甲基化修饰Shh/Bmp4信号通路调控大鼠肛肠畸形胎儿直肠末端肠神经系统发育不良的实验研究

Q4 Medicine

中华小儿外科杂志 Pub Date : 2019-11-15 DOI:10.3760/CMA.J.ISSN.0253-3006.2019.11.017

Ying Zhou, Yifan Yang, R. Dong

{"title":"DNA甲基化修饰Shh/Bmp4信号通路调控大鼠肛肠畸形胎儿直肠末端肠神经系统发育不良的实验研究","authors":"Ying Zhou, Yifan Yang, R. Dong","doi":"10.3760/CMA.J.ISSN.0253-3006.2019.11.017","DOIUrl":null,"url":null,"abstract":"Objective \nTo explore the impact of gene methylation in Shh/Bmp4 signaling pathway on the enteric nervous system (ENS) in rectal end of fetal mice with anorectal malformations (ARM). \n \n \nMethods \nA total of 15 Sprague Dawley pregnant rats were randomly divided into 3 groups of ETU, ETU+ 5-azaC and normal.At gestational Day 10, ETU and ETU+ 5-azaC groups received 1% ETU via gavage while ETU+ 5-azaC group had an intraperitoneal injection of 5-azaC (3 mg/kg). At gestational Day 12, cesarean section was performed.The incidence of ARMs, the content of DNA methyltransferase (DNMT1, DNMT3a & DNMT3b), the methylation status of Shh gene promoter region and the expression of key components in Shh/Bmp4 signaling pathway in rectal end of fetal mice were detected by polymerase chain reaction (PCR), immunohistochemistry and Western blot. \n \n \nResults \na) The incidence of ARMs was 98.3% and 64.9% in ETU and ETU+ 5-azaC groups respectively and there were statistical differences (P<0.0001); b) The expression of DNMTs (DNMT1, DNMT3a & DNMT3b) was significantly higher in rectal tissue of ETU/ETU+ 5-azaC group than that of control group (DNMT1: ETU vs normal, 3.63±0.26 vs 1.00±0.09, P<0.0001; ETU+ 5-azaC vs normal, 2.33±0.27 vs 1.00±0.09, P=0.0003; DNMT3a: ETU vs normal, 3.05±0.28 vs 1.00±0.29, P=0.0003; ETU+ 5-azaC vs normal, 2.20±0.07 vs 1.00±0.29, P<0.0001; DNMT3b: ETU vs normal, 2.62±0.49 vs 1.00±0.26, P=0.0005; ETU+ 5-azaC vs normal, 2.33±0.30 vs 1.00±0.26, P=0.0028). The expression of DNMT1/DNMT3a was significantly higher in rectal tissue of ETU group than that in ETU+ 5-azaC group (DNMT1: 3.63±0.26 vs 2.33±0.27, P=0.0025; DNMT3a: 3.05±0.28 vs 2.20±0.07, P=0.0033) while no significant inter-group difference existed in the expression level of DNMT3b; c) The methylation level of Shh gene promoter was significantly higher in rectal end of ETU group than that in ETU+ 5-azaC/control group (ETU vs ETU+ 5-azaC: 7.13±0.66 vs 5.20±1.21, P=0.0263; ETU vs normal: 7.13±0.66 vs 2.81±0.27, P<0.0001) and ETU+ 5-azaC group was obviously higher than control group (5.20±1.21 vs 2.81±0.27, P=0.0300); d) Shh/Bmp4 expression level was significantly lower in rectal end of ETU/ETU+ 5-azaC group than that of control group (Shh: ETU vs normal, 1.00±0.23 vs 3.81±0.25, P<0.0001; ETU+ 5-azaC vs normal, 2.19±0.60 vs 3.81±0.25, P=0.0121; Bmp4: ETU vs normal, 1.00±0.18 vs 4.91±0.36, P<0.0001; ETU+ 5-azaC vs normal, 2.16±0.23 vs 4.91±0.36, P<0.0001) and Shh/Bmp4 expression level in ETU group was also lower than that in ETU+ 5-azaC group (Shh: 1.00±0.23 vs 2.19±0.60, P=0.011; Bmp4: 1.00±0.18 vs 2.16±0.23, P=0.0019); e) S-100 labeled submucosa and intermuscular nerve plexus in rectal tissue of ETU group significantly decreased compared with that of normal group (P<0.0001) and ETU+ 5-azaC group was significantly different from that in ETU group (P<0.0001). The c-kit labeled Cajal stromal cells and NSE labeled ganglion cells were not obviously expressed in rectum of ETU/ETU+ 5-azaC group. \n \n \nConclusions \nThe methylation status of rectal end in ARM rat model may be changed by interventions.The low methylation level of Shh gene may promote the expression of key components in Shh/Bmp4 signaling pathway and the number of S-100 labeled nerve plexus significantly increases and promotes the development of rectal ENS. \n \n \nKey words: \nAnus, abnormalities; Rectum, abnormalities; DNA methylation; Shh/Bmp4 signaling pathway; Enteric nervous system","PeriodicalId":10157,"journal":{"name":"中华小儿外科杂志","volume":"137 1","pages":"1044-1049"},"PeriodicalIF":0.0000,"publicationDate":"2019-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Experimental study of DNA methylation modified Shh/Bmp4 signaling pathway in the regulation of enteric nervous system dysplasia in rectal end of rat fetus with anorectal malformations\",\"authors\":\"Ying Zhou, Yifan Yang, R. Dong\",\"doi\":\"10.3760/CMA.J.ISSN.0253-3006.2019.11.017\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective \\nTo explore the impact of gene methylation in Shh/Bmp4 signaling pathway on the enteric nervous system (ENS) in rectal end of fetal mice with anorectal malformations (ARM). \\n \\n \\nMethods \\nA total of 15 Sprague Dawley pregnant rats were randomly divided into 3 groups of ETU, ETU+ 5-azaC and normal.At gestational Day 10, ETU and ETU+ 5-azaC groups received 1% ETU via gavage while ETU+ 5-azaC group had an intraperitoneal injection of 5-azaC (3 mg/kg). At gestational Day 12, cesarean section was performed.The incidence of ARMs, the content of DNA methyltransferase (DNMT1, DNMT3a & DNMT3b), the methylation status of Shh gene promoter region and the expression of key components in Shh/Bmp4 signaling pathway in rectal end of fetal mice were detected by polymerase chain reaction (PCR), immunohistochemistry and Western blot. \\n \\n \\nResults \\na) The incidence of ARMs was 98.3% and 64.9% in ETU and ETU+ 5-azaC groups respectively and there were statistical differences (P<0.0001); b) The expression of DNMTs (DNMT1, DNMT3a & DNMT3b) was significantly higher in rectal tissue of ETU/ETU+ 5-azaC group than that of control group (DNMT1: ETU vs normal, 3.63±0.26 vs 1.00±0.09, P<0.0001; ETU+ 5-azaC vs normal, 2.33±0.27 vs 1.00±0.09, P=0.0003; DNMT3a: ETU vs normal, 3.05±0.28 vs 1.00±0.29, P=0.0003; ETU+ 5-azaC vs normal, 2.20±0.07 vs 1.00±0.29, P<0.0001; DNMT3b: ETU vs normal, 2.62±0.49 vs 1.00±0.26, P=0.0005; ETU+ 5-azaC vs normal, 2.33±0.30 vs 1.00±0.26, P=0.0028). The expression of DNMT1/DNMT3a was significantly higher in rectal tissue of ETU group than that in ETU+ 5-azaC group (DNMT1: 3.63±0.26 vs 2.33±0.27, P=0.0025; DNMT3a: 3.05±0.28 vs 2.20±0.07, P=0.0033) while no significant inter-group difference existed in the expression level of DNMT3b; c) The methylation level of Shh gene promoter was significantly higher in rectal end of ETU group than that in ETU+ 5-azaC/control group (ETU vs ETU+ 5-azaC: 7.13±0.66 vs 5.20±1.21, P=0.0263; ETU vs normal: 7.13±0.66 vs 2.81±0.27, P<0.0001) and ETU+ 5-azaC group was obviously higher than control group (5.20±1.21 vs 2.81±0.27, P=0.0300); d) Shh/Bmp4 expression level was significantly lower in rectal end of ETU/ETU+ 5-azaC group than that of control group (Shh: ETU vs normal, 1.00±0.23 vs 3.81±0.25, P<0.0001; ETU+ 5-azaC vs normal, 2.19±0.60 vs 3.81±0.25, P=0.0121; Bmp4: ETU vs normal, 1.00±0.18 vs 4.91±0.36, P<0.0001; ETU+ 5-azaC vs normal, 2.16±0.23 vs 4.91±0.36, P<0.0001) and Shh/Bmp4 expression level in ETU group was also lower than that in ETU+ 5-azaC group (Shh: 1.00±0.23 vs 2.19±0.60, P=0.011; Bmp4: 1.00±0.18 vs 2.16±0.23, P=0.0019); e) S-100 labeled submucosa and intermuscular nerve plexus in rectal tissue of ETU group significantly decreased compared with that of normal group (P<0.0001) and ETU+ 5-azaC group was significantly different from that in ETU group (P<0.0001). The c-kit labeled Cajal stromal cells and NSE labeled ganglion cells were not obviously expressed in rectum of ETU/ETU+ 5-azaC group. \\n \\n \\nConclusions \\nThe methylation status of rectal end in ARM rat model may be changed by interventions.The low methylation level of Shh gene may promote the expression of key components in Shh/Bmp4 signaling pathway and the number of S-100 labeled nerve plexus significantly increases and promotes the development of rectal ENS. \\n \\n \\nKey words: \\nAnus, abnormalities; Rectum, abnormalities; DNA methylation; Shh/Bmp4 signaling pathway; Enteric nervous system\",\"PeriodicalId\":10157,\"journal\":{\"name\":\"中华小儿外科杂志\",\"volume\":\"137 1\",\"pages\":\"1044-1049\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-11-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中华小儿外科杂志\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3760/CMA.J.ISSN.0253-3006.2019.11.017\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中华小儿外科杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3760/CMA.J.ISSN.0253-3006.2019.11.017","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Medicine","Score":null,"Total":0}

引用次数: 0

摘要

目的探讨Shh/Bmp4信号通路基因甲基化对肛肠畸形(ARM)胎鼠直肠末端肠神经系统(ENS)的影响。方法将15只Sprague Dawley妊娠大鼠随机分为ETU组、ETU+ 5-azaC组和正常组。妊娠第10天，ETU组和ETU+ 5-azaC组灌胃1%的ETU, ETU+ 5-azaC组腹腔注射5-azaC (3 mg/kg)。妊娠第12天行剖宫产。采用聚合酶链式反应(PCR)、免疫组织化学和Western blot检测胎鼠直肠末端ARMs发生率、DNA甲基转移酶(DNMT1、DNMT3a和DNMT3b)含量、Shh基因启动子区甲基化状态和Shh/Bmp4信号通路关键组分的表达。结果a) ETU组、ETU+ 5-azaC组的ARMs发生率分别为98.3%、64.9%，差异有统计学意义(P<0.0001);b) ETU/ETU+ 5-azaC组直肠组织中DNMT1、DNMT3a、DNMT3b的表达量显著高于对照组(DNMT1: ETU vs正常人，3.63±0.26 vs 1.00±0.09,P<0.0001;ETU+ 5-azaC vs正常人，2.33±0.27 vs 1.00±0.09,P=0.0003;DNMT3a: ETU vs正常人，3.05±0.28 vs 1.00±0.29,P=0.0003;ETU+ 5-azaC vs正常人，2.20±0.07 vs 1.00±0.29,P<0.0001;DNMT3b: ETU vs正常人，2.62±0.49 vs 1.00±0.26,P=0.0005;ETU+ 5-azaC vs正常人，2.33±0.30 vs 1.00±0.26,P=0.0028)。ETU组直肠组织中DNMT1/DNMT3a的表达明显高于ETU+ 5-azaC组(DNMT1: 3.63±0.26 vs 2.33±0.27,P=0.0025;DNMT3a: 3.05±0.28 vs 2.20±0.07,P=0.0033)， DNMT3b表达水平组间差异无统计学意义;c)直肠末端ETU组Shh基因启动子甲基化水平显著高于ETU+ 5-azaC/对照组(ETU vs ETU+ 5-azaC: 7.13±0.66 vs 5.20±1.21,P=0.0263;ETU vs正常人:7.13±0.66 vs 2.81±0.27,P<0.0001)， ETU+ 5-azaC组明显高于对照组(5.20±1.21 vs 2.81±0.27,P=0.0300);d) ETU/ETU+ 5-azaC组直肠末端Shh/Bmp4表达水平显著低于对照组(Shh: ETU vs正常人，1.00±0.23 vs 3.81±0.25,P<0.0001;ETU+ 5-azaC vs正常人，2.19±0.60 vs 3.81±0.25,P=0.0121;Bmp4: ETU vs正常人，1.00±0.18 vs 4.91±0.36,P<0.0001;ETU+ 5-azaC组与正常组相比(2.16±0.23 vs 4.91±0.36,P<0.0001)， ETU组Shh/Bmp4表达水平也低于ETU+ 5-azaC组(Shh: 1.00±0.23 vs 2.19±0.60,P=0.011;Bmp4: 1.00±0.18 vs 2.16±0.23,P=0.0019);e) ETU组直肠组织S-100标记粘膜下层和肌间神经丛与正常组相比显著减少(P<0.0001)， ETU+ 5-azaC组与ETU组相比差异显著(P<0.0001)。ETU/ETU+ 5-azaC组直肠中c-kit标记的Cajal间质细胞和NSE标记的神经节细胞表达不明显。结论干预可改变ARM大鼠直肠末端甲基化状态。低甲基化水平的Shh基因可促进Shh/Bmp4信号通路关键组分的表达，S-100标记的神经丛数量显著增加，促进直肠ens的发展。关键词:肛门，异常;直肠异常;DNA甲基化;Shh/Bmp4信号通路;肠神经系统

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Experimental study of DNA methylation modified Shh/Bmp4 signaling pathway in the regulation of enteric nervous system dysplasia in rectal end of rat fetus with anorectal malformations

Objective To explore the impact of gene methylation in Shh/Bmp4 signaling pathway on the enteric nervous system (ENS) in rectal end of fetal mice with anorectal malformations (ARM). Methods A total of 15 Sprague Dawley pregnant rats were randomly divided into 3 groups of ETU, ETU+ 5-azaC and normal.At gestational Day 10, ETU and ETU+ 5-azaC groups received 1% ETU via gavage while ETU+ 5-azaC group had an intraperitoneal injection of 5-azaC (3 mg/kg). At gestational Day 12, cesarean section was performed.The incidence of ARMs, the content of DNA methyltransferase (DNMT1, DNMT3a & DNMT3b), the methylation status of Shh gene promoter region and the expression of key components in Shh/Bmp4 signaling pathway in rectal end of fetal mice were detected by polymerase chain reaction (PCR), immunohistochemistry and Western blot. Results a) The incidence of ARMs was 98.3% and 64.9% in ETU and ETU+ 5-azaC groups respectively and there were statistical differences (P<0.0001); b) The expression of DNMTs (DNMT1, DNMT3a & DNMT3b) was significantly higher in rectal tissue of ETU/ETU+ 5-azaC group than that of control group (DNMT1: ETU vs normal, 3.63±0.26 vs 1.00±0.09, P<0.0001; ETU+ 5-azaC vs normal, 2.33±0.27 vs 1.00±0.09, P=0.0003; DNMT3a: ETU vs normal, 3.05±0.28 vs 1.00±0.29, P=0.0003; ETU+ 5-azaC vs normal, 2.20±0.07 vs 1.00±0.29, P<0.0001; DNMT3b: ETU vs normal, 2.62±0.49 vs 1.00±0.26, P=0.0005; ETU+ 5-azaC vs normal, 2.33±0.30 vs 1.00±0.26, P=0.0028). The expression of DNMT1/DNMT3a was significantly higher in rectal tissue of ETU group than that in ETU+ 5-azaC group (DNMT1: 3.63±0.26 vs 2.33±0.27, P=0.0025; DNMT3a: 3.05±0.28 vs 2.20±0.07, P=0.0033) while no significant inter-group difference existed in the expression level of DNMT3b; c) The methylation level of Shh gene promoter was significantly higher in rectal end of ETU group than that in ETU+ 5-azaC/control group (ETU vs ETU+ 5-azaC: 7.13±0.66 vs 5.20±1.21, P=0.0263; ETU vs normal: 7.13±0.66 vs 2.81±0.27, P<0.0001) and ETU+ 5-azaC group was obviously higher than control group (5.20±1.21 vs 2.81±0.27, P=0.0300); d) Shh/Bmp4 expression level was significantly lower in rectal end of ETU/ETU+ 5-azaC group than that of control group (Shh: ETU vs normal, 1.00±0.23 vs 3.81±0.25, P<0.0001; ETU+ 5-azaC vs normal, 2.19±0.60 vs 3.81±0.25, P=0.0121; Bmp4: ETU vs normal, 1.00±0.18 vs 4.91±0.36, P<0.0001; ETU+ 5-azaC vs normal, 2.16±0.23 vs 4.91±0.36, P<0.0001) and Shh/Bmp4 expression level in ETU group was also lower than that in ETU+ 5-azaC group (Shh: 1.00±0.23 vs 2.19±0.60, P=0.011; Bmp4: 1.00±0.18 vs 2.16±0.23, P=0.0019); e) S-100 labeled submucosa and intermuscular nerve plexus in rectal tissue of ETU group significantly decreased compared with that of normal group (P<0.0001) and ETU+ 5-azaC group was significantly different from that in ETU group (P<0.0001). The c-kit labeled Cajal stromal cells and NSE labeled ganglion cells were not obviously expressed in rectum of ETU/ETU+ 5-azaC group. Conclusions The methylation status of rectal end in ARM rat model may be changed by interventions.The low methylation level of Shh gene may promote the expression of key components in Shh/Bmp4 signaling pathway and the number of S-100 labeled nerve plexus significantly increases and promotes the development of rectal ENS. Key words: Anus, abnormalities; Rectum, abnormalities; DNA methylation; Shh/Bmp4 signaling pathway; Enteric nervous system

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

中华小儿外科杂志 Medicine-Pediatrics, Perinatology and Child Health

CiteScore

0.40

自引率

0.00%

发文量

8707

期刊介绍： Chinese Journal of Pediatric Surgery is an academic journal sponsored by the Chinese Medical Association. It mainly publishes original research papers, reviews and comments in this field. The journal was founded in 1980 and is included in well-known databases such as Peking University Journal (Chinese Journal of Humanities and Social Sciences) and CSCD Chinese Science Citation Database Source Journal (including extended version). It is one of the national key academic journals under the supervision of the China Association for Science and Technology. Chinese Journal of Pediatric Surgery enjoys a high reputation and influence in the academic community. The articles published in this journal have a high academic level and practical value, providing readers with a large number of practical cases and industry information, and have received widespread attention and citations from readers.