先天性肛门直肠畸形中DNA甲基化修饰Shh/Bmp4通路调控胎鼠肠管神经系统发育的实验研究

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

目的 研究Shh/Bmp4信号通路基因甲基化修饰对肛门直肠畸形(anorectal malforma-tion,ARM)胎鼠末端直肠神经系统(enteric nervous system,ENS)发育的影响.方法 SD孕鼠15只,按照数字表法随机分成模型组(ET U)、干预组(ET U+5-azaC)与正常组(NC).孕10 d,ET U组和ET U+5-azaC组均经胃管注入乙烯硫脲,ET U+5-azaC组同时腹腔注射5-azaC(3 mg/kg).孕20 d,剖宫取子鼠.观察ARM发生率,用PCR、免疫组化和Western blot等方法检测子鼠末端直肠组织中DNA甲基转移酶(DNM T1、DNM T3a、DNM T3b)含量及活性、Shh基因启动子区甲基化状态及Shh/Bmp4信号通路关键组分的表达水平.结果 ①ETU组和ETU+5-azaC组的ARM发生率分别为98.3% 、64.9%,两组之间差异有统计学意义(P<0.0001);②ET U组和ET U+5-azaC组末端直肠组织中DNMT1、DNMT3a、DNMT3b的表达水平较对照组均明显升高(P<0.01);ETU组DNMT1、DNMT3a的表达水平较ETU+5-azaC组明显升高(P<0.01),而两组DNMT3b表达水平未发现明显差异;③ET U组末端直肠组织中Shh启动子区甲基化水平明显高于ET U+5-azaC组和对照组(P<0.05),ETU+5-azaC组亦较对照组明显升高(P=0.05);④ETU组和ETU+5-azaC组末端直肠组织中Shh和Bmp4表达水平较对照组均明显降低(P<0.0001),同时ETU组较ETU+5-azaC组间相比Shh和Bmp4表达水平亦降低(P<0.05);⑤ETU组直肠组织中S100标记的黏膜下层、肌间神经丛明显较正常组表达减少(P<0.0001),ETU+5-azaC组与ETU组相比差异有统计学意义(P<0.0001);ETU组和ETU+5-azaC组直肠中c-kit标记的cajal间质细胞和NSE标记的神经节细胞未见明显表达.结论 ARM大鼠模型直肠末端组织甲基化状态可通过干预发生变化,Shh基因低甲基化可促使Shh/Bmp4通路关键组分表达上调,S-100标记的神经丛数量明显增多,可能介导直肠ENS的发育.

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 &DNM T3b) ,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;ET U+5-azaC vs normal ,2 .20 ± 0 .07 vs 1 .00 ± 0 .29 , P<0 .0001;DNM T3b:ET U vs normal ,2 .62 ± 0 .49 vs 1 .00 ± 0 .26 , P= 0 .0005;ET U+ 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 DNMT 3b;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:ET U 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 .