Shh/BMP4信号在先天性肛门直肠畸形中表达的研究
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摘要
先天性肛门直肠畸形是胚胎期后肠末端发育异常的一类疾病的总称,病变涉及肛门、直肠、尿道及外生殖器,是小儿最常见的消化道畸形,虽经治疗却常有不同程度的后遗症,严重危及患儿生命和生活质量,因此,对其发病机制的研究具有重要的意义。
发生肛门直肠畸形是一个复杂的病理过程,涉及许多基因的异常调控,但迄今为止致病基因尚未明确,研究仍处于探索阶段。近年来研究发现,Shh/BMP4信号在高等脊椎动物的胚胎发育中具有非常重要的作用,它参与多种组织结构的形成。在肠道发育中,它的作用更为广泛,在维持肠上皮的生长、肠壁层次的形成、肠管沿前后轴的区域性分化以及肠神经系统的建成等诸多方面起着重要的作用。这个信号通路中的成员一旦发生突变将导致不同畸形和疾病,如食道闭锁/食管气管瘘、肛门闭锁和青少年肠息肉病等的发生。转基因动物实验发现敲除Shh信号的鼠发生了从单纯的肛门狭窄到复杂的一穴肛等各种不同类型的肛门直肠畸形,说明肛门直肠的正常发育需要Shh信号,提示我们肛门直肠畸形发生可能与Shh/BMP4信号异常有关。
本研究包括动物实验和人体标本实验两部分内容:①ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究:用ETU致畸Wistar大鼠胚胎,观察畸形发生过程中的病理形态学变化并检测Shh/BMP4信号通路中的三个关键基因Shh、Gli2和BMP4在泄殖腔及直肠的表达模式和水平,从而探讨Shh/BMP4信号与肛门直肠畸形发生的关系。②Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究:检测肛门直肠畸形患儿直肠末端Shh/BMP4信号的表达状况,探讨这些基因与人类先天性肛门直肠畸形的关系,为寻找人类先天性肛门直肠畸形的致病基因提供线索。
材料与方法
一、实验材料
实验动物:Wistar大鼠由中国医科大学盛京医院动物实验中心提供。
病例标本:收集在我院手术治疗的肛门直肠畸形病人40例,其中低位畸形25例,高位畸形15例,男27例,女13例,平均年龄1.5个月。收集非胃肠道疾病死亡如新生儿窒息、颅内出血等10例为对照组,其中男6例,女4例,平均年龄1.0个月。标本处理:无菌条件下取直肠末端后壁肠组织,置于-75℃冰箱中保存。
试剂及实验仪器:①试剂:ETU购于Sigma公司,Shh、Gli2和BMP4多克隆抗体购于Santa Cruz公司,SP试剂盒购于迈新公司,TRJZOL试剂、RT-PCR试剂盒购于TaKaRa公司,引物由Invitrogen英骏生物技术有限公司合成。②仪器:NiKon Eclipse E_(800)光学显微镜,NIS-Elements F_(2.30)图像采集软件,UV-3000型紫外分光光度计,PTC-200~(TM)型PCR扩增仪,天能公司GIS-2020型凝胶成像分析系统。
二、实验方法
1、ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究。①动物模型的建立:实验动物随机分为ETU组和对照组。在妊娠第10天时给ETU组孕鼠经胃管注入1%ETU,用量为125 mg/kg;对照组给予等量的生理盐水。分别于妊娠第12.5、13.5、14、15和16天共5个时间点剖宫取仔。②Shh、Gli2和BMP4免疫组化染色:对照组和ETU组的各时间组各取3只孕鼠,胎仔置于4%多聚甲醛中固定过夜,常规酒精脱水、石蜡包埋,所得标本矢状位连续切片,切片厚度4μm,然后取正中矢状位切片行免疫组化染色。③RT-PCR实验:对照组和ETU组的各时间组各取2只孕鼠,检测胚胎泄殖腔部分Shh、Gli2和BMP4的表达水平。④Western蛋白印迹实验:胎龄16天时,对照组、ETU致畸形组和未致畸形组各为30只胎仔,取其直肠检测Shh、Gli2和BMP4的蛋白表达。
2、Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究。直肠标本的石蜡切片免疫组化染色、RT-PCR和Western蛋白印迹方法检测Shh、Gli2和BMP4基因在40例肛门直肠畸形病人和10例无畸形儿直肠末端的表达状态。
3、数据的统计学处理:数值以(?)±s表示,用SPSS 13.0 for windows软件进行统计学分析,组间比较用单因素方差分析或t检验,P＜0.05有统计学差异。
结果
1、ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究
免疫组化染色:组织切片观察ETU组63.2%胚胎发生肛门直肠畸形,对照组未见明显畸形,证实本组畸形稳定。鼠胚泄殖腔正常发育的形态学特点:胚胎12.5天到14天主要以泄殖腔形态变化为特征,同时可见生殖结节的迅速生长。胚胎14天到16天主要以尿直肠隔下降并与泄殖腔膜融合为主要特征。ETU组畸形鼠胚的泄殖腔发育主要有两个异常:一是在发育早期泄殖腔形态的异常,导致尿直肠隔的位置很高不能有效分隔泄殖腔;二是尿直肠隔下降障碍,未与泄殖腔膜融合,直肠和泌尿生殖道间存在共同通道而不能完全分离。Shh/BMP4信号在泄殖腔的表达模式:Shh和Gli2表达在泄殖腔、膀胱、尿道、直肠和肛门的上皮细胞,胞浆内呈浅棕色颗粒。BMP4的表达位于尿直肠隔内和泄殖腔、直肠、膀胱和尿道周围的间质细胞和胞外间质中,为胞浆及胞外间质染色,也呈浅棕色颗粒。在对照组胚胎14天,BMP4在尿直肠隔内及直肠周围表达最强。ETU致畸组Shh、Gli2和BMP4的表达较对照组明显减弱甚至缺乏。
RT-PCR实验:Shh、Gli2和BMP4 mRNA扩增产物经电泳后,分别于预期的187bp、158bp和493bp处见条带,提示有Shh、Gli2和BMP4的mRNA表达。β-actin在各反应体系中扩增结果一致,证实PCR反应基本真实地反映了各基因的表达。在对照组的胚胎12.5-14天,Shh/BMP4mRNA的表达水平较高,在胚胎15-16天时略有下降;在ETU组,Shh/BMP4mRNA的表达水平均低于对照组,其中Shh和Gli2在胚胎12.5-14天、BMP4在胚胎14-16天的差异有统计学意义(P＜0.05)。
Western蛋白印迹:对照组和ETU两组均出现被Shh、Gli2和BMP4抗体识别的显色带,其中对照组和ETU未致畸形组表达强度相似,对照组Shh、Gli2和BMP4蛋白的相对含量(1.127±0.13、1.527±0.16和0.980±0.05)与ETU未致畸形组(1.070±0.19、1.490±0.05和0.926±0.15)比较无统计学差异(P均大于0.05)。但ETU致畸形组Shh、Gli2和BMP4的条带明显减弱甚至消失,其蛋白的相对含量(0.886±0.09、1.060±0.08和0.913±0.04)与对照组和ETU未致畸形组比较有统计学意义(P值均小于0.05)。
2、Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究
免疫组化染色:Shh、Gli2表达在肠壁粘膜细胞的胞浆中,为棕黄色颗粒;BMP4表达在粘膜下层的间质细胞中,为胞核、胞浆着色,呈棕黄色颗粒。对照组和低位畸形组Shh、Gli2和BMP4均有明显染色,而在高位畸形组各基因的染色明显减弱。
RT-PCR检测:扩增产物经电泳后,显示条带亮度各不相同。在高位畸形组,Shh、Gli2和BMP4 mRNA扩增条带多呈弱带或无带,在低位畸形组及对照组多呈亮带。半定量分析Shh、Gli2和BMP4 mRNA含量:高位畸形组的各值(0.63±0.27,0.23±0.08和0.69±0.25)比较低位畸形组(0.89±0.39,0.81±0.32和0.98±0.33)和对照组(0.91±0.71,1.24±0.38和1.11±0.45)明显下降(P＜0.05);而在低位畸形组,除Gli2外,Shh和BMP4的表达与对照组无明显差异(P＞0.05)。
Western蛋白印迹:各组与Shh、Gli2和BMP4抗体产生免疫反应在硝酸纤维膜上均呈现出条带,但各组、各基因表达的信号强弱不同。Shh、Gli2和BMP4蛋白的相对含量在高位畸形组分别为0.920±0.09、1.106±0.11和1.070±0.10,在低位畸形组分别为1.317±0.13、1.210±0.10和1.137±0.14,在对照组分别为1.586±0.08、1.430±0.09和1.520±0.08。统计学分析显示:高位畸形组比较低位畸形组和对照组各基因的蛋白表达量均有显著性下降(P＜0.05),而低位畸形组与正常组比较,各基因的蛋白表达量无显著性差异(P＞0.05)。
结论
1、在大鼠胚胎发育过程中,Shh/BMP4信号表达在泄殖腔及直肠区域,调节泄殖腔及直肠的正常发育。
2、ETU组鼠胚的泄殖腔和直肠的Shh/BMP4信号表达下降表明ETU扰乱了Shh/BMP4信号表达。
3、在泄殖腔发育过程中,ETU组畸形鼠胚的泄殖腔区ShNBMP4信号表达水平下降,从而使泄殖腔不能正常发育,可能是导致肛门直肠畸形发生的重要因素之一。
4、正常人的直肠末端存在Shh/BMP4信号表达,说明人类肛门直肠的生长发育需要Shh/BMP4信号的调节。
5、高位肛门直肠畸形Shh/BMP4信号表达下调,提示人类高位肛门直肠畸形的发生可能与Shh/BMP4信号紊乱有关。
6、与高位肛门直肠畸形不同,低位畸形Shh/BMP4信号表达水平与对照组一致,说明高、低位畸形可能涉及不同的机制,还有其他发育信号的异常参与低位畸形的发生。
Introduction
Congenital anorectal malformations(ARMs) comprise a wide spectrum of diseases,which resulted from impeded hindgut development during embryonic stage, and involve the anus and rectum as well as the urogenital tracts.ARMs are the most common alimentary deformities and severely influence life quality of the patients. Some problems such as constipation and incontinence remain even after operation.
Pathogenesis of ARMs is too complex to be well understood.The abnormal regulations of several developmental genes have been implicated in the development of ARMs.However,the pathogenic genes of ARMs are still unlocated.Therefore,it is significant to investigate pathogenesis of ARMs on molecular level.Recent study has demonstrated that Shh/BMP4 signaling pathway is involved in the morphogenesis of many organ systems during embryonic development in higher vertebrates.In the gut development,Shh/BMP4 signaling pathway also effects on several aspects including the epithelial proliferation,the mesenchymal stratification,region-specific differentiation of the gut.Recent study in the human digestive tract indicated that mutations in members of the Shh/BMP4 signaling pathway are associated with different malformations such as imperforate anus,esophageal atresia /tracheoesophageal fistula,juvenile polyposis syndrome and etc.Experimental studies on Shh cascade mutant mice showed the mice develop imperforate anus that resemble humans and vary in severity from stenotic anus to complex cloacal malformations.On the basis of these findings,we hypothesized that the Shh/BMP4 pathway plays a role in the development of the ARMs.
This study includes:①The embryomorphogenesis and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:Rat embryos with ARMs were obtained by treating with administration of ethylenethiourea(ETU) on pregnant rats. The cloacal morphogenesis and the expression patterns and levels of Shh,Gli2 and BMP4,were continuously and dynamicly studied by immunohistochemistry,RT-PCR and Western-blotting to explore the mechanism of ARMs.②The expression of Shh/BMP signaling in human ARMs:Shh,Gli2 and BMP4 expression in terminal rectum of the patients with ARMs and control groups were studied to explore the relativity of ARMs to Shh/BMP4 signaling pathway and to investigate pathogenic genes.
Materials and Methods
Materials
1.Animals:Wistar rats were provided by Medical Animal Center in the Shengjing Hospital of China Medical University.
2.Samples:Among 40 cases with anorectal malformations(15 high type and 25 low type) operated in our department,27 were males,and 13 were females.The average age was 1.5 months.10 normal controls were obtained from the bodies died of neonatal asphyxia,and average aged 1.0 month.Tissues were removed from terminal pouch of rectum and stored immediately in -75℃refrigerator.
3.Reagents and instruments:
①Major reagents:ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc, USA).The The specific antibodies to Shh、Gli2 and BMP4 were bought from the Santa Cruz biological company.The SP immunohistochemistry kit was bought from the Maxin biological technique Ltd.in Fuzhou.RT-PCR Kit,Trizol(mRNA extraction system) were provided by TaKaBa Biotechnology(Dalian) Co.,Ltd.The primers for Shh、Gli2、BMP4 andβ-actin were made by Invitrogen Biotechnology Co.,Ltd.
②Major instruments:Nikon Eclipse E_(800) opticmicroscope;PTC-200~(TM) PCR extender;The image collect system is NIS-Elements F_(2.30).
Methods
1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:①Animal modle:The timed-pregnant rats were divided into two groups(the ETU treated group and the control group) in random.The ETU treated group females were gavage-fed with 1%ETU at 125 mg/kg b.w.on gestational days10(gD10).The control group received the same volume of Sodium Chloride on the same day.Embryos were harvested via caesarean section on gD12.5, 13.5,14,15 and gD16,respectively.②Immunohistochemistry staining:The whole embryos from 3 ETU treated females and 3 control females from gD12.5 to 16 were fixed in 4%formaldehyde for 24 hour,processed,embedded in paraffin wax and serially sliced at 4μm sagittally.The slices were stained with immunohistochemistry staining and were examined microscopically.③RT-PCR:We determined the expression levels of Shh,Gli2 and BMP4 mRNA in the cloaca of the embryos from 2 ETU treated females and 2 control females at different time point.④Western blotting: The fetuses on gD16 harvested from the control group and the ETU treated group were divided into three groups:the control group,the embryos without ARMs treated by ETU and the ARMs embryos treated by ETU.The protein expression of Shh,Gli2 and BMP4 in the rectum of the fetuses were detected and analyzed.
2.The expression of Shh/BMP4 signaling in human ARMs:The expression patterns and levels of Shh,Gli2 and BMP4 genes in rectal samples of 40 cases with ARMs(15 high type and 25 low type) and 10 normal controls were studied by immunohistochemistry staining,RT-PCR and Western blotting.
3.Statistical Analysis:All values are expressed as(?)±s,and the data were analyzed by One-way ANOVA or by t-test with SPSS13.0 for windows software,with P＜0.05 considered to indicated statistical significance.
Results
1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU.
Immunohistochemistry results:The embryos administered ETU showed high occurrence(63.2%) of ARMs and may used as a stable and reliable ARMs animal models.Histological observation of control embryos represented as follows:During normal cloacal developmental processes in rats,before gD 14,the genital tubercle developed and grew substantially,and the cloaca shifted ventrocaudally according to genital tubercle development.The cloaca configuration changes and obviously is divided into ventral urogenital sinus and dorsal primitive rectum.At the same time; from gD14 to gD16,urorectal septum descended with its distance to the cloacal membrane decreasing gradually,and fused with the cloacal membrane.Characters of cloaca development in ETU ARMs rat embryos were as follows:A.Cloacal configuration was abnormal.B.Urorectal septum descended incompletely,and never fused with cloaca membrane.In normal controls,immunoreactivity specific to Shh and Gli2 was detected most abundantly in the epithelium of the cloaca,urinary bladder, urethra,rectum and anus.The immunoreactivity for BMP4 was localized in the mesodermal cells and the mesenchyme beneath the epithelium of the cloaca and rectum and in urorectal septum.The most staining intensity of BMP4 occurred in the urorectal septum and in the mesenchyme surrounding the rectum on gD14.In ETU-treated embryos,the immunoreactivity specific to Shh,Gli2 and BMP4 were remarkably weak compared with those in the normal controls.From these histologic findings,teratogenic doses of ETU remarkably disturbed Shh/BMP4 signaling expression in the cloacal regions.
RT-PCR results:The PCR experiment successfully yielded the amplified fragments of expected size forβ-actin(690 bp),Shh(187 bp),Gli2(158bp) and BMP4 (493bp) in each sample.Intensities of the bands corresponding toβ-actin were similar among all the samples.This confirmed that Shh/BMP4 expressed in control and experimental samples throughout the cloaca developmental period(gD12.5-16). Semiquantitative results demonstrated that the top of Shh,Gli2 and BMP4 mRNA expression curve was on gD12.5-14,and Shh/BMP4 expression levels were different in the developing cloaca of ETU-treated rat embryos on gD12.5-16 from those of the control embryos.Shh and Gli2 expression levels were significantly decreased on gD12.5-14(BMP4 on gD14-16) in developing cloaca of ETU-treated rat embryos as compared to the controls(P＜0.05).
Western blotting results:Shh,Gli2 and BMP4 protein expression were evident in the rectums harvested from the control embryos and the embryos without ARMs treated by ETU on gD16.Shh,Gli2 and BMP4 expression levels(1.070±0.19, 1.490±0.05 and 0.926±0.15) of the embryos without ARMs treated by ETU diminished but did not reach the significant levels compared with controls(1.127±0.13, 1.527±0.16 and 0.980±0.05)(P＞0.05).Immunoblotting detected low levels of Shh/BMP4 protein in rectal tissues collected from the ARMs group treated by ETU. There were significant difference between the ARMs group and the embryos without ARMs treated by ETU(0.886±0.09、1.060±0.08 and 0.913±0.04) and the control group and the embryos without ARMs treated by ETU(P＜0.05).
2.Expression of Shh/BMP4 signaling in human samples.
Immunohistoc(?)emistry results:There were obvious immunostaining of Shh and Gli2 showed in the epithelium of the rectum,and positive staining for BMP4 showed in the mesenchyme and in the mesodermal cells underlying the epithelium and within the muscular layers of the gut.In the high ARMs,however,Shh,Gli2 and BMP4 expression were weaker than those in the control and low ARMs.
RT-PCR results:We found that expression levels of Shh,Gli2 and BMP4 mRNA in terminal rectum of high ARMs(0.63±0.27,0.23±0.08 and 0.69±0.25) was significantly lower than those in low ARMs(0.89±0.39,0.91±0.32 and 0.98±0.33) and in normal rectum(0.91±0.71,1.24±0.38 and 1.11±0.45)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group (except Gli2mRNA)(P＞0.05).
Western blotting results:Average gray value(G-value) represented the level of protein expression of Shh,Gli2 and BMP4.G-values of Shh,Gli2 and BMP4 in the high ARMs(0.920±0.09,1.106±0.11 and 1.070±0.10) were significantly lower than in the low ARMs(1.317±0.13,1.210±0.10 and 1.137±0.14 ) and the control(1.586±0.08, 1.430±0.09 and 1.520±0.08)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group in the expression levels of these genes.
Conclusions
1.This study showed that Shh/BMP4 was expressed in the developing cloaca of normal rat embryos,and morphogenic events in the cloaca were relative to Shh signal induction.
2.Expression levels of Shh/BMP4 signaling were reduced in the cloaca of ETU-treated embryos.It suggested that ETU affected the expression of Shh/BMP4.
3.During the critical stages of the clocal development,the aberrations in Shh/BMP4 signaling may be responsible for the abnormal morphogenesis of the cloaca. We presumed the reduced level of Shh/BMP4 expression may contribute to the ARMs.
4.Shh/BMP4 signaling expressed in the terminal pouch of the human rectum and regulated the normal anorectal development.
5.The expression level of Shh/BMP4 signaling in high ARMs was lower than that of the control group.Down-regulation of Shh/BMP4 signaling pathway was closely relative to the occurrence of high ARMs.
6.The expression pattern of Shh/BMP4 signaling of low ARMs was normal.The result suggested there were still some other mechanisms responsible for the occurrence of low ARMs.
发生肛门直肠畸形是一个复杂的病理过程,涉及许多基因的异常调控,但迄今为止致病基因尚未明确,研究仍处于探索阶段。近年来研究发现,Shh/BMP4信号在高等脊椎动物的胚胎发育中具有非常重要的作用,它参与多种组织结构的形成。在肠道发育中,它的作用更为广泛,在维持肠上皮的生长、肠壁层次的形成、肠管沿前后轴的区域性分化以及肠神经系统的建成等诸多方面起着重要的作用。这个信号通路中的成员一旦发生突变将导致不同畸形和疾病,如食道闭锁/食管气管瘘、肛门闭锁和青少年肠息肉病等的发生。转基因动物实验发现敲除Shh信号的鼠发生了从单纯的肛门狭窄到复杂的一穴肛等各种不同类型的肛门直肠畸形,说明肛门直肠的正常发育需要Shh信号,提示我们肛门直肠畸形发生可能与Shh/BMP4信号异常有关。
本研究包括动物实验和人体标本实验两部分内容:①ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究:用ETU致畸Wistar大鼠胚胎,观察畸形发生过程中的病理形态学变化并检测Shh/BMP4信号通路中的三个关键基因Shh、Gli2和BMP4在泄殖腔及直肠的表达模式和水平,从而探讨Shh/BMP4信号与肛门直肠畸形发生的关系。②Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究:检测肛门直肠畸形患儿直肠末端Shh/BMP4信号的表达状况,探讨这些基因与人类先天性肛门直肠畸形的关系,为寻找人类先天性肛门直肠畸形的致病基因提供线索。
材料与方法
一、实验材料
实验动物:Wistar大鼠由中国医科大学盛京医院动物实验中心提供。
病例标本:收集在我院手术治疗的肛门直肠畸形病人40例,其中低位畸形25例,高位畸形15例,男27例,女13例,平均年龄1.5个月。收集非胃肠道疾病死亡如新生儿窒息、颅内出血等10例为对照组,其中男6例,女4例,平均年龄1.0个月。标本处理:无菌条件下取直肠末端后壁肠组织,置于-75℃冰箱中保存。
试剂及实验仪器:①试剂:ETU购于Sigma公司,Shh、Gli2和BMP4多克隆抗体购于Santa Cruz公司,SP试剂盒购于迈新公司,TRJZOL试剂、RT-PCR试剂盒购于TaKaRa公司,引物由Invitrogen英骏生物技术有限公司合成。②仪器:NiKon Eclipse E_(800)光学显微镜,NIS-Elements F_(2.30)图像采集软件,UV-3000型紫外分光光度计,PTC-200~(TM)型PCR扩增仪,天能公司GIS-2020型凝胶成像分析系统。
二、实验方法
1、ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究。①动物模型的建立:实验动物随机分为ETU组和对照组。在妊娠第10天时给ETU组孕鼠经胃管注入1%ETU,用量为125 mg/kg;对照组给予等量的生理盐水。分别于妊娠第12.5、13.5、14、15和16天共5个时间点剖宫取仔。②Shh、Gli2和BMP4免疫组化染色:对照组和ETU组的各时间组各取3只孕鼠,胎仔置于4%多聚甲醛中固定过夜,常规酒精脱水、石蜡包埋,所得标本矢状位连续切片,切片厚度4μm,然后取正中矢状位切片行免疫组化染色。③RT-PCR实验:对照组和ETU组的各时间组各取2只孕鼠,检测胚胎泄殖腔部分Shh、Gli2和BMP4的表达水平。④Western蛋白印迹实验:胎龄16天时,对照组、ETU致畸形组和未致畸形组各为30只胎仔,取其直肠检测Shh、Gli2和BMP4的蛋白表达。
2、Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究。直肠标本的石蜡切片免疫组化染色、RT-PCR和Western蛋白印迹方法检测Shh、Gli2和BMP4基因在40例肛门直肠畸形病人和10例无畸形儿直肠末端的表达状态。
3、数据的统计学处理:数值以(?)±s表示,用SPSS 13.0 for windows软件进行统计学分析,组间比较用单因素方差分析或t检验,P＜0.05有统计学差异。
结果
1、ETU诱导大鼠胚胎肛门直肠畸形的演变与Shh/BMP4表达的关系的研究
免疫组化染色:组织切片观察ETU组63.2%胚胎发生肛门直肠畸形,对照组未见明显畸形,证实本组畸形稳定。鼠胚泄殖腔正常发育的形态学特点:胚胎12.5天到14天主要以泄殖腔形态变化为特征,同时可见生殖结节的迅速生长。胚胎14天到16天主要以尿直肠隔下降并与泄殖腔膜融合为主要特征。ETU组畸形鼠胚的泄殖腔发育主要有两个异常:一是在发育早期泄殖腔形态的异常,导致尿直肠隔的位置很高不能有效分隔泄殖腔;二是尿直肠隔下降障碍,未与泄殖腔膜融合,直肠和泌尿生殖道间存在共同通道而不能完全分离。Shh/BMP4信号在泄殖腔的表达模式:Shh和Gli2表达在泄殖腔、膀胱、尿道、直肠和肛门的上皮细胞,胞浆内呈浅棕色颗粒。BMP4的表达位于尿直肠隔内和泄殖腔、直肠、膀胱和尿道周围的间质细胞和胞外间质中,为胞浆及胞外间质染色,也呈浅棕色颗粒。在对照组胚胎14天,BMP4在尿直肠隔内及直肠周围表达最强。ETU致畸组Shh、Gli2和BMP4的表达较对照组明显减弱甚至缺乏。
RT-PCR实验:Shh、Gli2和BMP4 mRNA扩增产物经电泳后,分别于预期的187bp、158bp和493bp处见条带,提示有Shh、Gli2和BMP4的mRNA表达。β-actin在各反应体系中扩增结果一致,证实PCR反应基本真实地反映了各基因的表达。在对照组的胚胎12.5-14天,Shh/BMP4mRNA的表达水平较高,在胚胎15-16天时略有下降;在ETU组,Shh/BMP4mRNA的表达水平均低于对照组,其中Shh和Gli2在胚胎12.5-14天、BMP4在胚胎14-16天的差异有统计学意义(P＜0.05)。
Western蛋白印迹:对照组和ETU两组均出现被Shh、Gli2和BMP4抗体识别的显色带,其中对照组和ETU未致畸形组表达强度相似,对照组Shh、Gli2和BMP4蛋白的相对含量(1.127±0.13、1.527±0.16和0.980±0.05)与ETU未致畸形组(1.070±0.19、1.490±0.05和0.926±0.15)比较无统计学差异(P均大于0.05)。但ETU致畸形组Shh、Gli2和BMP4的条带明显减弱甚至消失,其蛋白的相对含量(0.886±0.09、1.060±0.08和0.913±0.04)与对照组和ETU未致畸形组比较有统计学意义(P值均小于0.05)。
2、Shh/BMP4信号在人类先天性肛门直肠畸形中表达的研究
免疫组化染色:Shh、Gli2表达在肠壁粘膜细胞的胞浆中,为棕黄色颗粒;BMP4表达在粘膜下层的间质细胞中,为胞核、胞浆着色,呈棕黄色颗粒。对照组和低位畸形组Shh、Gli2和BMP4均有明显染色,而在高位畸形组各基因的染色明显减弱。
RT-PCR检测:扩增产物经电泳后,显示条带亮度各不相同。在高位畸形组,Shh、Gli2和BMP4 mRNA扩增条带多呈弱带或无带,在低位畸形组及对照组多呈亮带。半定量分析Shh、Gli2和BMP4 mRNA含量:高位畸形组的各值(0.63±0.27,0.23±0.08和0.69±0.25)比较低位畸形组(0.89±0.39,0.81±0.32和0.98±0.33)和对照组(0.91±0.71,1.24±0.38和1.11±0.45)明显下降(P＜0.05);而在低位畸形组,除Gli2外,Shh和BMP4的表达与对照组无明显差异(P＞0.05)。
Western蛋白印迹:各组与Shh、Gli2和BMP4抗体产生免疫反应在硝酸纤维膜上均呈现出条带,但各组、各基因表达的信号强弱不同。Shh、Gli2和BMP4蛋白的相对含量在高位畸形组分别为0.920±0.09、1.106±0.11和1.070±0.10,在低位畸形组分别为1.317±0.13、1.210±0.10和1.137±0.14,在对照组分别为1.586±0.08、1.430±0.09和1.520±0.08。统计学分析显示:高位畸形组比较低位畸形组和对照组各基因的蛋白表达量均有显著性下降(P＜0.05),而低位畸形组与正常组比较,各基因的蛋白表达量无显著性差异(P＞0.05)。
结论
1、在大鼠胚胎发育过程中,Shh/BMP4信号表达在泄殖腔及直肠区域,调节泄殖腔及直肠的正常发育。
2、ETU组鼠胚的泄殖腔和直肠的Shh/BMP4信号表达下降表明ETU扰乱了Shh/BMP4信号表达。
3、在泄殖腔发育过程中,ETU组畸形鼠胚的泄殖腔区ShNBMP4信号表达水平下降,从而使泄殖腔不能正常发育,可能是导致肛门直肠畸形发生的重要因素之一。
4、正常人的直肠末端存在Shh/BMP4信号表达,说明人类肛门直肠的生长发育需要Shh/BMP4信号的调节。
5、高位肛门直肠畸形Shh/BMP4信号表达下调,提示人类高位肛门直肠畸形的发生可能与Shh/BMP4信号紊乱有关。
6、与高位肛门直肠畸形不同,低位畸形Shh/BMP4信号表达水平与对照组一致,说明高、低位畸形可能涉及不同的机制,还有其他发育信号的异常参与低位畸形的发生。
Introduction
Congenital anorectal malformations(ARMs) comprise a wide spectrum of diseases,which resulted from impeded hindgut development during embryonic stage, and involve the anus and rectum as well as the urogenital tracts.ARMs are the most common alimentary deformities and severely influence life quality of the patients. Some problems such as constipation and incontinence remain even after operation.
Pathogenesis of ARMs is too complex to be well understood.The abnormal regulations of several developmental genes have been implicated in the development of ARMs.However,the pathogenic genes of ARMs are still unlocated.Therefore,it is significant to investigate pathogenesis of ARMs on molecular level.Recent study has demonstrated that Shh/BMP4 signaling pathway is involved in the morphogenesis of many organ systems during embryonic development in higher vertebrates.In the gut development,Shh/BMP4 signaling pathway also effects on several aspects including the epithelial proliferation,the mesenchymal stratification,region-specific differentiation of the gut.Recent study in the human digestive tract indicated that mutations in members of the Shh/BMP4 signaling pathway are associated with different malformations such as imperforate anus,esophageal atresia /tracheoesophageal fistula,juvenile polyposis syndrome and etc.Experimental studies on Shh cascade mutant mice showed the mice develop imperforate anus that resemble humans and vary in severity from stenotic anus to complex cloacal malformations.On the basis of these findings,we hypothesized that the Shh/BMP4 pathway plays a role in the development of the ARMs.
This study includes:①The embryomorphogenesis and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:Rat embryos with ARMs were obtained by treating with administration of ethylenethiourea(ETU) on pregnant rats. The cloacal morphogenesis and the expression patterns and levels of Shh,Gli2 and BMP4,were continuously and dynamicly studied by immunohistochemistry,RT-PCR and Western-blotting to explore the mechanism of ARMs.②The expression of Shh/BMP signaling in human ARMs:Shh,Gli2 and BMP4 expression in terminal rectum of the patients with ARMs and control groups were studied to explore the relativity of ARMs to Shh/BMP4 signaling pathway and to investigate pathogenic genes.
Materials and Methods
Materials
1.Animals:Wistar rats were provided by Medical Animal Center in the Shengjing Hospital of China Medical University.
2.Samples:Among 40 cases with anorectal malformations(15 high type and 25 low type) operated in our department,27 were males,and 13 were females.The average age was 1.5 months.10 normal controls were obtained from the bodies died of neonatal asphyxia,and average aged 1.0 month.Tissues were removed from terminal pouch of rectum and stored immediately in -75℃refrigerator.
3.Reagents and instruments:
①Major reagents:ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc, USA).The The specific antibodies to Shh、Gli2 and BMP4 were bought from the Santa Cruz biological company.The SP immunohistochemistry kit was bought from the Maxin biological technique Ltd.in Fuzhou.RT-PCR Kit,Trizol(mRNA extraction system) were provided by TaKaBa Biotechnology(Dalian) Co.,Ltd.The primers for Shh、Gli2、BMP4 andβ-actin were made by Invitrogen Biotechnology Co.,Ltd.
②Major instruments:Nikon Eclipse E_(800) opticmicroscope;PTC-200~(TM) PCR extender;The image collect system is NIS-Elements F_(2.30).
Methods
1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:①Animal modle:The timed-pregnant rats were divided into two groups(the ETU treated group and the control group) in random.The ETU treated group females were gavage-fed with 1%ETU at 125 mg/kg b.w.on gestational days10(gD10).The control group received the same volume of Sodium Chloride on the same day.Embryos were harvested via caesarean section on gD12.5, 13.5,14,15 and gD16,respectively.②Immunohistochemistry staining:The whole embryos from 3 ETU treated females and 3 control females from gD12.5 to 16 were fixed in 4%formaldehyde for 24 hour,processed,embedded in paraffin wax and serially sliced at 4μm sagittally.The slices were stained with immunohistochemistry staining and were examined microscopically.③RT-PCR:We determined the expression levels of Shh,Gli2 and BMP4 mRNA in the cloaca of the embryos from 2 ETU treated females and 2 control females at different time point.④Western blotting: The fetuses on gD16 harvested from the control group and the ETU treated group were divided into three groups:the control group,the embryos without ARMs treated by ETU and the ARMs embryos treated by ETU.The protein expression of Shh,Gli2 and BMP4 in the rectum of the fetuses were detected and analyzed.
2.The expression of Shh/BMP4 signaling in human ARMs:The expression patterns and levels of Shh,Gli2 and BMP4 genes in rectal samples of 40 cases with ARMs(15 high type and 25 low type) and 10 normal controls were studied by immunohistochemistry staining,RT-PCR and Western blotting.
3.Statistical Analysis:All values are expressed as(?)±s,and the data were analyzed by One-way ANOVA or by t-test with SPSS13.0 for windows software,with P＜0.05 considered to indicated statistical significance.
Results
1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU.
Immunohistochemistry results:The embryos administered ETU showed high occurrence(63.2%) of ARMs and may used as a stable and reliable ARMs animal models.Histological observation of control embryos represented as follows:During normal cloacal developmental processes in rats,before gD 14,the genital tubercle developed and grew substantially,and the cloaca shifted ventrocaudally according to genital tubercle development.The cloaca configuration changes and obviously is divided into ventral urogenital sinus and dorsal primitive rectum.At the same time; from gD14 to gD16,urorectal septum descended with its distance to the cloacal membrane decreasing gradually,and fused with the cloacal membrane.Characters of cloaca development in ETU ARMs rat embryos were as follows:A.Cloacal configuration was abnormal.B.Urorectal septum descended incompletely,and never fused with cloaca membrane.In normal controls,immunoreactivity specific to Shh and Gli2 was detected most abundantly in the epithelium of the cloaca,urinary bladder, urethra,rectum and anus.The immunoreactivity for BMP4 was localized in the mesodermal cells and the mesenchyme beneath the epithelium of the cloaca and rectum and in urorectal septum.The most staining intensity of BMP4 occurred in the urorectal septum and in the mesenchyme surrounding the rectum on gD14.In ETU-treated embryos,the immunoreactivity specific to Shh,Gli2 and BMP4 were remarkably weak compared with those in the normal controls.From these histologic findings,teratogenic doses of ETU remarkably disturbed Shh/BMP4 signaling expression in the cloacal regions.
RT-PCR results:The PCR experiment successfully yielded the amplified fragments of expected size forβ-actin(690 bp),Shh(187 bp),Gli2(158bp) and BMP4 (493bp) in each sample.Intensities of the bands corresponding toβ-actin were similar among all the samples.This confirmed that Shh/BMP4 expressed in control and experimental samples throughout the cloaca developmental period(gD12.5-16). Semiquantitative results demonstrated that the top of Shh,Gli2 and BMP4 mRNA expression curve was on gD12.5-14,and Shh/BMP4 expression levels were different in the developing cloaca of ETU-treated rat embryos on gD12.5-16 from those of the control embryos.Shh and Gli2 expression levels were significantly decreased on gD12.5-14(BMP4 on gD14-16) in developing cloaca of ETU-treated rat embryos as compared to the controls(P＜0.05).
Western blotting results:Shh,Gli2 and BMP4 protein expression were evident in the rectums harvested from the control embryos and the embryos without ARMs treated by ETU on gD16.Shh,Gli2 and BMP4 expression levels(1.070±0.19, 1.490±0.05 and 0.926±0.15) of the embryos without ARMs treated by ETU diminished but did not reach the significant levels compared with controls(1.127±0.13, 1.527±0.16 and 0.980±0.05)(P＞0.05).Immunoblotting detected low levels of Shh/BMP4 protein in rectal tissues collected from the ARMs group treated by ETU. There were significant difference between the ARMs group and the embryos without ARMs treated by ETU(0.886±0.09、1.060±0.08 and 0.913±0.04) and the control group and the embryos without ARMs treated by ETU(P＜0.05).
2.Expression of Shh/BMP4 signaling in human samples.
Immunohistoc(?)emistry results:There were obvious immunostaining of Shh and Gli2 showed in the epithelium of the rectum,and positive staining for BMP4 showed in the mesenchyme and in the mesodermal cells underlying the epithelium and within the muscular layers of the gut.In the high ARMs,however,Shh,Gli2 and BMP4 expression were weaker than those in the control and low ARMs.
RT-PCR results:We found that expression levels of Shh,Gli2 and BMP4 mRNA in terminal rectum of high ARMs(0.63±0.27,0.23±0.08 and 0.69±0.25) was significantly lower than those in low ARMs(0.89±0.39,0.91±0.32 and 0.98±0.33) and in normal rectum(0.91±0.71,1.24±0.38 and 1.11±0.45)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group (except Gli2mRNA)(P＞0.05).
Western blotting results:Average gray value(G-value) represented the level of protein expression of Shh,Gli2 and BMP4.G-values of Shh,Gli2 and BMP4 in the high ARMs(0.920±0.09,1.106±0.11 and 1.070±0.10) were significantly lower than in the low ARMs(1.317±0.13,1.210±0.10 and 1.137±0.14 ) and the control(1.586±0.08, 1.430±0.09 and 1.520±0.08)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group in the expression levels of these genes.
Conclusions
1.This study showed that Shh/BMP4 was expressed in the developing cloaca of normal rat embryos,and morphogenic events in the cloaca were relative to Shh signal induction.
2.Expression levels of Shh/BMP4 signaling were reduced in the cloaca of ETU-treated embryos.It suggested that ETU affected the expression of Shh/BMP4.
3.During the critical stages of the clocal development,the aberrations in Shh/BMP4 signaling may be responsible for the abnormal morphogenesis of the cloaca. We presumed the reduced level of Shh/BMP4 expression may contribute to the ARMs.
4.Shh/BMP4 signaling expressed in the terminal pouch of the human rectum and regulated the normal anorectal development.
5.The expression level of Shh/BMP4 signaling in high ARMs was lower than that of the control group.Down-regulation of Shh/BMP4 signaling pathway was closely relative to the occurrence of high ARMs.
6.The expression pattern of Shh/BMP4 signaling of low ARMs was normal.The result suggested there were still some other mechanisms responsible for the occurrence of low ARMs.
引文
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15 Reddi AH. BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev.2005;16:249-50.
16 Batts LE, Polk DB, Dubois RN, et al. Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn.2006;235:1563-70.
17 Roberts DJ, Smith DM, Goff DJ, et al. Epithelial - mesenchymal signaling during the regionalization of the chick gut. Development. 1998; 125:2791-801.
18 Sukegawa A, Narita T, Kameda T, et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development.2000; 127:1971 -80.
19 De Santa Barbara P, Williams J, Goldstein AM, et al.Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn.2005;234:312-22.
20 Roberts DJ .Molecular mechanisms of development of the Gastrointestinal tract. Developmental Dynamics.2000;219:109-20.
21 Pyati UJ, Cooper MS, Davidson AJ, et al. Sustained Bmp signaling is essential for cloaca development in zebrafish. Development.2006;133:2275-84.
22 Liu G, Moro A, Zhang JJ, et al. The role of Shh transcription activator Gli2 in chick cloacal development.Dev Biol.2007;303:448-60.
23 Sasaki Y, Iwai N, Tsuda T, et al. Sonic hedgehog and bone morphogenetic protein4 expressions in the hindgut region of murine embryos with anorectal malformations. J Pediatr Surg.2004;39:170-3.
24 Mandhan P, Quan QB, Beasley S,et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg.2006;41:2041-5.
25 Nievelstein RA , Van der Werff J F , Verbeek FJ , et al. Normal and abnormal embryonic development of the anorectum in human embryos. Teratology. 1998; 57: 70-78
26 Paidas CN , Morreale RF , Holoski KM, et al. Separation and differentiation of the embryonic human cloaca. J Pediatr Surg.1999;34:877-884.
27 Kluth D, Hillen M , Lambrecht W. The principle of normal and abnormal hindgut Development.J Pediatr Surg.1995;30:1143-1147.
28 Hynes PJ, Fraher JP. The development of the male genitourinary system. I. The origin of the urorectal septum and the formation of the perineum. Br J Plast Surg. 2004;57:27-36.
29 Qi BQ, Beasley SW, Frizelle FA. Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg.2002;37:1305-12.
30 Haraguchi R, Mo R, Hui C,et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development.2001;128:4241-50.
31 Haraguchi R, Motoyama J, Sasaki H,et al. Molecular analysis of coordinated bladder and urogenital organ formation by Hedgehog signaling. Development.2007;134: 525-33.
32 Qi BQ, Williams A, Beasley S, et al. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg. 2000;35:1810-6.
33 Bai Y, Chen H, Yuan ZW, et al. Normal and abnormal embryonic development of the anorectum in rats.J Pediatr Surg.2004;39:587-90.
34 Roberts DJ, Johnson RL, Burke AC,et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development. 1995;121:3163-74.
35 梁娟,王艳萍,代礼,等．中国人直肠肛门畸形1262例分析[J]．中华小儿外科杂志,1999,20：9-11．
36 Bai Y, Yuan Z, Wang W, et al. Quality of life for children with fecal incontinence after surgically corrected anorectal malformation.J Pediatr Surg.2000;35:462- 4.
37 Davies MC, Creighton SM, Wilcox DT. Long-term outcomes of anorectal malformations. Pediatr Surg Int.2004,20:567-72.
38 Goyal A,Williams JM,Kenny SE,et al.Functional outcome and quality of life in anorectal malformations.J Pediatr Surg.2006;41:318-22.
39 Spilde TL,Bhatia AM,Mehta S,et al.Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula.Surgery.2003;134:345-50.
40 张志波,高红,王练英.先天性无肛畸形Gli2基因表达的研究.中华小儿外科杂志,2001,22:325-327
41 De Santa Barbara P,Van den Brink,G R,Roberts,D J,et al.Development and differentiation of the intestinal epithelium.Cell Mol Life Sci.2003;60:1322-32.
42 Howe JR,Bair JL,Sayed MG,et al.Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis.Nat Genet.2001;28:184-7.
43 Fougerousse F,Bullen P,Herasse M,et al.Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes.Hum Mol Genet.2000;9:165-73.
44 Cohen MM Jr.The hedgehog signaling network.Am J Med Genet A.2003;123:5-28.
45 Bitgood MJ,McMahon AP.Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo.Dev Biol.1995;172:126-38.
46 Penington EC,Hutson JM.The cloacal plate:the missing link in anorectal and urogenital development.BJU Int.2002;89:726-32.
47 Nebot-Cegarra J,Fabregas PJ,Sanchez-Perez I.Cellular proliferation in the urorectal septation complex of the human embryo at Carnegie stages 13-18:a nuclear area-based morphometric analysis.J Anat.2005;207:353-64.
48 Kromer P.Further study of the urorectal septum in staged human embryos.Folia Morphol.1999;58:53-63.
49 Rogers DS,Paidas CN,Morreale RF,et al.Septation of the anorectal and genitourinary tracts in the human embryo:crucial role of the catenoidal shape of the urorectal sulcus.Teratology.2002;66:144-52.
50 Van der Putte SC.Anal and ano-urogenital malformations:a histopathological study of "imperforate anus" with a reconstruction of the pathogenesis.Pediatr Dev Pathol.2006;9:280-96.
51 Mandhan P,Beasley S,Hale T.Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats.Pediatr Surg Int.2006;22:31-6.
52 李正,王慧贞,吉士俊.实用小儿外科学.北京:人民卫生出版社.491-500.
53 Spilde T,Bhatia A,Ostlie D,et al.A role for sonic hedgehog signaling in the pathogenesis of human tracheoesophageal fistula.J Pediatr Surg.2003;38:465-8.
54 Crowley AR,Mehta SS,Hembree MJ,et al.Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula.Pediatr Surg Int.2006;22:154-7.
55 Ratan SK,Rattan KN,Pandey RM,et al.Associated congenital anomalies in patients with anorectal malformations—a need for developing a uniform practical approach.J Pediatr Surg.2004;39:1706-11.
1 Tabata T, Takei Y. Morphogens, their identification and regulation. Development. 2004;131:703-12.
2 Ashe HL , Briscoe J.The interpretation of morphogen gradients. Development. 2006;133:385-94.
3 Cohen MM Jr. The hedgehog signaling network. Am J Med Genet A. 2003 15;123:5-28.
4 Nybakken K, Perrimon N. Hedgehog signal transduction: recent findings. Curr Opin Genet Dev.2002;12:503-11.
5 Murone M, Rosenthal A, De Sauvage FJ. Sonic hedgehog signaling by the patched-smoothened receptor complex.Curr Biol.1999;9:76-84.
6 Charytoniuk D, Porcel B, Rodriguez Gomez J,et al.Sonic Hedgehog signalling in the developing and adult brain.J Physiol Paris.2002;96:9-16.
7 Roberts DJ, Smith DM, Goff DJ,et al. Epithelial - mesenchymal signaling during the regionalization of the chick gut. Development. 1998;125:2791-801.
8 Sukegawa A, Narita T, Kameda T,et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development. 2000;127:1971-80.
9 Huelsken J , Birchmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev.2001;11:547-553.
10 Mao B , Wu W, Davidson G, et al . Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signaling. Nature.2002;417: 664-667.
11 Nusse R. Wnt signaling in disease and in development. Cell Res.2005;15:28-32. Review.
12 Theodosiou NA, Tabin CJ. Wnt signaling during development of the gastrointestinal tract.Dev Biol.2003;15;259:258-71.
13 Reddi AH. BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev. 2005; 16: 249-50.
14 Kawabata M, Imamura T, Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev.1998;9:49-61.
15 Kishigami S, Mishina Y.BMP signaling and early embryonic patterning. Cytokine Growth Factor Rev.2005;16:265-78.
16 Batts LE, Polk DB, Dubois RN, et al. Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn.2006;235:1563-70.
17 De Santa Barbara P, Williams J, Goldstein AM,et al.Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn.2005;234:312-22.
18 Thisse B, Thisse C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol.2005;287:390-402.
19 Mailleux AA, Spencer-Dene B, Dillon C, et al. Role of FGF10/ FGFR2b signaling during mammary gland development in the mouse embryo. Development.2002;129: 53-60.
20 Fairbanks TJ, Sala FG, Kanard R, et al. The fibroblast growth factor pathway serves a regulatory role in proliferation and apoptosis in the pathogenesis of intestinal atresia. J Pediatr Surg.2006;41:132-6.
21 Fairbanks TJ, Kanard RC, Del Moral PM, et al. Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway. J Pediatr Surg.2005;40:390-6.
22 Roberts DJ. Molecular mechanisms of development of the gastrointestinal tract. Dev Dyn.2000;219:109-20.
23 Ormestad M, Astorga J, Landgren H, et al. Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production. Development.2006;133:833-43.
24 Qi BQ, Beasley SW, Frizelle FA. Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus.J Pediatr Surg.2002;37:1305-12.
25 Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development.J Pediatr Surg.1995;30:1143-7.
26 Van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg.1986;21:434-40.
27 Mo R, Kim JH, Zhang J,et al. Anorectal malformations caused by defects in sonic hedgehog signaling.Am J Pathol.2001;159:765-74.
28 Kimmel SG, Mo R, Hui CC,et al. New mouse models of congenital anorectal malformations. J Pediatr Surg.2000;35:227-30.
29 Gregorieff A, Grosschedl R, Clevers H. Hindgut defects and transformation of the gastro-intestinal tract in Tcf4(-/-) / Tcf1 (-/-) embryos.EMBO J.2004;23: 1825-33.
30 Lickert H, Kispert A, Kutsch S,et al. Expression patterns of Wnt genes in mouse gut development. Mech Dev.2001;105:181-4.
31 Sasaki Y, Iwai N, Tsuda T,et al. Sonic hedgehog and bone morphogenetic protein 4 expressions in the hindgut region of murine embryos with anorectal malformations.J Pediatr Surg.2004;39:170-3.
32 Fairbanks TJ, De Langhe S, Sala FG,et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004;39:360-5.
33 Sala FG, Curtis JL, Veltmaat JM,et al. Fibroblast growth factor 10 is required for survival and proliferation but not differentiation of intestinal epithelial progenitor cells during murine colon development. Dev Biol.2006;299:373-85.
34 Freeman M, Gurdon JB. Regulatory principles of developmental signaling. Annu Rev Cell Dev Biol.2002;18:515-39.
2 Kiefer SM, Ohlemiller KK, Yang J, et al. Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects. Hum Mol Genet.2003;12:2221-7.
3 Fairbanks TJ, De Langhe S, Sala FG,et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004;39:360-5.
4 Liem KF Jr, Jessell TM, Briscoe J. Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites. Development.2000;127:4855-66.
5 Zhao M, Qiao M, Harris SE, et al. The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling. Mol Cell Biol.2006;26:6197-208.
6 Spilde TL, Bhatia AM, Mehta S, et al. Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula. Surgery.2003;134:345-50.
7 Zhang Y, Zhang Z, Zhao X, et al. A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ. Development. 2000; 127:1431-43.
8 Ishizuya-Oka A, Hasebe T, Shimizu K,et al. Shh/BMP-4 signaling pathway is essential for intestinal epithelial development during Xenopus larval-to-adult remodeling. Dev Dyn.2006;235:3240-9.
9 Kimmel SG, Mo R, Hui CC, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg.2000;35:227-30.
10 Mo R, Kim JH, Zhang J,et al. Anorectal malformations caused by defects in sonic hedgehog signalingAm J Pathol.2001;159:765-74.
11 Nybakken K, Perrimon N. Hedgehog signal transduction: recent findings. Curr Opin Genet Dev.2002;12:503-ll.
12 McMahon AP.More surprises in the Hedgehog signaling pathway.Cell.2000;100: 185-8.
13 Charytoniuk D, Porcel B, Rodriguez Gomez i,et al.Sonic Hedgehog signalling in the developing and adult brain.J Physiol Paris.2002;96:9-16.
14 Kishigami S, Mishina Y.BMP signaling and early embryonic patterning. Cytokine Growth Factor Rev.2005;16:265-78.
15 Reddi AH. BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev.2005;16:249-50.
16 Batts LE, Polk DB, Dubois RN, et al. Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn.2006;235:1563-70.
17 Roberts DJ, Smith DM, Goff DJ, et al. Epithelial - mesenchymal signaling during the regionalization of the chick gut. Development. 1998; 125:2791-801.
18 Sukegawa A, Narita T, Kameda T, et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development.2000; 127:1971 -80.
19 De Santa Barbara P, Williams J, Goldstein AM, et al.Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn.2005;234:312-22.
20 Roberts DJ .Molecular mechanisms of development of the Gastrointestinal tract. Developmental Dynamics.2000;219:109-20.
21 Pyati UJ, Cooper MS, Davidson AJ, et al. Sustained Bmp signaling is essential for cloaca development in zebrafish. Development.2006;133:2275-84.
22 Liu G, Moro A, Zhang JJ, et al. The role of Shh transcription activator Gli2 in chick cloacal development.Dev Biol.2007;303:448-60.
23 Sasaki Y, Iwai N, Tsuda T, et al. Sonic hedgehog and bone morphogenetic protein4 expressions in the hindgut region of murine embryos with anorectal malformations. J Pediatr Surg.2004;39:170-3.
24 Mandhan P, Quan QB, Beasley S,et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg.2006;41:2041-5.
25 Nievelstein RA , Van der Werff J F , Verbeek FJ , et al. Normal and abnormal embryonic development of the anorectum in human embryos. Teratology. 1998; 57: 70-78
26 Paidas CN , Morreale RF , Holoski KM, et al. Separation and differentiation of the embryonic human cloaca. J Pediatr Surg.1999;34:877-884.
27 Kluth D, Hillen M , Lambrecht W. The principle of normal and abnormal hindgut Development.J Pediatr Surg.1995;30:1143-1147.
28 Hynes PJ, Fraher JP. The development of the male genitourinary system. I. The origin of the urorectal septum and the formation of the perineum. Br J Plast Surg. 2004;57:27-36.
29 Qi BQ, Beasley SW, Frizelle FA. Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg.2002;37:1305-12.
30 Haraguchi R, Mo R, Hui C,et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development.2001;128:4241-50.
31 Haraguchi R, Motoyama J, Sasaki H,et al. Molecular analysis of coordinated bladder and urogenital organ formation by Hedgehog signaling. Development.2007;134: 525-33.
32 Qi BQ, Williams A, Beasley S, et al. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg. 2000;35:1810-6.
33 Bai Y, Chen H, Yuan ZW, et al. Normal and abnormal embryonic development of the anorectum in rats.J Pediatr Surg.2004;39:587-90.
34 Roberts DJ, Johnson RL, Burke AC,et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development. 1995;121:3163-74.
35 梁娟,王艳萍,代礼,等．中国人直肠肛门畸形1262例分析[J]．中华小儿外科杂志,1999,20：9-11．
36 Bai Y, Yuan Z, Wang W, et al. Quality of life for children with fecal incontinence after surgically corrected anorectal malformation.J Pediatr Surg.2000;35:462- 4.
37 Davies MC, Creighton SM, Wilcox DT. Long-term outcomes of anorectal malformations. Pediatr Surg Int.2004,20:567-72.
38 Goyal A,Williams JM,Kenny SE,et al.Functional outcome and quality of life in anorectal malformations.J Pediatr Surg.2006;41:318-22.
39 Spilde TL,Bhatia AM,Mehta S,et al.Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula.Surgery.2003;134:345-50.
40 张志波,高红,王练英.先天性无肛畸形Gli2基因表达的研究.中华小儿外科杂志,2001,22:325-327
41 De Santa Barbara P,Van den Brink,G R,Roberts,D J,et al.Development and differentiation of the intestinal epithelium.Cell Mol Life Sci.2003;60:1322-32.
42 Howe JR,Bair JL,Sayed MG,et al.Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis.Nat Genet.2001;28:184-7.
43 Fougerousse F,Bullen P,Herasse M,et al.Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes.Hum Mol Genet.2000;9:165-73.
44 Cohen MM Jr.The hedgehog signaling network.Am J Med Genet A.2003;123:5-28.
45 Bitgood MJ,McMahon AP.Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo.Dev Biol.1995;172:126-38.
46 Penington EC,Hutson JM.The cloacal plate:the missing link in anorectal and urogenital development.BJU Int.2002;89:726-32.
47 Nebot-Cegarra J,Fabregas PJ,Sanchez-Perez I.Cellular proliferation in the urorectal septation complex of the human embryo at Carnegie stages 13-18:a nuclear area-based morphometric analysis.J Anat.2005;207:353-64.
48 Kromer P.Further study of the urorectal septum in staged human embryos.Folia Morphol.1999;58:53-63.
49 Rogers DS,Paidas CN,Morreale RF,et al.Septation of the anorectal and genitourinary tracts in the human embryo:crucial role of the catenoidal shape of the urorectal sulcus.Teratology.2002;66:144-52.
50 Van der Putte SC.Anal and ano-urogenital malformations:a histopathological study of "imperforate anus" with a reconstruction of the pathogenesis.Pediatr Dev Pathol.2006;9:280-96.
51 Mandhan P,Beasley S,Hale T.Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats.Pediatr Surg Int.2006;22:31-6.
52 李正,王慧贞,吉士俊.实用小儿外科学.北京:人民卫生出版社.491-500.
53 Spilde T,Bhatia A,Ostlie D,et al.A role for sonic hedgehog signaling in the pathogenesis of human tracheoesophageal fistula.J Pediatr Surg.2003;38:465-8.
54 Crowley AR,Mehta SS,Hembree MJ,et al.Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula.Pediatr Surg Int.2006;22:154-7.
55 Ratan SK,Rattan KN,Pandey RM,et al.Associated congenital anomalies in patients with anorectal malformations—a need for developing a uniform practical approach.J Pediatr Surg.2004;39:1706-11.
1 Tabata T, Takei Y. Morphogens, their identification and regulation. Development. 2004;131:703-12.
2 Ashe HL , Briscoe J.The interpretation of morphogen gradients. Development. 2006;133:385-94.
3 Cohen MM Jr. The hedgehog signaling network. Am J Med Genet A. 2003 15;123:5-28.
4 Nybakken K, Perrimon N. Hedgehog signal transduction: recent findings. Curr Opin Genet Dev.2002;12:503-11.
5 Murone M, Rosenthal A, De Sauvage FJ. Sonic hedgehog signaling by the patched-smoothened receptor complex.Curr Biol.1999;9:76-84.
6 Charytoniuk D, Porcel B, Rodriguez Gomez J,et al.Sonic Hedgehog signalling in the developing and adult brain.J Physiol Paris.2002;96:9-16.
7 Roberts DJ, Smith DM, Goff DJ,et al. Epithelial - mesenchymal signaling during the regionalization of the chick gut. Development. 1998;125:2791-801.
8 Sukegawa A, Narita T, Kameda T,et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development. 2000;127:1971-80.
9 Huelsken J , Birchmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev.2001;11:547-553.
10 Mao B , Wu W, Davidson G, et al . Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signaling. Nature.2002;417: 664-667.
11 Nusse R. Wnt signaling in disease and in development. Cell Res.2005;15:28-32. Review.
12 Theodosiou NA, Tabin CJ. Wnt signaling during development of the gastrointestinal tract.Dev Biol.2003;15;259:258-71.
13 Reddi AH. BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev. 2005; 16: 249-50.
14 Kawabata M, Imamura T, Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev.1998;9:49-61.
15 Kishigami S, Mishina Y.BMP signaling and early embryonic patterning. Cytokine Growth Factor Rev.2005;16:265-78.
16 Batts LE, Polk DB, Dubois RN, et al. Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn.2006;235:1563-70.
17 De Santa Barbara P, Williams J, Goldstein AM,et al.Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn.2005;234:312-22.
18 Thisse B, Thisse C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol.2005;287:390-402.
19 Mailleux AA, Spencer-Dene B, Dillon C, et al. Role of FGF10/ FGFR2b signaling during mammary gland development in the mouse embryo. Development.2002;129: 53-60.
20 Fairbanks TJ, Sala FG, Kanard R, et al. The fibroblast growth factor pathway serves a regulatory role in proliferation and apoptosis in the pathogenesis of intestinal atresia. J Pediatr Surg.2006;41:132-6.
21 Fairbanks TJ, Kanard RC, Del Moral PM, et al. Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway. J Pediatr Surg.2005;40:390-6.
22 Roberts DJ. Molecular mechanisms of development of the gastrointestinal tract. Dev Dyn.2000;219:109-20.
23 Ormestad M, Astorga J, Landgren H, et al. Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production. Development.2006;133:833-43.
24 Qi BQ, Beasley SW, Frizelle FA. Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus.J Pediatr Surg.2002;37:1305-12.
25 Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development.J Pediatr Surg.1995;30:1143-7.
26 Van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg.1986;21:434-40.
27 Mo R, Kim JH, Zhang J,et al. Anorectal malformations caused by defects in sonic hedgehog signaling.Am J Pathol.2001;159:765-74.
28 Kimmel SG, Mo R, Hui CC,et al. New mouse models of congenital anorectal malformations. J Pediatr Surg.2000;35:227-30.
29 Gregorieff A, Grosschedl R, Clevers H. Hindgut defects and transformation of the gastro-intestinal tract in Tcf4(-/-) / Tcf1 (-/-) embryos.EMBO J.2004;23: 1825-33.
30 Lickert H, Kispert A, Kutsch S,et al. Expression patterns of Wnt genes in mouse gut development. Mech Dev.2001;105:181-4.
31 Sasaki Y, Iwai N, Tsuda T,et al. Sonic hedgehog and bone morphogenetic protein 4 expressions in the hindgut region of murine embryos with anorectal malformations.J Pediatr Surg.2004;39:170-3.
32 Fairbanks TJ, De Langhe S, Sala FG,et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004;39:360-5.
33 Sala FG, Curtis JL, Veltmaat JM,et al. Fibroblast growth factor 10 is required for survival and proliferation but not differentiation of intestinal epithelial progenitor cells during murine colon development. Dev Biol.2006;299:373-85.
34 Freeman M, Gurdon JB. Regulatory principles of developmental signaling. Annu Rev Cell Dev Biol.2002;18:515-39.