正常及先天性肛门直肠畸形大鼠及人类肠神经系统发育及调控的对比研究
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摘要
前言
先天性肛门直肠畸形(Anorectal Malformations,ARMs)是小儿常见的消化道畸形,是世界卫生组织常规监测的先天畸形之一,占消化道畸形的1/4,发病率为1/5000～1/1500。尽管长期以来人们对肛门直肠畸形的手术方式进行着不断的改进,但中、高位ARMs的术后长期随访显示,仍有许多患者存在不同程度的排便、控便功能障碍。术后肛门直肠功能障碍是盆底神经肌肉发育不良所致,而肠道动力无疑是影响直肠肛管控便屏障最重要的因素之一,研究ARMs肠神经系统、肠道肌肉及神经肌肉联系的发育及其可能的调控机制已经很有必要。
既往研究显示,在中、高位肛门直肠畸形患者中,其骶髓中枢及直肠末端的神经系统有不同程度的改变,但迄今为止,对其胚胎发育过程和详细的病理改变尚不清楚。
胚胎学的研究进展已初步阐明肠神经系统胚胎学发生的机制。肠神经系统的胚胎发育来源于迷走神经嵴及骶神经嵴细胞,循肠管头尾方向发育增殖。ARMs在畸形发生后,是否伴随着神经嵴细胞迁移异常呢?抑或为迁移至肠壁后发育增殖异常呢?这些都是尚未解决的问题。先天性肛门直肠畸形的病因至今仍不明了。目前认为,肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。最近国外学者利用肛门直肠畸形动物实验研究发现,Shh、Hox、Fgf10、Wnt5a等多种基因在消化道末端发育中发挥重要作用。当上述基因异常均可导致动物出现肛门闭锁、肛门开口异位等畸形。在ARMs发生后,这些信号分子是否仍然继续在肠道发育中发挥重要作用并且在胚胎发育过程中的表达是否存在异常呢?
在上述众多的信号分子中,高度保守的Wnt信号蛋白家族在胚胎发育中发挥着尤其重要的作用,已经引起了许多学者的注意。Wnt蛋白通过不同信号分子间的相互作用,触发了调节细胞生长、迁移、分化及发育等多方面的复杂信号级联反应,其中对Wnt5a的研究正在逐渐增多,但多是涉及人体各个系统发生的常见肿瘤,对于其在调控肠道发育胚胎发育的研究刚刚起步,尤其对于ARMs肠神经肌肉发育调控的机制目前尚不清楚。
本文应用乙烯硫脲(ethylenethiourea,ETU)致畸的Wistar大鼠的ARMs动物模型,利用神经元、突触囊泡蛋白及氮能神经元的发育作为评价肠神经系统及肠道神经肌肉发育的标志物,应用相应的抗体即蛋白基因产物(protein gene product9.5,PGP9.5)、突触素(synaptophysin,SYP)及一氧化氮合成酶(nitric oxidesynthases,NOs)观察上述指标在胚胎发育过程中的表达及变化;同时分析Wnt5a及其受体Frizzled-1、TCF4蛋白和mRNA在正常及ARMs大鼠结肠的表达及变化情况,探讨正常及ARMs大鼠与排便控制密切相关的肠神经系统及肠道平滑肌的胚胎期发育的可能调控机制。另外,我们采用ARMs患儿的直肠末端组织对Wnt5a的表达进行了进一步验证,以期得到一些更为有益的研究启示。
实验材料
1、实验动物:
体重250～300克的Wistar大白鼠,由中国医科大学附属盛京医院动物部提供。
2、病例标本:
ARMs患儿直肠末端标本20例:男14例,女6例,平均年龄8.5个月;其中高位畸形5例,中位畸形7例,低位畸形8例。7例死于非胃肠道疾病患儿的直肠末端标本为对照组:男5例,女2例,平均年龄7.3个月。
3、实验试剂:
①PGP9.5单克隆抗体(购自美国Abcam公司);②SYP单克隆抗体、NOs、Wnt5a、Frizzled-1和TCF4多克隆抗体(购自美国Santa Cruz公司);③免疫组化Ultra Sensitive~(TM)Sp超敏试剂盒(购自福州迈新生物技术开发有限公司);二步法山羊免疫组化检测试剂盒(购自北京中杉金桥公司);④乙烯硫脲(Ethylenethiourea,ETU)(购自德国Sigma-Aldrich公司);⑤逆转录试剂盒(购自大连宝生物工程有限公司);⑥总蛋白抽提试剂盒(购自赛驰公司)。
实验方法
在成熟健康Wistar孕鼠妊娠第10天(E10),按125mg/kg经胃管给予孕鼠灌入ETU(浓度为1%,以生理盐水做溶剂),制作ARMs大鼠动物模型;对照组给予等量的生理盐水。分别在大鼠E15-E21时剖宫取胎,部分取完整胚胎或胚胎盆部,经4%多聚甲醛磷酸盐缓冲液固定、脱水后制成蜡块,行矢状面、横断面的连续切片,分别进行如下实验:①PGP9.5、SYP及NOs免疫组化/荧光染色,以其作为胚胎期ENS发育的标志物,观察正常和ARMs大鼠ENS的胚胎发育过程;②进行Wnt5a、Frizzled-1、TCF4的免疫组化染色来大体比较上述因子在直肠壁内的表达情况。另一部分鼠胚通过显微镜下取材获得末端直肠标本,部分进行Wnt5a、Frizzled-1及TCF4 mRNA的RT-PCR实验,其余部分标本进行PGP9.5、SYP、NOs、Wnt5a、Frizzled-1及TCF4的western blot实验进行蛋白定量分析,G:BOXSYNGENE凝胶分析系统拍照,Kodark Digital Science 1.0 DNA分析软件进行半定量分析。所有数据均以(?)表示,差异比较采用SPSS 13.0统计软件进行各胎龄正常组与ARMs组的t检验,P＜0.05表示差异有统计学意义。
人类正常及ARMs直肠末端标本进行Wnt5a的免疫组化及Western blot实验,对其表达进行定位和定量,并比较分析。
结果
1、正常与ARMs的直肠内ENS的胚胎发育过程及比较
①PGP9.5标记的神经元的在直肠内的迁移发育情况:通过免疫组化结果显示,正常对照组,E16时:神经嵴细胞已经定植于整个后肠,直肠壁内可见散在阳性细胞,这时环肌层刚刚开始分化;从E16到E18,PGP9.5阳性细胞出现在环肌浆膜侧即肌间神经丛部位,成散在单个出现,E17-18时的直肠末端的纵肌纤维分布仍然稀疏,粘膜下PGP9.5阳性细胞少见;E20时,可以观察到黄褐色团块状的PGP9.5阳性反应的神经节细胞,呈团簇状,粘膜下也可见阳性细胞,E21天更为明显。而在同张切片中观察,同期的中肠肠壁的神经发育较后肠明显成熟。而在ARMs组:E16-17时神经嵴细胞在畸形组直肠中也可显示,与正常组定位无明显差异,但在直肠尿道瘘管周围未见阳性细胞;E18-19后近直肠盲端处仍可见阳性细胞,但分布与正常组相比,阳性细胞的分布较稀疏,瘘管周围仍未见阳性细胞分布;E20-21的直肠盲端阳性细胞分布与上位正常段结肠及正常组相比,密度减低,分布更为稀疏,并未见明显的神经细胞簇形成,发育滞后更明显。
②NOs标记的氮能神经元发育情况:通过免疫荧光结果显示,与神经嵴细胞迁移发育过程伴随,氮能神经元的发育与之大致同步,但大约晚于PGP9.5阳性细胞出现在直肠区域一天以后,即出现在大鼠E17后的直肠壁内;E18时,直肠壁肌间出现清晰的阳性神经元,胞浆处有明显的荧光标记,出生前阳性神经元发育更趋于成熟;ARMs组与正常组相比,发育明显滞后;胎龄越大,差别越明显。
③SYP的表达情况:正常对照组观察到:在E17时,直肠上端结肠壁内只能见散在零星分布荧光显示,但非常微弱;E18开始,结直肠壁内可见清晰强绿色荧光,主要分布在肠壁肌间位置,连续成条索状,上端结肠较直肠更为清晰,到E20以及出生前,荧光强度逐渐增强。在ARMs组:E16-17时结直肠大部分未见荧光显示,上位结肠与直肠处相比,偶可见极弱的零星分布的荧光;E18后上位结肠的发育更为成熟,近直肠盲端处仍可见绿色荧光,与上位正常段结肠及正常组相比,强度减弱,差别更明显,瘘管周围未见荧光显示。
④PGP9.5、SYP和NOs的western blot结果显示上述因子的蛋白定量结果在E19、E21时两组均存在差别,具有统计学意义。
2、Wnt5a等发育相关因子在正常和ARMs大鼠直肠壁胚胎发育期的表达对比
①Wnt5a、Frizzled-1、TCF4免疫组化染色结果:正常组从E16～E21,肌间神经丛及肠道平滑肌及直肠粘膜层部位均观察到上述因子的表达,但是表达强度存在细微差别,随着肠管的发育成熟,其表达逐渐增强,不同部位间表达强度的差异也更明显,神经丛及肠道平滑肌的表达更强一些。ARMs组:E16时,未见阳性反应的细胞;E17-E21,阳性细胞逐渐显现,与正常胎龄的直肠同区域相比,表达减弱,在瘘管周围无阳性反应细胞。
②Wnt5a、Frizzled-1和TCF4的western blot结果更清晰的显示了两组间三种蛋白定量的差别。
③Wnt5a、Frizzled-1及TCF4 mRNA的RT-PCR实验结果显示,E17、19、21时,Wnt5a、Frizzled-1及TCF4在正常组表达水平较ARMs组高,具有统计学差异(p＜0.05)。与免疫组化及western blot结果基本相符。
3、Wnt5a在正常和ARMs人类直肠壁表达的对比结果
Wnt5a蛋白表达为棕黄色颗粒状,表达于人直肠壁肌间神经丛、粘膜层和粘膜下层,呈弥漫性全浆表达,肌层表达较弱。ARMs患儿的直肠壁并未观察到阳性表达细胞。Western蛋白印迹结果显示,ARMs高位组、中位组和低位组的蛋白表达水平均明显低于后天瘘组和正常对照组(P＜0.05)。不同畸形之间,高位组和中位组之间差异无统计学意义(P=0.11),但二者明显低于低位组(P＜0.01)。后天瘘组与正常对照组比较没有明显差异(P=0.67)
结论
1、在正常及ARMs大鼠胚胎,在E16左右,神经嵴细胞迁移到直肠区域,但直肠末端神经发育很差,随着胚胎的进一步发育,直肠区域的肠神经系统发育逐渐完善,逐渐形成神经丛状结构,生后发育更加完善。ARMs大鼠胚胎的ENS在E16时,其形态与正常组无明显差异,从E18起,可观察到发育异常的ENS,随着胎龄的加大,与正常相比,发育差异更加显著。
2、氮能神经元作为重要的神经元亚型,其发育过程是伴随神经嵴细胞的迁移而发育的,但稍迟一些,推测NO可能在肠神经系统发育过程中也起着重要作用,ARMs大鼠胚胎直肠末端区域的突触素的发育较正常组差,可能也是导致肠道动力改变的一个原因。
3、E16～E21,Wnt5a及其受体Frizzled-1及Wnt经典通路的关键因子TCF4在ARMs组的肠壁粘膜、肌层及肠神经系统的表达较正常组减弱,可能与ARMs直肠神经系统及直肠末端壁平滑肌发育不良的调控相关。
4、Wnt5a主要表达在人类直肠壁肌间神经丛部位,在肛门直肠畸形患者的直肠末端表达水平减低,推测Wnt5a在人类先天性肛门直肠畸形发生后的直肠神经系统进一步的发育中可能具有重要调节作用。
Introduction
Anorectal malformations(ARMs)are one of the most common digestive disorders, affecting 1/5000 to 1/1500 live births,one kind of malformations supervised by World Health Organization.In spite of the recent advances in operative techniques,the long-term prognosis in intermediate and high type ARMs remains poor.Some patients with ARMs continued to suffer from postoperative anal dysfunctions.The anorectal dysfunction was caused by the dysplasia of the neuromuscular in the pelvic floor. Undoubtedly,the intestinal motility is the focal point of the organism of continence. The research of the development of the enteric nervous system,intestinal muscle and nerve-muscle contact and the possible control mechanisms in ARMs has been necessary.
In previous studies,it has been shown that in the middle and high ARMs,sacral cord and the enteric nervous system of the rectal end have changed differently,but so far the course of their embryonic development and detailed pathological changes are unclear.
With the progress of molecular biology,the embryogenesis of the enteric nervous system has been illuminated initially.Vagal and sacral neural crest cells give rise to enteric neurons and glial cells throughout the entire gastrointestinal tract.In ARMs, after the defect appears,there has been a question that if the abnormity of the enteric nervous system accompanied the ARMs.The etiopathogenisis of ARMs continues poorly understood and controversial.In current study,it has been only demonstrated that ARMs resulted from hereditary factors and environmental factors in the development of embryogenesis.During the research of epidemiology and animal experiments,it is suggested that the hereditary factors play a key role in the development of ARMs.Recently,it was found that Shh、Hox、Fgf10 and Wnt5a play crucial roles during the development of the termination of digestive tract.Once the foresaid genes dysfunction,there may be ARMs,such as anal atresia,preternatural anus et al.
In recent years research has shown that the highly conserved Wnt signaling protein family in embryonic development play an important role in signaling through different molecular interactions,Wnt proteins trigger the regulation of cell growth,migration, differentiation and development,and many other complex signaling cascade reaction, Wnt5a is an important parterner in this family.Most of its researches involving human subjects are common in all systems tumor,at the same time it play an important role in the embryogenesis of intestinal tract,but the its regulation of intestinal smooth muscle and enteric nervous system during embryonic development have not been reported, especially it is not clear for the mechanism of the intestinal neuromuscular dysplasia in ARMs.
In this paper,we utilize the Wistar rat animal model of ARMs induced by ETU (ethylenethiourea).We used PGP9.5(protein gene product 9.5)and SYP (synaptophysin)and NOs(nitric oxide synthases)for evaluating the enteric nervous system and neuromuscular development of intestinal tract,at the same time we analyzed the expression of Wnt5a and its receptor Frizzled-1,TCF4 protein and mRNA in the rectum of normal and ARMs to explore the possible regulatory mechanism of enteric nervous system and gastro-intestinal smooth muscle of the embryonic period,which is the important role in participate in defecation.In addition, we used the clinical specimens in children with ARMs to explore the Wnt5a protein expression.
Materials
Animals:Wistar rats(250-300g)were provided by Medical Animal Center in Shengjing Hospital of China Medical University.
Clinical specimens:the case group:20 ARMs infants operated in our department. Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at 1 cm superior to dentate line in acquired anal fistula and rectal traumas infants.
Reagents:①The specific antibody to PGP9.5 was purchased from Abcam Corporation.②The specific antibodies to SYP,Nos,Wnt5a,Frizzled-1,TCF4 were purchased from Santa Cruz Corporation.③The UltraSensitiveTMSP kits for immuhistochmistry were purchased from MaiXin biological limited company,FuJian Province.Two-step immunohistochemical goat detection kit was purchased from Beijing Zhong Shan Golden Bridge Company;④Ethylenethiourea,ETU was purchased from Sigma-Aldrich Inc.,Germany;⑤Reverse transcriptase kit was purchased from Takara Bio-engineering Co.,Ltd;⑥Total protein extraction kit was purchased from Seitz Corporation.
Methods
Time-mated pregnant Wistar rats were gavage-fed a single dose of either 125 mg/kg of 1%ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc,Germany) or an equal dose of saline on gestational day 10(E0—sperm in vaginal smear after overnight mating).The embryos were harvested via cesarean section on E15 to E21. The embryos were fixed in 4%paraformaldehyde/0.1 M phosphate buffered saline (PBS)for 12 to 24 hours depending on their size.Then the embryos from each age group were dehydrated,embedded in paraffin and sectioned serially transversely,and sagittally at 4μm thickness.①Immunohistochemistry and fluorescence staining of PGP9.5 and SYP,NOs(embryonic development ENS markers)were to observe normal and ARMs rat embryonic development of the ENS.②Immunohistochemical staining of Wnt5a,Frizzled-1 and TCF4 were compared to roughly to position at the intestinal wall.Another part of rectum samples of rats were drawn by microscope,we evaluated the expression of Wnt5a,Frizzled-1 and TCF4 mRNA by RT-PCR and we evaluated the expression of PGP9.5,SYP,NOs,Wnt5a,Frizzeled-1,TCF4 prtein by the western blot quantitative analysis.G:BOX SYNGENE photographed gel analysis system and Kodark Digital Science 1.0 DNA analysis software to carry out semi-quantitative analysis.T-test was used to compare the differences of foresaid genes expression levels between the normal and ARMs groups,P＜0.05 indicates that the difference has statistical significance.③Immunohistochemical staining and western blot analysis of Wnt5a were used to compare the expression level in the the case group and the control group.
Results
1.The embryonic development of the ENS in normal and ARMs rat embryos
①PGP9.5 labeled neurons in the post-intestinal migration and development:
Immunohistochemical results showed that in normal control group,at E16:neural crest cells already colonized in the whole intestine and scattered positive cells were seen in the rectum,when the circular muscles has just begun to differentiate;From E16 to E18,PGP9.5-positive cells appeared in the myenteric plexus sites poradically;At E17-18,the terminal of the longitudinal intestinal muscle fiber distribution is still sparse and there were rare PGP9.5 positive cells in the submucosa;At E20:it can be observed that the yellowish-brown mass of PGP9.5-positive ganglion cells,showing cluster-like.The positive cells also can be seen in the submucosa.At E21,it become more obvious.While at the same sections observed in the same period in the intestinal wall,the development of ENS grew earlierly.In ARMs Group,at E16-17,neural crest cells at the terminal of hindgut malformation group could also be seen and there was no significant difference between the two groups,but there was no positive cells around rectal urethral fistula;At E18-19,more positive cells were still visible near the rectum terminus,but the distribution compared with the normal group is sparse,yet no positive cells were observed around the fistula;At E20-21,compared with the normal and upper colon,the density of positive cells reduced in the rectal terminus,and no obvious clusters of nerve cells were seen.The development of ENS in ARMs lags behind the normal group.
②The development of nitrergic neurons marked by NOs:
The immunofluorescence results showed the development of nitrergic neurons accompanied neural crest cell migration during embrygenesis,but it was a little later than the emergence of PGP9.5.They appeared at around E17 in the rectum.At E18,the clear positive neurons appeared between the muscles of the rectum.The cytoplasm has full of obvious green fluorescence.Before birth the development of positive neurons have become more sophisticated.But in ARMs group compared with the normal group, the growth of nitrergic neurons have lagged.The older,the greater of the difference between the two groups.
③Immunofluorescence results of SYP:
In normal control group,only scattered fluorescent display were observed in the top of the colon wall at E16,but they are very weak;From E18,strong green fluorescence can be seen clearly within in the intestinal myenteric position.It is straight into a cord-like and was more clear in the upper colon than the rectum.To E20,as well as before birth,fluorescence intensity gradually increased.But in ARMs Group,at E16-17,no fluorescence were shown in the majority of colorectal.Only a very weak visible fluorescence could be found in the upper colon.At E18,the development of SYP in the upper colon became more sophisticated,but in the blind-side near the rectum green fluorescence was still visible.Compared with the upper segment of normal colon and normal group,the intensity of fluorescence in ARMs decreased,there were no fluorescence around the fistula.
④The western blot analysis of PGP9.5,SYP and NOs
The results showed that the protein of the above-mentioned factor had significant differences between the two groups on E19-21.
2.Expression of Wnt 5a and other development-related factors in normal and ARMs rat embryos
①Immunohistochemical staining results of Wnt5a,Frizzled-land TCF4:in the normal group from the E16～E21,the expression of the above-mentioned factors were observed in the myenteric plexus,the mucosa and intestinal smooth muscle of the rectum,but the intensity of expression were different.As the development of the rectum,the expression become more increased.In ARMs Group,at E16 there were no positive cells.From E17-E21,the positive cells gradually becoming apparent,with the normal gestational age of the rectum.But compared to the same region of the normal group,the expression decreased,no positive cells appeared around the fistula.
②The western blot results of Wnt5a,Frizzled-1 and TCF4 showed on E17,there was no significant difference between the two groups;on E19-E21,the expression levels of Wnt5a,Frizzled-1 and TCF4 were higher in the normal group than in ARMs with significant difference(p＜0.05).
③RT-PCR results of Wnt5a,Frizzled-1 and TCF4 mRNA showed that there have been a significant difference between the two groups.
3.Expression of Wnt5a in normal and ARMs
In the anorectal tissues of the human of the normal case,the expression of wnt5a was mainly located in the cytoplasm of the myenteric plexus by immunohistochemistry. But in the ARMs group,we did not found positive cells in the myenteric plexus of the the blind part of the rectum espesially in high ARMs.There was no significant difference of wnt5a protein expression level between high and intermediate ARMs by western blot analysis(P=0.33).However,the expression levels of wnt5a protein in high and intermediate ARMs were significantly lower than that in low group(P＜0.05). There was no significant difference of wnt5a protein expression level between the infected recto-vestibular fistula and the control group(P=0.67).
Conclusions
1.In normal and ARMs rat embryos,on E16,neural crest cells migrate to the rectal area,but the development of rectal terminus is worse.With the further development of rat embryos,the enteric nervous system in rectal region improved gradually,plexus-like structure has gradually formed,and postnatal development became more perfect.In ARMs group,on E16,neural crest cells could also be seen in the terminal of hindgut,there was no significant difference between the two groups. From E18,ARMs group compared with the normal group,the development of ENS has lagged;the older,the greater the difference between the two groups.
2.The developmental course of the nitrergic neurons is accompanied by neural crest cell migration and development,but it is some later.It is suggested that NO may play an important role during the development of enteric nervous system.In ARMs group,there was significant difference of synaptophysin and NOs expression between the two groups in the rectal terminal region,which may lead to change to intestinal motility.
3.E16～E21,compared with in the normal group,it is in ARMs group,the expression of Wnt5a,its receptor Frizzled-1 and TCF4 which is a classical key factor of Wnt pathways in intestinal smooth muscle and nervous system became significantly weakened.It is suggested that Wnt 5a,which may play a role by the classical pathway and may be related with the developmental regulation of enteric nervous system and smooth muscle of intestinal tract in ARMs.
4.Wnt 5a may play a role in the developmental regulation of the ENS in ARMs.
先天性肛门直肠畸形(Anorectal Malformations,ARMs)是小儿常见的消化道畸形,是世界卫生组织常规监测的先天畸形之一,占消化道畸形的1/4,发病率为1/5000～1/1500。尽管长期以来人们对肛门直肠畸形的手术方式进行着不断的改进,但中、高位ARMs的术后长期随访显示,仍有许多患者存在不同程度的排便、控便功能障碍。术后肛门直肠功能障碍是盆底神经肌肉发育不良所致,而肠道动力无疑是影响直肠肛管控便屏障最重要的因素之一,研究ARMs肠神经系统、肠道肌肉及神经肌肉联系的发育及其可能的调控机制已经很有必要。
既往研究显示,在中、高位肛门直肠畸形患者中,其骶髓中枢及直肠末端的神经系统有不同程度的改变,但迄今为止,对其胚胎发育过程和详细的病理改变尚不清楚。
胚胎学的研究进展已初步阐明肠神经系统胚胎学发生的机制。肠神经系统的胚胎发育来源于迷走神经嵴及骶神经嵴细胞,循肠管头尾方向发育增殖。ARMs在畸形发生后,是否伴随着神经嵴细胞迁移异常呢?抑或为迁移至肠壁后发育增殖异常呢?这些都是尚未解决的问题。先天性肛门直肠畸形的病因至今仍不明了。目前认为,肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。最近国外学者利用肛门直肠畸形动物实验研究发现,Shh、Hox、Fgf10、Wnt5a等多种基因在消化道末端发育中发挥重要作用。当上述基因异常均可导致动物出现肛门闭锁、肛门开口异位等畸形。在ARMs发生后,这些信号分子是否仍然继续在肠道发育中发挥重要作用并且在胚胎发育过程中的表达是否存在异常呢?
在上述众多的信号分子中,高度保守的Wnt信号蛋白家族在胚胎发育中发挥着尤其重要的作用,已经引起了许多学者的注意。Wnt蛋白通过不同信号分子间的相互作用,触发了调节细胞生长、迁移、分化及发育等多方面的复杂信号级联反应,其中对Wnt5a的研究正在逐渐增多,但多是涉及人体各个系统发生的常见肿瘤,对于其在调控肠道发育胚胎发育的研究刚刚起步,尤其对于ARMs肠神经肌肉发育调控的机制目前尚不清楚。
本文应用乙烯硫脲(ethylenethiourea,ETU)致畸的Wistar大鼠的ARMs动物模型,利用神经元、突触囊泡蛋白及氮能神经元的发育作为评价肠神经系统及肠道神经肌肉发育的标志物,应用相应的抗体即蛋白基因产物(protein gene product9.5,PGP9.5)、突触素(synaptophysin,SYP)及一氧化氮合成酶(nitric oxidesynthases,NOs)观察上述指标在胚胎发育过程中的表达及变化;同时分析Wnt5a及其受体Frizzled-1、TCF4蛋白和mRNA在正常及ARMs大鼠结肠的表达及变化情况,探讨正常及ARMs大鼠与排便控制密切相关的肠神经系统及肠道平滑肌的胚胎期发育的可能调控机制。另外,我们采用ARMs患儿的直肠末端组织对Wnt5a的表达进行了进一步验证,以期得到一些更为有益的研究启示。
实验材料
1、实验动物:
体重250～300克的Wistar大白鼠,由中国医科大学附属盛京医院动物部提供。
2、病例标本:
ARMs患儿直肠末端标本20例:男14例,女6例,平均年龄8.5个月;其中高位畸形5例,中位畸形7例,低位畸形8例。7例死于非胃肠道疾病患儿的直肠末端标本为对照组:男5例,女2例,平均年龄7.3个月。
3、实验试剂:
①PGP9.5单克隆抗体(购自美国Abcam公司);②SYP单克隆抗体、NOs、Wnt5a、Frizzled-1和TCF4多克隆抗体(购自美国Santa Cruz公司);③免疫组化Ultra Sensitive~(TM)Sp超敏试剂盒(购自福州迈新生物技术开发有限公司);二步法山羊免疫组化检测试剂盒(购自北京中杉金桥公司);④乙烯硫脲(Ethylenethiourea,ETU)(购自德国Sigma-Aldrich公司);⑤逆转录试剂盒(购自大连宝生物工程有限公司);⑥总蛋白抽提试剂盒(购自赛驰公司)。
实验方法
在成熟健康Wistar孕鼠妊娠第10天(E10),按125mg/kg经胃管给予孕鼠灌入ETU(浓度为1%,以生理盐水做溶剂),制作ARMs大鼠动物模型;对照组给予等量的生理盐水。分别在大鼠E15-E21时剖宫取胎,部分取完整胚胎或胚胎盆部,经4%多聚甲醛磷酸盐缓冲液固定、脱水后制成蜡块,行矢状面、横断面的连续切片,分别进行如下实验:①PGP9.5、SYP及NOs免疫组化/荧光染色,以其作为胚胎期ENS发育的标志物,观察正常和ARMs大鼠ENS的胚胎发育过程;②进行Wnt5a、Frizzled-1、TCF4的免疫组化染色来大体比较上述因子在直肠壁内的表达情况。另一部分鼠胚通过显微镜下取材获得末端直肠标本,部分进行Wnt5a、Frizzled-1及TCF4 mRNA的RT-PCR实验,其余部分标本进行PGP9.5、SYP、NOs、Wnt5a、Frizzled-1及TCF4的western blot实验进行蛋白定量分析,G:BOXSYNGENE凝胶分析系统拍照,Kodark Digital Science 1.0 DNA分析软件进行半定量分析。所有数据均以(?)表示,差异比较采用SPSS 13.0统计软件进行各胎龄正常组与ARMs组的t检验,P＜0.05表示差异有统计学意义。
人类正常及ARMs直肠末端标本进行Wnt5a的免疫组化及Western blot实验,对其表达进行定位和定量,并比较分析。
结果
1、正常与ARMs的直肠内ENS的胚胎发育过程及比较
①PGP9.5标记的神经元的在直肠内的迁移发育情况:通过免疫组化结果显示,正常对照组,E16时:神经嵴细胞已经定植于整个后肠,直肠壁内可见散在阳性细胞,这时环肌层刚刚开始分化;从E16到E18,PGP9.5阳性细胞出现在环肌浆膜侧即肌间神经丛部位,成散在单个出现,E17-18时的直肠末端的纵肌纤维分布仍然稀疏,粘膜下PGP9.5阳性细胞少见;E20时,可以观察到黄褐色团块状的PGP9.5阳性反应的神经节细胞,呈团簇状,粘膜下也可见阳性细胞,E21天更为明显。而在同张切片中观察,同期的中肠肠壁的神经发育较后肠明显成熟。而在ARMs组:E16-17时神经嵴细胞在畸形组直肠中也可显示,与正常组定位无明显差异,但在直肠尿道瘘管周围未见阳性细胞;E18-19后近直肠盲端处仍可见阳性细胞,但分布与正常组相比,阳性细胞的分布较稀疏,瘘管周围仍未见阳性细胞分布;E20-21的直肠盲端阳性细胞分布与上位正常段结肠及正常组相比,密度减低,分布更为稀疏,并未见明显的神经细胞簇形成,发育滞后更明显。
②NOs标记的氮能神经元发育情况:通过免疫荧光结果显示,与神经嵴细胞迁移发育过程伴随,氮能神经元的发育与之大致同步,但大约晚于PGP9.5阳性细胞出现在直肠区域一天以后,即出现在大鼠E17后的直肠壁内;E18时,直肠壁肌间出现清晰的阳性神经元,胞浆处有明显的荧光标记,出生前阳性神经元发育更趋于成熟;ARMs组与正常组相比,发育明显滞后;胎龄越大,差别越明显。
③SYP的表达情况:正常对照组观察到:在E17时,直肠上端结肠壁内只能见散在零星分布荧光显示,但非常微弱;E18开始,结直肠壁内可见清晰强绿色荧光,主要分布在肠壁肌间位置,连续成条索状,上端结肠较直肠更为清晰,到E20以及出生前,荧光强度逐渐增强。在ARMs组:E16-17时结直肠大部分未见荧光显示,上位结肠与直肠处相比,偶可见极弱的零星分布的荧光;E18后上位结肠的发育更为成熟,近直肠盲端处仍可见绿色荧光,与上位正常段结肠及正常组相比,强度减弱,差别更明显,瘘管周围未见荧光显示。
④PGP9.5、SYP和NOs的western blot结果显示上述因子的蛋白定量结果在E19、E21时两组均存在差别,具有统计学意义。
2、Wnt5a等发育相关因子在正常和ARMs大鼠直肠壁胚胎发育期的表达对比
①Wnt5a、Frizzled-1、TCF4免疫组化染色结果:正常组从E16～E21,肌间神经丛及肠道平滑肌及直肠粘膜层部位均观察到上述因子的表达,但是表达强度存在细微差别,随着肠管的发育成熟,其表达逐渐增强,不同部位间表达强度的差异也更明显,神经丛及肠道平滑肌的表达更强一些。ARMs组:E16时,未见阳性反应的细胞;E17-E21,阳性细胞逐渐显现,与正常胎龄的直肠同区域相比,表达减弱,在瘘管周围无阳性反应细胞。
②Wnt5a、Frizzled-1和TCF4的western blot结果更清晰的显示了两组间三种蛋白定量的差别。
③Wnt5a、Frizzled-1及TCF4 mRNA的RT-PCR实验结果显示,E17、19、21时,Wnt5a、Frizzled-1及TCF4在正常组表达水平较ARMs组高,具有统计学差异(p＜0.05)。与免疫组化及western blot结果基本相符。
3、Wnt5a在正常和ARMs人类直肠壁表达的对比结果
Wnt5a蛋白表达为棕黄色颗粒状,表达于人直肠壁肌间神经丛、粘膜层和粘膜下层,呈弥漫性全浆表达,肌层表达较弱。ARMs患儿的直肠壁并未观察到阳性表达细胞。Western蛋白印迹结果显示,ARMs高位组、中位组和低位组的蛋白表达水平均明显低于后天瘘组和正常对照组(P＜0.05)。不同畸形之间,高位组和中位组之间差异无统计学意义(P=0.11),但二者明显低于低位组(P＜0.01)。后天瘘组与正常对照组比较没有明显差异(P=0.67)
结论
1、在正常及ARMs大鼠胚胎,在E16左右,神经嵴细胞迁移到直肠区域,但直肠末端神经发育很差,随着胚胎的进一步发育,直肠区域的肠神经系统发育逐渐完善,逐渐形成神经丛状结构,生后发育更加完善。ARMs大鼠胚胎的ENS在E16时,其形态与正常组无明显差异,从E18起,可观察到发育异常的ENS,随着胎龄的加大,与正常相比,发育差异更加显著。
2、氮能神经元作为重要的神经元亚型,其发育过程是伴随神经嵴细胞的迁移而发育的,但稍迟一些,推测NO可能在肠神经系统发育过程中也起着重要作用,ARMs大鼠胚胎直肠末端区域的突触素的发育较正常组差,可能也是导致肠道动力改变的一个原因。
3、E16～E21,Wnt5a及其受体Frizzled-1及Wnt经典通路的关键因子TCF4在ARMs组的肠壁粘膜、肌层及肠神经系统的表达较正常组减弱,可能与ARMs直肠神经系统及直肠末端壁平滑肌发育不良的调控相关。
4、Wnt5a主要表达在人类直肠壁肌间神经丛部位,在肛门直肠畸形患者的直肠末端表达水平减低,推测Wnt5a在人类先天性肛门直肠畸形发生后的直肠神经系统进一步的发育中可能具有重要调节作用。
Introduction
Anorectal malformations(ARMs)are one of the most common digestive disorders, affecting 1/5000 to 1/1500 live births,one kind of malformations supervised by World Health Organization.In spite of the recent advances in operative techniques,the long-term prognosis in intermediate and high type ARMs remains poor.Some patients with ARMs continued to suffer from postoperative anal dysfunctions.The anorectal dysfunction was caused by the dysplasia of the neuromuscular in the pelvic floor. Undoubtedly,the intestinal motility is the focal point of the organism of continence. The research of the development of the enteric nervous system,intestinal muscle and nerve-muscle contact and the possible control mechanisms in ARMs has been necessary.
In previous studies,it has been shown that in the middle and high ARMs,sacral cord and the enteric nervous system of the rectal end have changed differently,but so far the course of their embryonic development and detailed pathological changes are unclear.
With the progress of molecular biology,the embryogenesis of the enteric nervous system has been illuminated initially.Vagal and sacral neural crest cells give rise to enteric neurons and glial cells throughout the entire gastrointestinal tract.In ARMs, after the defect appears,there has been a question that if the abnormity of the enteric nervous system accompanied the ARMs.The etiopathogenisis of ARMs continues poorly understood and controversial.In current study,it has been only demonstrated that ARMs resulted from hereditary factors and environmental factors in the development of embryogenesis.During the research of epidemiology and animal experiments,it is suggested that the hereditary factors play a key role in the development of ARMs.Recently,it was found that Shh、Hox、Fgf10 and Wnt5a play crucial roles during the development of the termination of digestive tract.Once the foresaid genes dysfunction,there may be ARMs,such as anal atresia,preternatural anus et al.
In recent years research has shown that the highly conserved Wnt signaling protein family in embryonic development play an important role in signaling through different molecular interactions,Wnt proteins trigger the regulation of cell growth,migration, differentiation and development,and many other complex signaling cascade reaction, Wnt5a is an important parterner in this family.Most of its researches involving human subjects are common in all systems tumor,at the same time it play an important role in the embryogenesis of intestinal tract,but the its regulation of intestinal smooth muscle and enteric nervous system during embryonic development have not been reported, especially it is not clear for the mechanism of the intestinal neuromuscular dysplasia in ARMs.
In this paper,we utilize the Wistar rat animal model of ARMs induced by ETU (ethylenethiourea).We used PGP9.5(protein gene product 9.5)and SYP (synaptophysin)and NOs(nitric oxide synthases)for evaluating the enteric nervous system and neuromuscular development of intestinal tract,at the same time we analyzed the expression of Wnt5a and its receptor Frizzled-1,TCF4 protein and mRNA in the rectum of normal and ARMs to explore the possible regulatory mechanism of enteric nervous system and gastro-intestinal smooth muscle of the embryonic period,which is the important role in participate in defecation.In addition, we used the clinical specimens in children with ARMs to explore the Wnt5a protein expression.
Materials
Animals:Wistar rats(250-300g)were provided by Medical Animal Center in Shengjing Hospital of China Medical University.
Clinical specimens:the case group:20 ARMs infants operated in our department. Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at 1 cm superior to dentate line in acquired anal fistula and rectal traumas infants.
Reagents:①The specific antibody to PGP9.5 was purchased from Abcam Corporation.②The specific antibodies to SYP,Nos,Wnt5a,Frizzled-1,TCF4 were purchased from Santa Cruz Corporation.③The UltraSensitiveTMSP kits for immuhistochmistry were purchased from MaiXin biological limited company,FuJian Province.Two-step immunohistochemical goat detection kit was purchased from Beijing Zhong Shan Golden Bridge Company;④Ethylenethiourea,ETU was purchased from Sigma-Aldrich Inc.,Germany;⑤Reverse transcriptase kit was purchased from Takara Bio-engineering Co.,Ltd;⑥Total protein extraction kit was purchased from Seitz Corporation.
Methods
Time-mated pregnant Wistar rats were gavage-fed a single dose of either 125 mg/kg of 1%ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc,Germany) or an equal dose of saline on gestational day 10(E0—sperm in vaginal smear after overnight mating).The embryos were harvested via cesarean section on E15 to E21. The embryos were fixed in 4%paraformaldehyde/0.1 M phosphate buffered saline (PBS)for 12 to 24 hours depending on their size.Then the embryos from each age group were dehydrated,embedded in paraffin and sectioned serially transversely,and sagittally at 4μm thickness.①Immunohistochemistry and fluorescence staining of PGP9.5 and SYP,NOs(embryonic development ENS markers)were to observe normal and ARMs rat embryonic development of the ENS.②Immunohistochemical staining of Wnt5a,Frizzled-1 and TCF4 were compared to roughly to position at the intestinal wall.Another part of rectum samples of rats were drawn by microscope,we evaluated the expression of Wnt5a,Frizzled-1 and TCF4 mRNA by RT-PCR and we evaluated the expression of PGP9.5,SYP,NOs,Wnt5a,Frizzeled-1,TCF4 prtein by the western blot quantitative analysis.G:BOX SYNGENE photographed gel analysis system and Kodark Digital Science 1.0 DNA analysis software to carry out semi-quantitative analysis.T-test was used to compare the differences of foresaid genes expression levels between the normal and ARMs groups,P＜0.05 indicates that the difference has statistical significance.③Immunohistochemical staining and western blot analysis of Wnt5a were used to compare the expression level in the the case group and the control group.
Results
1.The embryonic development of the ENS in normal and ARMs rat embryos
①PGP9.5 labeled neurons in the post-intestinal migration and development:
Immunohistochemical results showed that in normal control group,at E16:neural crest cells already colonized in the whole intestine and scattered positive cells were seen in the rectum,when the circular muscles has just begun to differentiate;From E16 to E18,PGP9.5-positive cells appeared in the myenteric plexus sites poradically;At E17-18,the terminal of the longitudinal intestinal muscle fiber distribution is still sparse and there were rare PGP9.5 positive cells in the submucosa;At E20:it can be observed that the yellowish-brown mass of PGP9.5-positive ganglion cells,showing cluster-like.The positive cells also can be seen in the submucosa.At E21,it become more obvious.While at the same sections observed in the same period in the intestinal wall,the development of ENS grew earlierly.In ARMs Group,at E16-17,neural crest cells at the terminal of hindgut malformation group could also be seen and there was no significant difference between the two groups,but there was no positive cells around rectal urethral fistula;At E18-19,more positive cells were still visible near the rectum terminus,but the distribution compared with the normal group is sparse,yet no positive cells were observed around the fistula;At E20-21,compared with the normal and upper colon,the density of positive cells reduced in the rectal terminus,and no obvious clusters of nerve cells were seen.The development of ENS in ARMs lags behind the normal group.
②The development of nitrergic neurons marked by NOs:
The immunofluorescence results showed the development of nitrergic neurons accompanied neural crest cell migration during embrygenesis,but it was a little later than the emergence of PGP9.5.They appeared at around E17 in the rectum.At E18,the clear positive neurons appeared between the muscles of the rectum.The cytoplasm has full of obvious green fluorescence.Before birth the development of positive neurons have become more sophisticated.But in ARMs group compared with the normal group, the growth of nitrergic neurons have lagged.The older,the greater of the difference between the two groups.
③Immunofluorescence results of SYP:
In normal control group,only scattered fluorescent display were observed in the top of the colon wall at E16,but they are very weak;From E18,strong green fluorescence can be seen clearly within in the intestinal myenteric position.It is straight into a cord-like and was more clear in the upper colon than the rectum.To E20,as well as before birth,fluorescence intensity gradually increased.But in ARMs Group,at E16-17,no fluorescence were shown in the majority of colorectal.Only a very weak visible fluorescence could be found in the upper colon.At E18,the development of SYP in the upper colon became more sophisticated,but in the blind-side near the rectum green fluorescence was still visible.Compared with the upper segment of normal colon and normal group,the intensity of fluorescence in ARMs decreased,there were no fluorescence around the fistula.
④The western blot analysis of PGP9.5,SYP and NOs
The results showed that the protein of the above-mentioned factor had significant differences between the two groups on E19-21.
2.Expression of Wnt 5a and other development-related factors in normal and ARMs rat embryos
①Immunohistochemical staining results of Wnt5a,Frizzled-land TCF4:in the normal group from the E16～E21,the expression of the above-mentioned factors were observed in the myenteric plexus,the mucosa and intestinal smooth muscle of the rectum,but the intensity of expression were different.As the development of the rectum,the expression become more increased.In ARMs Group,at E16 there were no positive cells.From E17-E21,the positive cells gradually becoming apparent,with the normal gestational age of the rectum.But compared to the same region of the normal group,the expression decreased,no positive cells appeared around the fistula.
②The western blot results of Wnt5a,Frizzled-1 and TCF4 showed on E17,there was no significant difference between the two groups;on E19-E21,the expression levels of Wnt5a,Frizzled-1 and TCF4 were higher in the normal group than in ARMs with significant difference(p＜0.05).
③RT-PCR results of Wnt5a,Frizzled-1 and TCF4 mRNA showed that there have been a significant difference between the two groups.
3.Expression of Wnt5a in normal and ARMs
In the anorectal tissues of the human of the normal case,the expression of wnt5a was mainly located in the cytoplasm of the myenteric plexus by immunohistochemistry. But in the ARMs group,we did not found positive cells in the myenteric plexus of the the blind part of the rectum espesially in high ARMs.There was no significant difference of wnt5a protein expression level between high and intermediate ARMs by western blot analysis(P=0.33).However,the expression levels of wnt5a protein in high and intermediate ARMs were significantly lower than that in low group(P＜0.05). There was no significant difference of wnt5a protein expression level between the infected recto-vestibular fistula and the control group(P=0.67).
Conclusions
1.In normal and ARMs rat embryos,on E16,neural crest cells migrate to the rectal area,but the development of rectal terminus is worse.With the further development of rat embryos,the enteric nervous system in rectal region improved gradually,plexus-like structure has gradually formed,and postnatal development became more perfect.In ARMs group,on E16,neural crest cells could also be seen in the terminal of hindgut,there was no significant difference between the two groups. From E18,ARMs group compared with the normal group,the development of ENS has lagged;the older,the greater the difference between the two groups.
2.The developmental course of the nitrergic neurons is accompanied by neural crest cell migration and development,but it is some later.It is suggested that NO may play an important role during the development of enteric nervous system.In ARMs group,there was significant difference of synaptophysin and NOs expression between the two groups in the rectal terminal region,which may lead to change to intestinal motility.
3.E16～E21,compared with in the normal group,it is in ARMs group,the expression of Wnt5a,its receptor Frizzled-1 and TCF4 which is a classical key factor of Wnt pathways in intestinal smooth muscle and nervous system became significantly weakened.It is suggested that Wnt 5a,which may play a role by the classical pathway and may be related with the developmental regulation of enteric nervous system and smooth muscle of intestinal tract in ARMs.
4.Wnt 5a may play a role in the developmental regulation of the ENS in ARMs.
引文
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