胶质细胞源性神经营养因子、Cajal间质细胞和缝隙连接蛋白43在先天性巨结肠中的表达分布研究

摘要 目的 探讨胶质细胞源性神经营养因子（GDNF） mRNA、Cajal间质细胞（ICC）及缝隙连接蛋白43（Cx43）与先天性巨结肠(HD)发病的关系 。方法 依据纳入和排除标准选择2006年8月～2007年9月经病理诊断为HD的患儿，取手术切除结肠标本作为HD组，根据取材位置不同分为狭窄段（又分为短段型和常见型）、移行段和扩张段亚组。应用半定量RT PCR及免疫组化技术检测结肠组织GDNF mRNA水平和ICC、Cx43的分布，以肠套叠患儿手术结肠标本作为对照组。结果 研究期间HD组纳入42例，对照组纳入5例。①狭窄段亚组GDNF mRNA表达较扩张段亚组和对照组明显降低（P＜0.05）；扩张段亚组和对照组GDNF mRNA表达差异无统计学意义（P＞0.05）。狭窄段亚组中短段型较常见型GDNF mRNA表达低（P＜0.05）。② ICC在对照组和扩张段亚组主要分布于黏膜下丛和肌间丛，呈现连续性分布，相互连接形成网络状结构。ICC在狭窄段亚组结肠组织内的分布显著减少或消失，与对照组和扩张段亚组差异有极显著统计学意义（P＜0.001），肌间丛的网络状结构完全破坏，残存ICC形态异常；移行段亚组结肠组织内ICC的分布较对照组和扩张段亚组减少（P＜0.05），但较狭窄段亚组显著增加（P＜0.001），其形态部分接近正常，但肌间丛缺乏连续性分布，未能形成正常的网络状结构。③狭窄段亚组肠壁肌层内Cx43表达缺失，各层中几乎未见Cx43表达。移行段亚组肠壁环肌层与纵肌层交界处Cx43有中等强度表达。扩张段亚组肠壁环肌层与纵肌层交界处Cx43呈强阳性分布，黏膜下丛和肌间丛未见或少见Cx43表达。结论 GDNF mRNA表达异常、ICC分布减少和形态异常、Cx43表达缺失或减少和缝隙连接结构的破坏可能引起细胞间物质和电信号的传递障碍，而导致HD发病的原因之一。     

Cajal间质细胞在先天性巨结肠不同肠段的分布

目的 检测先天性巨结肠(HD)患儿结肠手术标本组织Cajal间质细胞(ICC)的分布及形态,分析并统计其在不同肠段的分布数量,为研究HD的发病机制和功能障碍提供必要的实验依据.方法 收集42例经病理诊断为HD的标本.男33例,女9例;年龄2个月～10岁.实验标本均为散发病例,全部取材经组织病理学检测符合HD诊断.应用免疫组织化学技术检测42例HD患儿手术标本狭窄段(狭窄段组)、移行段(移行段组)和扩张段(扩张段组)组织ICC的分布及数量,及5例肠套叠患儿(对照组,男4例,女1例;年龄30 d～8岁)结肠手术标本组织ICC的分布及数量.结果 1.对照组结肠组织ICC主要分布于黏膜下丛(IC-SM)和肌间从(IC-MY),在环肌层和纵肌层(IC-IM)也有分布.IC-SM、IC-MY在切面上呈连续性分布,相互连接形成网络状结构.2.结肠狭窄段组结肠组织ICC的分布显著减少或消失,较对照组和扩张段组均有显著性差异(Pa＜0.001),IC-MY的嘲络状结构完全破坏,残存ICC形态异常;另狭窄段组2例ICC形态、分布及数量与对照组或扩张段组比较均无显著性差异;移行段组结肠组织内ICC的分布较对照组和扩张段组均显著减少(Pa＜0.05),较狭窄段组显著增加(P＜0.001),其形态部分接近正常,但IC-MY缺乏连续性分布,未能形成止常的网络状结构;扩张段组与对照组结肠组织内ICC分布相当,无显著性差异(P＞0.05).结论 HD患儿结肠的不同肠段ICC的数量、形态、分布不同.在狭窄段IC-MY偶见残存的ICC,其形态变钝、突起减少或消失.ICC分布减少、形态异常可能是HD胃肠动力障碍的原因之一.     

先天性巨结肠肠壁Cx43蛋白和Cajal间质细胞的分布

目的：观察缝隙连接蛋白43（Cx43）及Cajal 间质细胞(ICC)在先天性巨结肠(HD)肠壁中的分布,探讨Cx43和ICC与HD发病的关系。方法：运用免疫组织化学方法观察42例经病理诊断为HD的肠壁标本内各层之间Cx43蛋白和ICC的分布情况,其中男33例,女9例（年龄2个月至10岁）,实验标本均为散发病例,全部取材经组织病理学检测符合HD诊断,包括常见型30例,短段型12例,并取5例肠套叠患儿（30 d至8岁,男4例,女1例）作为对照组。结果：HD狭窄段(无神经节细胞肠段)肠壁肌层内Cx43和ICC表达缺失,各层中几乎未见Cx43表达和ICC分布,与HD扩张段（有神经节细胞肠段）及正常对照组之间的差异有显著性意义(P<0.01)。HD移行段肠壁Cx43和ICC有中等强度表达,与扩张段、狭窄段之间差异有显著性(P<0.01)。HD扩张段肠壁Cx43和ICC呈强阳性分布,黏膜下层和纵肌层未见或少见Cx43表达。HD扩张段和正常对照组肠壁内Cx43和ICC表达差异无显著性。结论：Cx43表达缺失或减少、缝隙连接结构的破坏及ICC结构、分布异常,导致细胞间物质、电信号的传递障碍,可能是HD发病的原因之一。［中国当代儿科杂志,2009,11（3）：213-216］     

先天性巨结肠HuD蛋白表达的实验研究

目的 HuD是神经元胚胎发育中生长锥、突起形成延伸及成熟神经元存活所必须的蛋白,其在肠神经系统发育及先天性巨结肠发生中作用机制尚不明确,国内外均未见相关报道。本文探讨HuD蛋白在先天性巨结肠不同部位肠壁组织中的表达,以了解HuD在先天性巨结肠发病中作用机制。方法分别取15例先天性巨结肠患儿手术切除的病变段、移行段、扩张段结肠壁全层及5例人正常结肠组织,采用免疫组织化学、Western免疫印迹分析对不同部位肠壁HuD蛋白表达进行检测,利用图象分析系统及统计软件进行结果分析。结果先天性巨结肠狭窄段粘膜下和肌间抗HuD蛋白抗体染色缺如,移行段显色程度及表达量都明显减少于扩张段和正常对照组（P〈0．01）;狭窄段和移行段的HuD蛋白水平明显少于扩张段和正常段（P〈O．01）。结论HuD在先天性巨结肠病变段粘膜下、肌间神经丛不表达提示HuD蛋白减少可能是使肠神经系统胚胎发育或成熟神经元存活出现障碍而导致先天性肠无神经节细胞的原因之一。     

神经营养因子3及其受体在先天性巨结肠中的表达

目的比较NT-3及其受体TrkC在先天性巨结肠（HD）中不同节段的表达情况，以探讨HD的发病机制。方法收集我院20例经巨结肠根治术治疗的HD标本。以正常段肠管组织为对照组，以痉挛段和移行段肠管组织为实验组，应用Western blot方法，对标本内的NT-3及TrkC蛋白表达进行半定量分析比较。结果NT-3及TrkC在正常段肠壁表达明显，在狭窄段肠壁无表达，两者之间的差异具有统计学意义（P〈0．05）。在移行段肠壁内，NT-3没有表达，TrkC表达明显减弱，与正常段比较，差异具有统计学意义（P〈0．05）。结论HD痉挛段肠壁内NT-3和TrkC的异常表达，可能与HD的发生有关。     

SOX10mRNA在先天性巨结肠肠壁中的表达

目的研究性别决定区Y基因相关高可变区基因10（SRY related high mobilitvgroup—BoX gene10，SOX10）在先天性巨结肠（Hirschsprung’s disease，HD）肠壁中的表达情况，以进一步了解HD在分子基础上的发病机制。方法分别取12例先天性巨结肠病例的手术标本狭窄段、移行段及扩张段，随机取12例非巨结肠手术病例（如结肠造瘘或关瘘手术）作为对照组，提取平滑肌组织总RNA，应用逆转录多聚酶链反应（RT-PCR）扩增目的基因和看家基因片段，观察病变段和正常段SOX10mRNA的表达，并与看家基因（B—actin）在病变段和正常段的表达比较，进行统计学分析。结果SOX10mRNA在HD患儿痉挛段呈低表达，在扩张段及正常对照组呈高表达。痉挛段SOX10mRNA的表达量与移行段、扩张段及正常对照组比较，差异均有统计学意义；而移行段、扩张段及对照组比较，差异无统计学意义。结论HD患儿结肠SOX10mRNA的异常分布显示SOXIO基因是出生后肠神经系统维持正常功能所必需的，SOX10mRNA表达减少可引起肠管痉挛，肠腔狭窄，造成肠功能障碍。     

先天性巨结肠患儿肠道内Smoothelin的表达

目的 本研究旨在探讨先天性巨结肠(Hirschsprung's disease,HD)肠壁中Smoothelin(SM)的变化和临床意义. 方法 自2007年1月至2013年1月,随机选取53例经病理证实先天性巨结肠患儿术中切除的肠段样本(狭窄段即无神经节细胞段、移行段和扩张段即有神经节细胞段).男33例,女20例,年龄3月龄~4岁(平均年龄1.3岁).取材分别为狭窄段、移行段和扩张段肠壁组织各1.0cm3大小,迅速用pH7.4的PBS液冲洗,不同浓度的蔗糖溶液中脱水,-80℃保存.同时留取相同部位的肠壁组织做组织HE染色病理学检查外,辅以PGP9.5、神经元特异性烯醇化酶(NSE)及S-100免疫组织化学染色,证实符合HD诊断.采用RealTime-PCR和免疫蛋白印记法方法从RNA和蛋白水平对SM在HD患儿肠道组织内的表达进行检测,并分析其参与HD发病过程的可能机制. 结果 SM mRNA表达在先天性巨结肠病扩张段、移行段和狭窄段分别为(27.13±16.44)×10-3、(21.60±10.02)×10-3和(19.66±9.92)×10-3;蛋白表达分别为43.13±12.44、36.83±9.43、23.63±4.97.移行段和狭窄段SM mRNA和蛋白均低于扩张段(P<0.05).结论 SM可能参与HD的发病过程.     

先天性巨结肠肠壁S-100蛋白的表达和神经元型一氧化氮合酶阳性神经的异常分布

目的探讨先天性巨结肠(HD)肠壁S-100蛋白的表达和神经元型一氧化氮合酶(nNOS)阳性神经的异常分布。方法对25例HD的病变肠段行S-100蛋白和nNOS染色,比较其染色结果和分布特点。结果扩张段及狭窄段结肠壁S-100在神经丛内的神经纤维、施万细胞及周围细胞均有阳性表达,而在神经节细胞则无阳性表达;扩张段nNOS阳性神经节细胞和神经纤维形态分布良好,狭窄段无nNOS阳性神经节细胞,散在分布细小nNOS阳性神经纤维以黏膜层为多。扩张段及狭窄段中神经纤维数密度无明显差异(t=1.251 P>0.05),但前者神经纤维直径明显较后者大(t=4.492 P<0.01)。结论神经分布异常是HD发病的机制之一,S-100与nNOS的协同检测可作为HD明确诊断的重要手段。     

The expression and significance of calretinin in the colon of patients with Hirschsprung's disease

目的 本研究应用免疫组织化学染色方法,观察对照组肠壁、先天性巨结肠症(Hirschsprung's disease,HD)患儿有神经节细胞段和无神经节细胞段肠壁神经组织中钙视网膜蛋白(Calretinin,CR)的表达结果,目的在于了解HD的病理生理改变以及寻找诊断先天性巨结肠的简便有效的方法.方法 收集苏州大学附属儿童医院小儿外科2005至2008年手术切除HD标本54例,包括HD扩张段与痉挛段.以15例年龄与之相符的无HD患儿的手术切除结肠标本作为对照组.分别对HD痉挛段、扩张段、对照组肠壁组织切片进行CR的免疫组织化学染色和HE染色,计算机成像系统照相存盘,用图像分析软件(Image-Pro-Plus)分别判定CR在HD扩张段与痉挛段神经丛中阳性染色面积百分比.所得数据用SPSS 12.0统计软件包进行处理分析.结果 ①无论是HE或免疫组化染色HD的扩张段神经节细胞皆存在,肌层神经纤维有不同程度的排列改变,神经节细胞大小不等;痉挛段均未见神经节细胞;②在正常结肠及HD扩张段肠壁免疫组化染色可见钙视网膜蛋白在肌间及黏膜下神经丛中对神经节细胞呈强阳性表达,神经纤维也呈阳性反应;而HD痉挛段肠壁的免疫组化染色则可见钙视网膜蛋白在肌间神经丛及黏膜下神经丛大多表达阴性(90.7％),仅少量呈弱阳性表达(9.3％);③定量分析:CR分别在HD痉挛段之间神经丛中阳性染色面积百分率(0.00665±().00387)与其在HD扩张段神经丛中阳性染色面积百分率(0.26483±0.14626)存在差异,有显著统计学意义(P＜0.01).结论 钙视网膜蛋白免疫组化染色可很好的显示正常结肠及HD扩张段肠壁的神经节细胞及神经纤维,而在HD痉挛段该指标免疫组化染色结果呈阴性或弱阳性表达.钙视网膜蛋白可能作为诊断HD的神经标志物之一.     

胶质细胞源性神经营养因子在先天性巨结肠中的表达

目的 检测胶质细胞源性神经营养因子(GDNF)基因mRNA在先天性巨结肠(HD)患儿不同肠段中的表达.方法 收集42例经病理诊断为HD的标本(每例患儿的无神经节细胞肠段和神经节细胞正常肠段全层肠组织).男33例,女9例;年龄2个月～10岁,实验标本均为散发病例,全部取材经组织病理学检测符合HD诊断,包括常见型30例,短段型12例.应用半定量RT-PCR技术检测42例HD患儿手术标本狭窄段、移行段和扩张段组织中GDNF mRNA水平及5例肠套叠患儿(对照组30 d～8岁;男4例,女1例)结肠手术标本组织中GDNF mRNA水平,分析并统计其与临床病理特点之间的关系.结果 对照组肠管GDNF mRNA均表达阳性(100%);42例新鲜标本中,狭窄段11例GDNF mRNA表达阳性(26.2%),移行段15例GDNF mRNA表达阳性(35.7%),扩张段40例GDNF mRNA表达阳性(95.2%).与扩张段和对照结肠比较,狭窄段结肠组织中GDNF mRNA水平明显降低,具有显著性差异(Pa＜0.05);扩张段结肠和对照组GDNF mRNA水平相当,无显著性差异(P＞0.05).短段型较常见型狭窄段组GDNF mRNA表达低,有显著性差异(P＜0.05).结论 HD狭窄段和移行段肠管GDNF mRNA表达异常; GDNF mRNA与HD病理类型有相关性;提示GDNF可能与HD的发病有关.     

先天性巨结肠RET基因转录水平的研究

目的 探讨ＲＥＴ基因的表达情况与先天性巨结肠发生的关系。方法 采用ＲＴ－ＰＣＲ技术对１２例先天性巨结肠的狭窄段,移行段及扩张段分别做ＲＥＴ基因的表达,并以Ｇ３ＰＤＨ作内参标,观察ＲＥＴ基因的表达情况。结果 发现ＲＥＴ基因的表达从扩张段→移行段→狭窄段是逐渐减低的。结论 提示ＲＥＴ基因与先天性巨结肠的发生有一定的关系。     

Mast-cells distribution and colonic mucin composition in Hirschsprung's disease and intestinal neuronal dysplasia

 Intestinal neuronal dysplasia (IND) is a malformation of the enteric plexus that clinically resembles Hirschsprung's disease (HD). In HD, the aganglionic bowel is characterized by the presence of hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic nerve fibers, whereas IND type B (IND-B) is characterized by dysplasia of parasympathetic nerves, hyperganglionosis, and gaint ganglia. The aims of this study were to investigate the relationship between the distribution of mast cells (MC) and abnormal neuronal innervation and the impact of abnormal neuronal innervation on colonic epithelial differentiation in relation to the typical innervation abnormalities seen in HD and IND. Full-thickness rectal-biopsy or resected surgical specimens from 15 patients (7 HD, 4 IND, 4 control) were examined by conventional hematoxylin-eosin, periodic acid-Schiff Alcian blue (PAS-AB), toluidine blue staining. The aganglionic and IND-B segments had larger numbers of MC in all layers than the ganglionic and control segments. There was a close relationship between the hypertrophic nerve fibers and the distribution of the MC. In contrast to the aganglionic segments, the mucin composition of the IND-B segments was normal. This finding suggests that innervation anomalies do not reflect epithelial differentiation to the same extent. Accepted: 10 March 2000     

先天性巨结肠c-kit+肠间质细胞分布的免疫组织化学研究

目的：了解先天性巨结肠（HD）肠壁内神经节细胞与肠间质细胞（ICCs）分布的关系。以进一步研究HD的发病机制。方法：采用兔抗人多克隆c-kit抗体，通过免疫组化SABC法观察了58例HD患儿及12例正常儿结、直肠壁内c-kit^ ICCs的分布情况，患儿年龄3个月－10岁，男46例，女12例，短段型HD7例，常见型30例，长段型16例，全结肠型5例。结果：对照组肠肌层内及肌间神经节周围分布有中等量至大量的c-kit^ ICCs，并连接成网络状结构，而在肌间神经节和粘膜下神经内均无c-kit免疫反应性表达，在HD有神经节细胞段肠壁内c-kit^ ICCs的数目和分布与正常对照组无显著差异（P＞0．05），而在HD有神经节细胞段及正常对照组相比，差异有显著性意义（P＜0．01），结论：c-kit^ ICCs的异常分布可能与HD肠动力障碍的发生机制有关。     

Interstitial cells of Cajal are normally distributed in both ganglionated and aganglionic bowel in Hirschsprung’s disease

Surgery for Hirschsprung's disease is often complicated by post-operative bowel motility disorders. The impact of intestinal neural histology on the surgical outcome has been previously studied, but no information is available concerning the influence of the distribution of interstitial cells of Cajal (ICC) on these complications. These cells are considered to be pacemakers in the gastrointestinal tract. The aim of this study was to assess the distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease and confront these results with the clinical outcome. Using immunohistochemistry for light microscopy, we compared the pattern of distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease with that in normal colon. We correlated these results with the corresponding neural intestinal histology determined by CD56 and the protein gene product 9.5 immunohistochemistry. The distribution of ICC in the proximal segment of resected bowel is identical to that of normal colon, regardless of normal or abnormal colon innervation. ICC distribution does not seem to contribute to post-operative bowel motility disorders in patients operated for Hirschsprung's disease     

Universal distribution of c-kit-positive cells in different types of Hirschsprung's disease

Interstitial cells of Cajal (ICCs) have been reported to play the role of a pacemaker in regulating bowel motility. The relationship between neurons and ICCs, however, remains unclear. Hirschsprung's disease (HD) is an ideal model for investigating this relationship. The operated specimens obtained from 6 short and 3 long segment aganglionosis patients and 3 controls were used as the subject materials in this study. ICCs were immunohistochemically identified using a specific antiserum c-kit, a tyrosine kinase receptor expressing ICCs. Nitrergic nerves were demonstrated by NADPH-diaphorase (NADPH-d) histochemistry. C-kit immunohistochemistry was also combined with protein gene product 9.5 (PGP 9.5; as a general neuronal marker). In the normoganglionic segment of HD, numerous c-kit-positive cells and NADPH-d positive neurons were found in the proper muscle layer, including Auerbach's plexus. In the oligoganglionic segment, the number of c-kit-positive cells and NADPH-d neurons slightly decreased. In the inner border of the circular muscle layer (IBCM), the c-kit-positive cell networks and NADPH-d activities remained in short segment cases, while both of them were absent in the long segment cases. In the aganglionic segment, c-kit positive cells were present universally but the number of them was slightly decreased in the proper muscle layer. The c-kit-positive cell networks of IBCM were seen where extrinsic neurons were present, while they were almost completely absent where extrinsic neurons were absent in the proximal zone of the long segment cases. C-kit positive cells were present universally in the oligoganglionic as well as aganglionic segments of HD. The distribution and properties of c-kit positive cells were related to the presence of extrinsic neurons in aganglionic segment. Based on these findings, aperistalsis is considered not to relate with c-kit positive cells, and c-kit positive cells are not supposed to have a neurogenic origin and can develop without neurons, however the lack of enteric neurons may influence the full differentiation of ICCs.     

神经生长因子受体在先天性巨结肠表达的意义

目的 探讨神经生长因子受体（ＮＧＦＲ）在先天性巨结肠（ＨＤ）中的分布情况及其意义。方法 应用ＮＧＦＲ免疫组化方法对１０例３周￣２岁的ＨＤ患儿及８例对照组患儿结肠进行染色观察。结果 丰富的ＮＧＦＲ染色阳性神经纤维分布于正常结肠环肌层及粘膜下层,少量分布于纵肌层,ＮＧＦＲ染色阳性神经元分布于肌间神经丛及粘膜下神经丛;ＮＧＦＲ染色阳性神经纤维在ＨＤ无肌间神经节细胞肠段肌层及粘膜下层内明显减少或缺如,而肌     

Histopathological differences between recto-sigmoid Hirschsprung's disease and total colonic aganglionosis

Total colonic aganglionosis (TCA) is a severe form of ultra long Hirschsprung's disease with an incidence of 2 to 14% among all forms of intestinal aganglionosis. C-kit positive interstitial cells of Cajal (ICCs) are pacemaker cells that play a key role in the motility function of the bowel. The aim of this study was to compare the innervation and ICCs distribution in total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from four children with TCA, ten with recto-sigmoid HD and four controls. Single immunohistochemistry using peripherin, neuronal nitric oxide synthase (nNOS) and c-kit antibody was performed and analysed in light microscopy. Additionally, whole-mount preparations were stained using anti c-kit immunohistochemistry and NADPH-diaphorase. In the ganglionic bowel of TCA, recto-sigmoid HD and control patients there was a strong nNOS and peripherin immunoreactivity (IR) in ganglia of myenteric and submucous plexus and in thin nerve fibres in the muscle layers. In the TCA there was weak or lack of nNOS IR in the sparse, short nerve trunks of the myenteric and submucous plexuses and muscle layers, whereas nNOS weakly positive nerve trunks were observed in the recto-sigmoid HD bowel. Peripherin IR was markedly reduced in the TCA specimens compared to recto-sigmoid HD. In the TCA specimens there was a lack of ICCs-MY in the smooth muscle layer in all the specimens, whereas in the recto-sigmoid aganglionic bowel ICCs-MY were markedly reduced. Whole-mount preparations showed lack of ICCs-MY and a markedly reduced number of NADPH-positive nerve trunks in TCA. Our findings demonstrate clear histopathological differences between rectosigmoid Hirschsprung's disease and total colonic aganglionosis.     

血红素氧合酶-2基因表达与先天性巨结肠发病关系的初步探讨

目的：探讨血红素氧合酶－2（HO－2）基因表达水平与先天性巨结肠症（HD）发病机制的关系。方法：2001年8月－2002年1月收治的15例HD患儿（男12例，女3例），年龄21d-6岁（平均1．8岁），其中短段型3型，常见型9例，长段型3例，标本于术中发别取自狭窄段和正常近心端肠段，提取肠组织总RNA后利用逆转录－聚合酶链反应（RT－PCR）技术进行HO－2mRNA表达的半定量分析。结果：发现HO－2 mRNA在正常近心肠段均有正常表达，狭窄段则表达缺失，结论：HD患儿狭窄肠段HO－2表达缺失，提示HO－2基因表达缺可能与HD的发病有关。