先天性巨结肠免疫组织化学的表达

目的探讨蛋白基因产物(PGP 9.5)、组织蛋白酶D(CAD)和S100蛋白(S100)在先天性巨结肠(HD)中的表达及其意义。方法采用PGP 9.5、CAD及S100对35例正常结肠和30例HD狭窄段结肠行免疫组织化学染色。利用图文分析系统观察染色表达情况。结果1.对照组结肠中,CAD对神经节细胞特异性表达。其他神经组织包括神经胶质细胞、雪旺细胞与神经纤维都不表达CAD,但对PGP 9.5和S100均阳性表达。PGP 9.5对神经节细胞强阳性着色,表现为棕褐色细胞状团块。S100对神经节细胞阴性表达,在神经丛中形成细胞状空白区。2.HD狭窄段组结肠中,免疫组织化学表达情况与对照组明显不同。CAD对肠神经组织表达缺失。PGP 9.5染色中棕褐色的细胞状团块及S100染色中的细胞状空白区均未观察到。扭曲增生的神经纤维对PGP 9.5与S100强阳性着色。S100染色的神经纤维着色强度[(146.70±10.87)灰度值]及直径(57.81±14.99)μm与对照组神经纤维的着色强度[(169.25±9.53)灰度值]及直径(23.15±5.56)μm比较,差异显著(P均<0.01)。结论PGP 9.5、CAD、S100在HD狭窄段的异常表达证实肠神经组织发育异常是HD主要病理改变。3种抗体的免疫组织化学染色标记肠神经组织对HD的诊断有重要帮助。     

先天性巨结肠免疫组织化学研究

为观察神经标志物PGP9．5和S－100蛋白在先天性巨结肠（HD）中的表达,采用一抗为抗PGP9．5和抗S－100蛋白的PAP免疫组织化学方法,探讨其在临床诊断中的意义。结果：（1）在对照组结肠壁神经丛中可见染色深浅不一的PGP9．5免疫反应阳性神经节细胞,神经纤维均匀分布在肠壁各层;神经节细胞胞体不表达S-100蛋白,表现为细胞状“空白区”。（2）HD结肠壁神经发育异常,PGP9．5和S－100蛋白免疫反应性神经纤维明显增生,分布紊乱,未见有PGP9．5阳性神经节细胞;在神经丛中,增生的S－100蛋白阳性神经纤维中偶见有细胞状的“空白”区。提示结肠壁神经发育异常是HD的主要病理生理变化,神经丛中PGP9．5阳性反应的细胞团块和S－100蛋白染色的神经丛中细胞状“空白”区,可特征性地提示神经节细胞的存在,用于HD的临床诊断敏感度高。     

S-100蛋白与Cathepsin D在先天性巨结肠诊断中的应用

目的观察S-100蛋白(S-100)和组织蛋白酶D(cathepsin D，CAD)在先天性巨结肠(Hirschspnmg’s disease，HD)中的表达。方法应用免疫组织化学染色观察25例HD患儿及10例对照组儿童结肠。结果(1)在正常对照组与HD扩张段组肠壁肌间和黏膜下层可见CAD只对神经丛中神经节细胞阳性表达，对神经纤维与神经胶质细胞均不表达。S-100染色与CAD染色相反，神经纤维与神经胶质细胞均阳性表达，神经节细胞阴性表达，表现为阳性神经丛中细胞状“空白区”。(2)在HD狭窄段组肠壁神经丛中缺乏CAD阳性表达。S-100染色神经丛中细胞状“空白区”未观察到；S-100强阳性表达的神经纤维束显著增生，扭曲呈波浪状。结论S-100和CAD的免疫组化染色对诊断HD具有重要意义。     

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.     

先天性巨结肠症及其同源病神经标志物的表达以及图形测量定量研究

目的免疫组织化学方法检测4种肠道神经标志物在正常结肠、先天性巨结肠症（HD）、神经元发育不良-B（IND-B）及神经节细胞减少症（HG）病变结肠的表达特点，并采用图像分析技术对肠道神经系统进行定量分析。方法组织蛋白酶D、蛋白基因产物9．5、S-100蛋白和外周蛋白4种抗体，对10例正常结肠、20例HD、8例IND-B型及20例FIG的患儿结肠石蜡标本切片采用过氧化酶标记的链霉卵白素免疫组织化学染色。ImageJ图像处理软件对所有数码图像（×400）中神经丛、节细胞计数以及节细胞形态进行定量测量。结果组织蛋白酶D仅在神经节细胞表达阳性，不表达于神经纤维；PGP9．5和外周蛋白在肠壁肌间、黏膜下神经丛及神经节细胞中均有阳性表达；S-100在神经节细胞中表达阴性。定量分析：HD病变肠段缺乏神经节细胞；IND-B病变肠段肌间神经节细胞与正常比较计数明显增多，节细胞直径以及细胞核直径分别为正常的1．22倍和1．34倍（P〈0．01）；HG病变肠段神经丛内节细胞数量明显减少（P〈0．05）。结论免疫组织化学染色图像分析技术使不同病变间的形态差异得到定量分析，有助于对HD和同源病的病理学研究以及临床诊断和鉴别诊断。     

全结肠巨结肠肠壁内神经标志物表达及神经支配的研究

目的通过免疫组织化学染色研究不同神经标志物在全结肠型无神经节细胞症（TCA）和常见型巨结肠（HD）肠壁内表达的差异。方法复旦大学附属儿科医院1996年1月～2005年12月间收治TCA确诊病例18例，收集结肠、回肠全层标本。对照组为常见型HD及肛门直肠畸形各10例。采用免疫组织化学染色技术对肠壁内神经标记物：S-100蛋白、外周蛋白、蛋白基因产物9．5（PGP9．5）、神经元特异性烯醇化酶（NSE）的不同程度的表达进行比较。数据采用t检验分析。结果PGP9．5、S-100蛋白、NSE和外周蛋白在对照组和TCA组、HD组近端肠壁内表达差异无统计学意义（P〉0．05）；移行段内可见少数成熟和不成熟神经节细胞，神经标志物表达低于HD组（P〈0．01）；18例TCA远端肠壁内均无神经节细胞，粗大的神经干较HD少见，神经标志物表达明显低于HD组（P〈0．01），以PGP9．5最明显。结论研究表明TCA和常见型HD病变肠段中存在明显的神经标记物阳性表达差异，提示全结肠巨结肠肠壁内神经支配异常不同与常见型巨结肠。     

AQP1在先天性巨结肠肠壁的表达及其意义

目的：本研究应用免疫组织化学染色方法,观察先天性巨结肠症（Hir －schsprunffs dis-ease,HD）患儿有神经节细胞肠段、无神经节细胞肠段及非 HD 小儿肠壁（对照组）中水通道蛋白—1（AQP1）的表达结果,探讨 AQP—1在先天性巨结肠症的表达意义及其在 HD 发病机理中可能起到的作用。方法收集江西省儿童医院小儿外科2009—2014年经手术切除的 HD 肠组织标本65例,包括扩张段与痉挛段;将21例年龄与之相仿的非 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 HE 染色和 AQP1、组织蛋白酶 D 和 S100的免疫组织化学染色,通过计算机成像系统照相存盘,分别判定 AQP1在各组肠组织黏膜下、肌间神经丛中阳性细胞染色面积的百分比,所得数据用 SPSS 19．0统计软件包进行处理分析。结果①对照组、HD 的扩张段经 HE 和组织蛋白酶 D 和 S100免疫组化染色,均证实神经节细胞存在;痉挛段未见神经节细胞（图1,2）;②对照组、HD 痉挛段、扩张段肠组织中见 AQP1表达于所有血管内皮细胞;③在正常结肠、HD 扩张段肠组织黏膜下丛、肌间神经丛中,神经节细胞和雪旺细胞均可见 AQP1呈阳性表达,HD 痉挛段相应部位 AQP1表达大多呈阴性（90．2％）,仅少量呈弱阳性表达;④半定量分析：AQP1在神经丛中阳性染色评分,HD痉挛段评分低于 HD 扩张段评分,二者间差异具有统计学意义（P ＜0．01）。结论AQP1在 HD 痉挛段和正常结肠、HD 扩张段肠壁神经丛的表达有显著差异,我们猜测在肠道神经系统发育过程中,AQP1对神经节细胞迁移具有一定的调控作用,在 HD 的发病机理中扮演了一定角色并可能成为一种新的诊断 HD 的神经标志物。     

小儿神经节细胞减少症肠壁肌间神经丛形态定量研究

目的研究神经节细胞减少症的形态定量特征。方法对15例小儿节细胞减少症、14例无神经节细胞症及14例正常神经分布的肠壁组织进行常规石蜡切片,并作HE染色、NSE及S-100免疫组织化学染色,在光学显微镜图像分析系统下对三组标本的肠壁肌间神经丛组织进行形态定量测定并对各组测量参数进行统计学的分析比较。结果节细胞减少症肠肌神经丛中每毫米肠壁的平均神经节细胞数是1.52个(仅为正常组的24.3%),其每毫米肠壁的平均神经丛面积是(5.61×103)μm2(为正常组的17.3%),无神经节细胞症神经丛面积亦有明显减少,数量更少,NSE、S-100免疫组织化学染色可帮助正确辨别神经节细胞。结论肠壁肌间神经丛中神经节细胞数量减少的同时伴有神经丛面积的减小是节细胞减少症的特征性改变,肠肌神经丛的形态定量测定可作为神经节细胞减少症的客观诊断依据,彻底切除节细胞减少的肠段对于根治此症具有重要意义。     

梅克尔憩室肠壁神经和平滑肌细胞的分布研究

目的 了解梅克尔憩室(MD)肠壁神经和平滑肌病理组织学改变,探讨MD及其并发症的发病机制.方法 收集2004年1月至2006年7月手术切除的44例MD标本,另取6例死于与肠道和神经系统无关疾病的足月新生儿尸检小肠标本为对照,采用常规HE染色计数肠壁肌间神经丛和丛内神经节细胞数,S-100蛋白、神经元特异性烯醇化酶(NSE)和平滑肌肌动蛋白(α-SMA)采用免疫组织化学染色(SABC法).结果 MD的肠壁肌间神经丛密度与对照组相比无统计学意义(P＞0.05),但丛内神经节细胞数、单个肌间神经丛的平均面积及其平均神经丛长轴长度均较对照组减少,而平均光密度值比对照组增加.肌间神经丛的平均面积与其平均吸光度呈负相关(r=一0.215,P＜0.05),且上述病理改变以症状组和伴有异位胃黏膜的MD尤为突出.MD的肠壁环肌层和纵肌层α-SMA表达减低,环肌层肥厚;而距憩室5 cm处肠壁肌间神经丛的各项指标的改变均减轻,但是环肌层仍明显肥厚,α-SMA染色亦减轻.结论 肠壁神经丛和平滑肌细胞的变化可能参与了MD及其并发症的发生.     

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。     

PERIPHERIN: A Novel Marker for the Immunohistochemical Study of Malformations of the Enteric Nervous System

Peripherin is a 57-kD type III intermediate filament that is a specific marker for peripheral neurons, including enteric ganglion cells (GCs). Hence antibodies to peripherin may be used to demonstrate abnormalities of the enteric nervous system (ENS). Serial longitudinal histologic sections of formalinfixed paraffin-embedded colons from 15 patients were immunostained for peripherin, neuron-specific enolase (NSE), neurofilaments, S-100, and synaptophysin. Ten patients had variable degrees of colonic aganglionosis (Hirschsprung's disease), three were premature infants, and two were controls. Peripherin labeling yielded the highest number of recognizable GCs. Overall, 56%, 78%, and 80% of the peripherin-positive GCs in the myenteric plexus and 55%, 75%, and 73% of the peripherinpositive GCs in the submucosal plexus were identified by staining for neurofilaments, NSE, and S-100, respectively. Intramucosal GCs were detected in 4 of 10 cases of Hirschsprung's disease (HD), none of which had been evident by routine histology. The other neuronal markers were less specific for intramucosal GCs than peripherin, because they also labeled enterochromaffin cells. Peripherin immunohistochemistry also allowed exact quantification of GC density expressed as GCs/mm colon, which is important for the diagnosis of HD-related disorders. In three cases of HD the GC density of the transition zone was markedly elevated compared with more proximal ganglionic bowel segments, consistent with neuronal intestinal dysplasia type B, and two cases of HD showed low GC density within the transition zone. Hence peripherin immunolabeling may prove to be a valuable aid in the diagnosis and classification of congenital malformations of the ENS.     

新生儿全结肠巨结肠阑尾壁内神经元标记物和神经支配分布的评价

目的 对新生儿全结肠巨结肠(TCA)阑尾神经分布的免疫组化进行研究,比较TCA阑尾和正常新生儿阑尾肠神经分布的差异.方法 收集本院新生儿TCA患儿阑尾10例,将常见型HD近端正常乙状结肠组织和因肠旋转不良行阑尾切除术的10例新生儿设为对照组.采用免疫组化染色技术对S100蛋白、外周蛋白和蛋白基因产物9.5(PGP9...     

先天性巨结肠RET蛋白免疫组化研究

目的 了解RET蛋白与先天性巨结肠(HD)组织改变之间的关系,以探讨HD的发病机理.方法 利用抗RET蛋白的三种抗体,以及抗磷酸酪氨酸抗体采用免疫组织化学方法,对42例散发性HD肠组织中RET蛋白表达情况进行研究,10例正常结肠作对照。结果 抗RET蛋白的三种抗体在HD扩张段、移行段、对照组均显强阳性染色,P>O.05;在狭窄段大多数表现阴性染色,少数可见强阳性或弱阳性染色,与前三者比较PO.05;HD移行段与狭窄段大多数呈现阴性或弱阳性染色,P>O.05。前两者与后两者比较P相似文献   

Significance of immunohistologic methods in the differential diagnosis of solid tumors in childhood

We have investigated 56 histologic and 13 cytologic specimens of malignant round cell tumors of childhood. The immunohistological detection of intermediate filament polypeptides, neuron specific enolase (NSE) as well as leukocyte common antigen (LCA) allows the histological classification of such tumors. Neuroblastomas demonstrate a positive reaction with antibodies to neurofilaments and NSE independent of differentiation. Rhabdomyosarcomas could be labeled by the desmin antibody, while Ewing sarcomas, malignant lymphomas as well as nonmuscular sarcomas only express vimentin. In nephroblastomas the intermediate filament specific antibodies reveal expression of keratin and vimentin in blastema cells, while tubules are only labeled by the keratin antibody. In undifferentiated nephroblastomas, which lack formation of tubules blastema cells are keratin negative and vimentin positive. Thus antibodies to intermediate filaments, LCA and NSE seem to be useful tools to distinguish the so called "round cell tumors" of childhood.     

神经细胞特异性烯醇化酶和S_（100）蛋白在先天性巨结肠症诊断中的应用

对疑有先天性巨结肠症的６１例患儿行直肠粘膜吸引活检，标本应用神经细胞特异性烯醇化酶（ＮＳＥ）及Ｓ（１００）蛋白免疫组织化学法染色检查，诊断为先天性巨结肠症的３３例，全部经手术后全层病理检查证实，诊断符合率达１００％。研究结果表明，ＮＳＥ结合Ｓ（１００）蛋白的免疫组化染色对直肠粘膜下神经丛中神经节细胞不同侧面的特征性表达，对先天性巨结肠的诊断具有重要价值，尤其在新生儿期。     

A Case of Small Round Cell Tumor of the Thoracopulmonary Region with Myogenic and Neurogenic Elements

We here report a unique case of a young boy with an intrathoracic tumor which consisted of neurogenic and myogenic elements. The initial pathological diagnosis was alveolar rhabdomyosarcoma. The tumor tissue from surgical resection was composed of three parts, each showing a different histological appearance, i.e. a monotonous small cell area, an alveolar area, and an area consisting of pleomorphic rhabdomyoblasts. The small round cells in the monotonous area were immunoreactive with the antibodies for Leu7, neuron-specific enolase (NSE), neurofilament proteins (NFP), and β₂ microglobulin, but not with the antibody for desmin. These cells also had dense core granules. The tumor cells in the alveolar area were immunoreactive with the antibodies for Leu7 and desmin, but not with the antibody for NFP. Pleomorphic rhabdomyoblasts were immunoreactive with the antibody for desmin, but not with the antibodies for Leu7 and NFP. These findings imply that this tumor consisted of neurogenic and myogenic elements and is considered to be a special type of rhabdomyosarcoma.     

The innervation of human bowel mucosa and its alterations in Hirschsprung's disease using a whole-mount preparation technique

 The innervation of the human bowel wall and its structural and functional changes in Hirschsprung's disease (HD) are well-recognised. The luminal surface of the bowel acts as a multifunctional barrier, and modifications in its physiochemical properties can result in serious complications such as enterocolitis (EC). The whole-mount preparation (WMP) technique produces a three-dimensional (3D) picture to better demonstrate the neuronal networks and the relationship of branching and interconnecting nerve fibres to each other. The aim of this study was to investigate the innervation of the mucosal layer in normal and HD bowel using a WMP immunohistochemistry technique in order to better understand the pathophysiology of HD. Full-thickness bowel specimens were collected from 9 HD patients at pull-through operation. Normal control small- and large-bowel specimens were collected from 10 patients at the time of bladder augmentation. Suction rectal biopsies from 8 patients with chronic constipation and 2 patients with HD were also included in this study. A WMP of the mucosal layer was made and stained with various neuronal markers (S100, PGP 9.5, and LlCAM) using fluorescein immunohistochemistry. PGP 9.5, S100, and LlCAM immunofluorescence staining of the normal mucosa demonstrated a characteristic 3D meshlike neuronal network of uniform thickness surrounding the crypts. In the aganglionic bowel S100, PGP 9.5, and LlCAM-positive meshlike networks were replaced by thick nerve trunks in the muscosa without any interconnecting network. The present study demonstrates for the first time the 3D morphology of mucosal innervation in normal and aganglionic bowel. The WMP technique clearly demonstrated that the mucosal innervation in HD is morphologically abnormal, and this may adversely influence secretory and absorptive functions of the bowel. WMPs using suction rectal biopsy specimens may be a useful additional technique to diagnose HD.     

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.     

巨结肠类缘病病理形态学诊断的探讨

目的采用NSE和S-100蛋白免疫组化染色方法,探讨巨结肠类缘病的病理形态学特征,提高其诊断率。方法对97例行巨结肠根治术后的病变肠段进行常规HE染色,并对其中21例采用NSE和S-100蛋白免疫组化,观察肠神经元及神经节细胞形态和数量的变化并与正常组进行比较。结果巨结肠类缘病(alliedHD)标本的神经元与神经节细胞数与正常组及巨结肠症(HD)比较差异有显著性(P<0.01)。结论巨结肠类缘病的诊断主要根据病理特征而定,在HE染色基础上结合NSE及S-100蛋白免疫组织化学染色能提高确诊率。