慢传输性便秘乙状结肠Cajal间质细胞分布的免疫组化观察

目的：Cajal间质细胞（interstitial cells of Cajal，ICC）对结肠动力有重要的调控作用。研究旨在初步观察ICC在STC乙状结肠的分布情况。方法：用ckit免疫组织化学技术观察了12例慢传输性便秘（slow transit constipation，STC）患者和8例对照组患者乙状结肠ICC形态和分布情况，并进行半定量分析。结果：ICC广泛分布于结肠肌层中，包括纵肌层（LM）、肌间丛（MP）、环肌层（CM）和黏膜下环肌表面（SMB）。ICC在形态上主要表现为双极细胞和多突起细胞两种类型。ICC—MP和ICC—CM相对多于ICC—SMB和ICC—LM。与对照组相比，STC患者各个区域ICC均明显减少（P〈0．01）。部分病例ICC—SMB几乎消失。结论：STC结肠ICC数量显著减少，但ICC减少是原发性的还是继发于某种其他损害仍有待探讨。     

慢传输型便秘患者结肠壁内Cajal细胞的形态学研究

目的　慢传输型便秘 (STC)患者乙状结肠壁内Cajal细胞 (ICC)的形态学研究。方法　全层铺片、冰冻切片的免疫细胞化学染色及透射电镜观察。结果　正常成人乙状结肠壁内ICC主要分布在环肌内侧面与粘膜下层之间 (ICC SM)、环肌层内 (ICC CM)、纵肌层内 (ICC LM)及肌间神经丛周围 (ICC MP)。STC乙状结肠壁内ICC的数量均较正常对照组减少 ,其中 ,ICC SM和ICC CM减少尤为显著 ,约减少 6 0 %。铺片显示ICC -MP不仅数量减少 ,且突起的分支亦减少 ,彼此间不能形成完整的细胞网络。电镜观察可见上述部位的Cajal细胞内溶酶体聚集、脂质沉积 ,ICC SM突起间的缝隙连接较小、数量减少。结论　本研究结果提示ICC的这些病理改变可能与STC的发生、发展有关 ,但是 ,ICC的减少是该病的原因还是继发性损害的结果仍有待进一步研究     

慢传输型便秘患者结肠壁内Cajal细胞的形态学研究

目的 慢传输型便秘(STC）患者乙状结肠壁内cajal细胞(ICC)的形态学研究。方法 全层铺片、冰冻切片的免疫细胞化学染色及透射电镜观察。结果 正常成人乙状结肠壁内ICC主要分布在环肌内侧面与粘膜下层之间(ICC-SM)、环肌层内(ICC-CM)、纵肌层内(ICC-LM)及肌间神经丛周围(ICC-MP)。STC乙状结肠壁内ICC的数量均较正常对照组减少，其中，ICC-SM和ICC-CM减少尤为显著，约减少60％。铺片显示ICC-MP不仅数量减少，且突起的分支亦减少，彼此间不能形成完整的细胞网络。电镜观察可见上述部位的cajal细胞内溶酶体聚集、脂质沉积，ICC-SM突起间的缝隙连接较小、数量减少。结论 本研究结果提示ICC的这些病理改变可能与STC的发生、发展有关，但是，ICC的减少是该病的原因还是继发性损害的结果仍有待进一步研究。     

Cajal间质细胞在先天性巨结肠分布的研究

目的：通过观察cajal间质细胞（interstitial cells of cajal，ICC）在正常结肠及先天性巨结肠先天性巨结肠（hirschsprung’s disease，HD）患者痉挛段、移行段、扩张段的分布，探讨HD的发病机制。方法：收集25例HD患儿标本，于术中分别选取扩张段、移行段、痉挛段肠壁的全层组织，另取6例手术患儿的正常结肠全层组织标本，常规固定石蜡包埋组织切片备用。对标本行c-Kit免疫组织化学染色。光镜观察ICC的分布，计数并进行统计学分析。结果：正常结肠ICC主要分布在环肌内侧面与黏膜下层之间即黏膜下ICC（submucosal ICC．ICC—SM）、环肌与纵肌之间的肌间神经从周围即肌间ICC（myenteric ICC，ICC—MY）以及环肌与纵肌内。HD患儿痉挛段ICC—SM、ICC—IM细胞数较扩张段和正常对照组明显减少（P〈0．01），且ICC的细胞突起的分支亦减少，彼此之间不能形成完整的细胞网络。而扩张段ICC与正常对照组比较无明显差异（P〉0．05）。结论：HD患儿结肠ICC的异常分布，可能是HD发病、肠管蠕动障碍以及排便异常的原因之一。     

雌激素受体β在女性慢传输性便秘患者乙状结肠壁内的分布及其蛋白表达

目的研究女性慢传输性便秘(slow transit constipation,STC)患者雌激素受体β(estrogen receptor β,ERβ)在乙状结肠组织的分布及其蛋白表达。方法采用免疫组织化学及Western blot方法检测20例STC患者以及20例对照组患者乙状结肠ERβ的分布及其蛋白表达。结果免疫组化结果显示ERβ在STC组及对照组乙状结肠黏膜、肌间神经丛及黏膜下神经丛均有表达,肌层未见表达。与对照组相比,STC组ERβ表达均明显降低,差异有统计学意义(P0.05)。Western blot结果显示STC组乙状结肠ERβ蛋白表达较对照组明显降低,差异有统计学意义(P0.01)。结论肌间及黏膜下神经丛ERβ蛋白表达降低可能参与STC发病。     

慢传输性便秘结肠平滑肌肌动蛋白改变

目的：深入地了解慢传输性便秘的发病机理和病理生理改变。方法：以免疫组化方法检测了STC患者结肠平滑肌收缩蛋白－肌动蛋白的变化。结果：与正常对照组相比,慢传输性便秘患者结肠黏膜下肌层、纵肌层内α肌动蛋白量明显减少（P＜0．01）,而环肌层内α肌动蛋白变化不明显（P＞0．05）。结论：推测这种平滑肌收缩蛋白的减少和分布异常导致了慢传输性便秘病人结肠运动功能的紊乱。     

Cajal间质细胞在大鼠"泻剂结肠"结肠肌电变化中的作用

目的　研究长期服用接触性泻剂对大鼠结肠肌电的影响 ,探讨Cajal间质细胞 (ICC)在“泻剂结肠”肌电变化中的作用。方法　 32只大鼠随机分为 2组 ,实验组饲以含酚酞饲料 ,3个月后测定结肠慢波频率及振幅 ;用碘化锌 锇酸法 (ZIO)观察肌间丛ICC变化 ,透射电镜观察肌间丛神经和ICC的超微结构变化。结果　“泻剂结肠”结肠慢波频率明显减慢 (P <0 .0 5 ) ,肌间丛ICC分布不均匀 ,突起连接杂乱 ;电镜下见肌间丛神经轴突空化 ,ICC样细胞变性。结论　长期服用酚酞可导致结肠慢波频率减慢 ,其可能机理为肌间丛神经及ICC变性所引起     

结肠慢传输型便秘的研究现状及展望(摘要)

结肠慢传输型便秘(STC)是临床上常见的、以腹胀及便意淡漠为主要症状的慢性顽固性便秘。近年来大量的临床和实验研究发现。(1)STC病人的结肠壁变薄、肌细胞空泡变性或脂肪变性、环肌萎缩,病变呈进行性过程。(2)肠壁问神经节细胞数量减少,排列紊乱,形态皱缩或轻度水肿,空泡变性;神经微丝和微管数量减少、排列紊乱。(3)肠壁内兴奋性神经递质(Ach、SP)减少,抑制性递质(VIP)含量有增高现象。(4)Cajal间质细胞(ICC)的分布和功能异常与肠动力障碍有密切关系,详细机理尚不清楚。根据研究资料,肠壁肌细胞和肌问神经丛的损害、神经递质的改变以及ICC的分布和功能异常是STC发病的关键环节,究竟什么原因引起这些病理改变,目前尚不清楚。因此,目的STC的预防尚无良策,治疗仍是一般性的保守治疗,包括:①粗纤维饮食;②在结肠高动力期(早晨起床后或早餐后)训练排便运动,改善排便体位(蹲位最佳);③加强腹肌和膈肌锻炼;①适当给予粪便软化剂和润肠剂;⑤上述治疗无效者给予低渗性药物、水灌肠或油剂保留灌肠。对保守治疗无效者给予手术治疗,目的理想的术式为全结肠切除,回一直肠吻合术和次伞结肠切除,盲一直肠吻合术。作者提出。在STC确诊后先用结肠壁活组织病理检查或电生理等检查。根据肠壁肌、肌间神经、ICC及神经递质的     

HCN通道在大鼠膀胱中的分布及表达

目的:探讨HCN通道在大鼠膀胱中的分布,及与膀胱ICC细胞之间的关系。方法:选择c-kit标记ICC细胞,利用免疫组化分别对大鼠膀胱黏膜、黏膜下层、肌层中HCN通道的表达情况进行检测,同时利用western blot对大鼠膀胱肌层HCN各通道蛋白的表达水平进行定量分析。结果:c-kit标记ICC细胞显示良好,免疫组化示4种HCN通道亚型在大鼠膀胱黏膜、黏膜下层及肌层均有表达,HCN1、2、3、4各组间差异有统计学意义(P0.01),组间分析结果显示HCN4通道亚型的表达明显高于其他通道;western blot示大鼠膀胱肌层HCN1、2、3、4各组间差异有统计学意义(P0.01),HCN4通道亚型表达水平最高。结论:HCN通道在膀胱黏膜、黏膜下层及肌层均有表达,以HCN4通道亚型表达最强;在黏膜下层主要表达在ICC细胞,肌层主要存在于肌肉间隙;HCN通道在ICC细胞中的表达可能参与了膀胱自发性收缩。     

Cajal间质细胞在先天性巨结肠和巨结肠同源病结肠中的分布研究

目的研究先天性巨结肠（Hirschsprung’s disease，HD）和巨结肠同源病（allied Hirschsprung’s disease，AHD）肠壁内Cajal间质细胞（interstitial cells of Cajal，ICCs）的分布状态，探讨HD和AHD的发病机制。方法选择确诊为HD和AHD的患者各20例，取巨结肠根治术吻合口远端的全层肠壁作为实验组，另取16例正常结肠标本作为对照组。用鼠抗人c—kit单克隆抗体（CD117）标记ICCs，Image Pro-Plus图像分析系统检测ICCs。结果对照组中大量ICCs分布在肌间神经丛周围和环纵肌层内，ICCs包绕神经丛周围，肌层间ICCs连续分布；HD组远端肠管中肌层间和各肌层内ICCs明显减少甚至缺如，与对照组比较差异有统计学意义，P〈0．01；AHD组远端肠管中神经丛大小不一，ICCs分布差异大，大多数神经丛区ICCs减少，环肌层内ICCs明显减少，与AHD组和对照组比较差异有统计学意义，P〈0.01。HD组远端结肠中ICCs减少比AHD组显著，差异有统计学意义，P〈0．01。结论HD、AHD病变肠管中除了神经节细胞的异常外，同时存在ICCs异常；ICCs在HD和AHD的分布不同可能与两者临床症状差异有关；肠管中ICCs数量可能与临床症状及预后有一定关系。     

Assessing interstitial cells of Cajal in slow transit constipation using CD117 is a useful diagnostic test

Slow transit constipation (STC) is a colonic motility disorder that is characterized by measurably delayed movement of stools through the colon. The pathophysiology of STC is unclear and both the interstitial cells of Cajal (ICC) and cells of the enteric nervous system are believed to play an important role. The aim of this study was to compare the number and distribution of ICC and cells of the enteric nervous system in patients with a control group by means of immunohistochemistry. Formalin-fixed paraffin-embedded colonic sections were obtained from 15 patients, aged between 23 and 52 (mean age=37 y), who underwent colectomy for STC. Forty-five cases of normal colon from age and sex-matched nonobstructive colorectal cancer patients were selected as controls. By using c-kit (CD117) and PGP 9.5 immunohistochemical studies, ICC and enteric neurofilaments were demonstrated, respectively. The number of cells were counted under 40 x high-power field (HPF) in 3 layers of the colonic muscularis propria, that is, the inner circular muscle layer, the myenteric plexus, and the outer longitudinal muscle layer in both test and control groups. The mean number of ICC and enteric neurofilaments were significantly reduced in all 3 layers of the muscularis propria from STC patients compared with controls. This reduction was most significant in the inner circular muscle layer (P<0.0001). A cutoff value of 7 ICC per HPF in the inner circular muscle layer can be used as a further confirmation to the clinical diagnosis of STC in resected specimens.     

豚鼠阴茎海绵体中ICC和NO信号通路的形态学关系

目的初步探讨豚鼠阴茎海绵体组织中cajal间质细胞（ICC）和神经型一氧化氮合成酶（nNOS）阳性神经元的分布及两者的组织学关系。方法①制作成年豚鼠阴茎海绵体组织冰冻切片,利用c-kit/nNOS免疫荧光双染技术,激光共聚焦扫描显微镜（LSCM）下观察阴茎海绵体ICC及nNOS阳性神经元的形态及分布情况。②酶消化法体外培养阴茎海绵体ICC,c-kit/nNOS免疫荧光双染,LSCM下观察细胞形态及c-kit和nNOS的表达情况。结果 ICC分布在阴茎海绵体平滑肌小梁边缘及平滑肌肌间,纺锤形,具有长的突起,胞体呈圆形或椭圆形,并且表达nNOS;阴茎海绵体平滑肌小梁边缘及平滑肌肌间同时分布有ICC和nNOS阳性神经元,局部nNOS阳性神经主干附近可见较多ICC分布。结论豚鼠阴茎海绵体ICC与nNOS阳性神经元组织联系紧密,ICC可能参与阴茎海绵体内NO神经信号的传递。     

Origin, course, and endings of abnormal enteric nerve fibres in Hirschsprung's disease defined by whole-mount immunohistochemistry

Accurate delineation of the intramural pathway of abnormal enteric nerve fibres in Hirschsprung's disease has previously proved impossible because the neural network is invariably transected in conventional histological sections. With the technique of wholemount immunohistochemistry (WI), the bowel segment is converted into a rectangular sheet and the serosa, long muscle (LM), circular muscle (CM), submucosa, and mucosa are separated into layers to allow each nerve plexus to be examined intact and neural pathways traced. The entire resected bowel specimens of nine HD infants and five infants serving as controls were investigated, using neuron-specific enolase and vasoactive intestinal peptide (VIP) for WI. The major new findings are (1) More VIP fibres were observed in aganglionic bowel with WI than with conventional sections; (2) Thick nerve trunks in aganglionic bowel do not descend from intrinsic neurons of oligoganglionic bowel as previously suggested, but have an extrinsic origin, accompanying blood vessels as small nerves initially, expanding subsequently, and ending blindly in submucosa; (3) CM nerve fibres follow muscle fibres concentrically for long distances in aganglionic bowel; and (4) LM nerve fibres meander in spirals in aganglionic bowel instead of running straight. This study shows that (1) WI is highly sensitive; (2) nerve fibres in aganglionic bowel have an extrinsic origin; and (3) innervation abnormalities in Hirschsprung's disease are not only quantitative but qualitative.     

Loss of CD117 (c-kit)- and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction

Chronic idiopathic intestinal pseudo-obstruction is a syndrome in which symptoms of intestinal obstruction are present in the absence of mechanical obstruction. Lack of normal pacemaker activity, usually generated by the interstitial cells of Cajal (ICC), could account for the apparent obstruction. ICC are normally located around and between the myenteric plexus ganglia and within muscle and also in the deep muscular plexus of the small bowel and the submuscular plexus of the large intestine, just within the circular muscle. ICC can be demonstrated immunohistochemically with CD117 (c-kit) as well as with CD34, although this is less specific. CD34 also stains a population of fibroblasts that are intimately associated with ICC. To determine whether there is a relative deficiency of ICC and CD34-positive fibroblasts in patients with chronic idiopathic intestinal pseudo-obstruction, tissue from 30 patients of large intestine and eight patients with small intestine pseudo-obstruction was obtained. Controls (large intestinal specimens from 12 patients, small intestinal specimens from six patients) were chosen from resections for Crohn's disease and colorectal neoplasia, both with and without dilatation. Examination of pseudo-obstruction cases identified 10 patients (nine large intestinal and one small intestinal) in which both CD117 and CD34 were absent or severely reduced in all three of the examined areas. In contrast, the control cases, including those with preobstructive dilatation, showed relatively constant ICC staining. These results suggest that there is a proportion of pseudo-obstruction cases in which the ICC are markedly reduced. These results also demonstrate that, in these cases, loss of the kit immunoreactivity is correlated with the loss of CD34 staining: this indicates that both the ICC and the CD34-positive fibroblasts associated with the ICC are absent. These findings will allow surgical pathologists to identify this subpopulation of patients with CIIP using tissue obtained by laparoscopic biopsy of the muscularis propria or surgical resection.     

Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons

PURPOSE: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells. MATERIALS AND METHODS: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy. RESULTS: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive. CONCLUSIONS: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.     

Kit positive cells in the guinea pig bladder

PURPOSE: We describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder. MATERIALS AND METHODS: Bladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy. RESULTS: Anti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active. CONCLUSIONS: ICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.     

大鼠小肠Cajal间质细胞的体外分离、培养及鉴定

目的：探讨大鼠小肠Cajal间质细胞（ICC）的原代分离、培养及鉴定方法。 方法：出生后5~10 d的SD乳大鼠,颈椎脱臼处死。无菌条件下取出小肠,在解剖显微镜下剥去小肠系膜、肠黏膜和黏膜下层,将小肠平滑肌层组织剪成小块后接种于含有干细胞因子的DMEM培养基中进行培养。倒置显微镜下连续观察组织块周围细胞的游出情况及细胞的形态,用酪氨酸蛋白激酶受体c-kit特异性抗体免疫荧光染色鉴定细胞类型。 结果：培养1周后,倒置显微镜下可见组织块周围长出细胞,呈梭形、三角形,有多个短突起;随着培养时间的延长,突起细长化并彼此相互连接形成网络。该类细胞c-kit抗体免疫荧光染色呈阳性。 结论：该研究成功建立了一套由大鼠小肠平滑肌组织块原代培养ICC的方法,为ICC的生物学特性及其与胃肠道动力障碍性疾病关系的研究奠定了基础。     

Growth pattern in the muscular layer reflects the biological behaviour of colorectal cancer

Objective  To determine the clinical value of evaluating the cancer morphology in muscularis propria (MP) for colorectal cancer (CRC) patients.
Method  A total of 994 patients with advanced CRC were reviewed in terms of two distinctive growth patterns in the MP: (i) horizontal spread between the circular and longitudinal muscle layers (H-spread) and (ii) 'streaming' spread between the muscle bundles of the circular muscle layer (S-spread).
Results  The incidence of H-spread ( n  = 153) and S-spread ( n  = 150) showed a positive correlation with tumour-node-metastasis (TNM) stage and both exerted a negative impact on postoperative survival. Adverse morphology in the MP (H-spread and/or S-spread) was consistent with a high grade of vascular invasion and budding in the extramural layer, as also with unfavourable fibrotic stromas in the reactive fibrous zone; the 5-year survival rate in patients with such features was 64.2%, which was lower than that in those without (86.5%, P  < 0.0001). Multivariate analysis demonstrated that adverse morphology was an independent prognostic determinant, along with T- and N -stage. As the mode of H-spread, perineural invasion in the myenteric plexus was found to be predominant over lymphatic spread on the basis of S100 and CD34 immunostaining, but neural cell adhesion molecule expression, whether on cancer cells or on neural cells, was not significant for this growth pattern.
Conclusion  A particular group of CRCs ingeniously utilizes the thin space between muscle fascicles for development in the MP. Although the biological mechanism remains unknown, this distinctive growth pattern could be a useful indicator to identify CRC patients at high risk of recurrence.