Cajal间质细胞的研究进展

Cajal间质细胞(interstitial cells of Cajal,ICC)是一类主要分布于胃肠道的间质细胞,是胃肠道的起搏细胞(pacemaker cell)和信号传导细胞,与肌细胞以及末梢神经元有着紧密的关系,具有激发和促进胃肠蠕动的作用.借助于电子显微镜技术,清楚观察到了ICC位置分布和内部精细结构;应用免疫荧光等生化技术,发现了其特殊表达的C-kit蛋白;利用电生理技术,得知多种胃肠动力障碍疾病也与其异常有关.多年来,学者逐渐在胃肠道、胆道、膀胱、子宫等部位发现了ICC的踪迹,并试图阐述其与某些疾病的发生机制.本文就ICC的起源、形态学、受体和功能、以及与其相关的疾病等作一综述.     

Cajal间质细胞与胃肠动力关系的研究进展

Cajal间质细胞(interstitial cells of Cajal,ICC)是胃肠慢波的起搏细胞,具有产生慢波、传导慢波电位、调节神经递质等功能,是调节胃肠动力的重要环节。ICC在维持正常胃肠动力方面发挥着重要作用,同时其形态、数量及分布的改变会导致多种胃肠动力障碍性疾病。一些以ICC为靶向治疗胃肠动力性疾病药物的研究也取得一定进展。本文就近年来ICC与胃肠动力关系的研究进展作一概述。     

消化道Cajal间质细胞及其受体的研究进展

Cajal间质细胞（interstitial cells of Cajal，ICCs）是胃肠起搏细胞，主要参与胃肠基本电节律（BER）调控和神经递质信号转导，近年来进行的一系列研究表明这种细胞在胃肠电及动力发生和运动障碍机制中有重要作用，同时关于消化道ICCs受体的研究也越来越被人们所关注。因此了解Cajal间质细胞及其受体的特点、功能具有重要意义，现主要对消化道ICCs及其受体的研究现状做一综述。     

成年C57BL／6小鼠空肠Cajal间质细胞的分离、培养及鉴定

目的尝试优化体外培养成年小鼠小肠Cajal间质细胞的操作方法,为深入研究该细胞的生理功能提供一定细胞模型。方法6～8周龄C57BL／6小鼠禁食24h,无菌条件下截取3～5cm中段空肠,分离平滑肌肌条并剪至小块后使用Ⅱ型胶原酶消化20min,离心后进行组织块原代培养,观察不同时期细胞形态。使用c—Kit免疫荧光染色鉴定细胞表型是否为Cajal间质细胞。Fluo-3AM染色细胞内钙,观察细胞能否自发产生钙离子振荡。结果分离所得组织不含空肠上皮和固有层组织,仅为小肠肌层。联合使用胶原酶消化和组织块原代培养能够较方便地培养出原代细胞,该细胞呈梭形,且具有细胞突起,彼此交织成网状。该细胞免疫荧光染色呈c—Kit阳性,平滑肌标志物SMA阴性。钙成像分析显示其能够产生钙离子振荡。结论联合使用酶消化和组织块培养能够较方便地培养出成年小鼠的空肠Cajal间质细胞,该方法为后续探讨Cajal间质细胞的生理功能及机制提供了一定的细胞模型。     

糖尿病大鼠胃肠功能紊乱时结肠组织中Cajal间质细胞的表达

目的观察糖尿病大鼠胃肠功能紊乱时结肠组织内Cajal间质细胞的分布及表达变化,探讨Cajal间质细胞在糖尿病胃肠功能紊乱发病机制中的作用。方法30只SD大鼠随机分为两组,糖尿病组20只,正常对照组10只。糖尿病组大鼠用链脲佐菌素单剂量腹腔注射建立糖尿病模型,对照组注射等量枸橼酸缓冲液。两组大鼠饲养6周后处死,计算胃肠推进率并且收集结肠组织标本。用免疫组化方法观察Cajal间质细胞在两组大鼠结肠组织内的分布和表达,用W estern b lot方法检测c-k it蛋白在两组大鼠结肠内的表达。结果糖尿病组大鼠胃肠推进率较对照组明显降低（P〈0.05）。免疫组化和W estern b lot检测都显示糖尿病大鼠结肠组织内Cajal间质细胞的表达较正常大鼠明显减少（P均〈0.05）。结论糖尿病大鼠结肠组织内Cajal间质细胞表达减少,推测与糖尿病大鼠胃肠功能紊乱有一定相关性。     

小鼠空肠cajal间质细胞的分离与培养

目的研究CD1小鼠空肠cajal间质细胞(ICC)的分离和培养方法,为进一步研究其生理学特征提供基础。方法无菌条件下取小鼠空肠组织,分离出平滑肌肌条,采取组织块培养法,在24孔培养板中进行原代培养,光镜下观察其形态,用荧光标记的特异性C_Kit抗体进行鉴定。结果倒置显微镜下观察ICC有多个突起,并有次级分支,相互之间形成网络。免疫荧光法可见ICC呈阳性。结论组织块培养法可以简便、有效地培养ICC。所培养的ICC细胞生物学特性有待进一步研究。     

小鼠不全性肠梗阻对Cajal间质细胞表型的影响

目的:探讨小鼠小肠慢性不全性肠梗阻导致自发性节律性收缩运动、电活动变化与Cajal间质细胞(ICC)表型转化的关系.方法:通过外科手术方法在小鼠回肠套入硅胶管,建立小鼠不全性肠梗阻模型.利用苏木精-伊红(H&E)染色,研究小肠平滑肌层形态学改变;利用常规生理和电生理技术观察环行平滑肌肌条自发性节律性收缩和慢波变化规律;利用全层平滑肌免疫组织化学方法,观察ICC表型标志酪氨酸激酶受体(c-kit)表达的变化.结果:小鼠不全性肠梗阻模型形成后14d,梗阻近端肠管显著扩张、小肠平滑肌层明显增生肥厚;小肠平滑肌自发性节律性收缩节律紊乱、幅度不规则、频率变慢;平滑肌静息膜电位去极化、慢波幅度变小、频率变慢;ICC表型标志c-kit表达显著减弱、甚至消失.结论:小鼠不全性肠梗阻导致机械和电活动紊乱与ICC表型转化导致的ICC数量减少和网络破坏有关.该模型为进一步研究ICC表型改变的细胞/分子机理提供了一个良好的实验模型.     

Dissociation, culture and morphologic changes of interstitial cells of Cajal in vitro

AIM: To study the method of dissociation, culture and investigate its morphologic changes in vitro of interstitial cells of Cajal (ICC). METHODS: Enzymatic digestion and Ficoll density centrifugation were used to dissociate ICC from the ileal segment of mice. Factors including contamination, Ca~(2+), Mg~(2+) and collagenase, and stem cell factor, etc., were investigated. ACK2, the antibody of c-kit, was used to identify the cultured ICC. Both light microscope and fluorescence microscope were used to observe the changes of ICC in vitro. RESULTS: The method for dissociation and culture of ICC in vitro was successfully established. After 24 h, cultured ICC exhibited a few axis-cylinders, and longer axis-cylinders were observed to form synapse of each other after 3 d. More widespread connections formed within 7 d in vitro. The changes of its morphologic character were obvious within 7 d; however, there were no obvious morphologic changes after 30 d. CONCLUSION: Many factors can influence the dissociation and culture of ICC.     

Interstitial cells of Cajal, the Maestro in health and disease

Interstitial cells of Cajal (ICC) are important players in the symphony of gut motility. They have a very signif icant physiological role orchestrating the normal peristaltic activity of the digestive system. They are the pacemaker cells in gastrointestinal (GI) muscles. Absence, reduction in number or altered integrity of the ICC network may have a dramatic effect on GI system motility. More understanding of ICC physiology will foster advances in physiology of gut motility which will help in a future break...     

Differential changes in intrinsic innervation and interstitial cells of Cajal in small bowel atresia in newborns

AIM: To investigate morphological changes of the enteric nervous system (ENS) and the interstitial cells of Cajal (ICCs) in small bowel atresia.METHODS: Resected small bowel specimens from affected patients (n = 7) were divided into three parts (proximal, atretic, distal). Standard histology and enzyme immunohistochemistry anti-S100, anti-protein gene product (PGP) 9.5, anti-neurofilament (NF), antic-kit-receptor (CD117) was carried out on conventional paraffin sections of the proximal and distal part. RESULTS: The neuronal and glial markers (PGP 9.5, NF, S-100) were expressed in hypertrophied ganglia and nerve fibres within the myenteric and submucosal plexuses. Furthermore, the submucous plexus contained typical giant ganglia. The innervation pattern of the proximal bowel resembled intestinal neuronal dysplasia. The density of myenteric ICCs was clearly reduced in the proximal bowel, whereas a moderate number of muscular ICCs were found. The anti-CD117 immunore- action revealed additional numerous mast cells. The distal bowel demonstrated normal morphology and density of the ENS, the ICCs and the mast cells.CONCLUSION: The proximal and distal bowel in small bowel atresia revealed clear changes in morphology and density of the ENS and ICCs.     

谷氨酸诱导大鼠结肠Cajal间质细胞构建自噬模型

目的 通过谷氨酸诱导离体大鼠结肠Cajal间质细胞(ICC)构建细胞自噬模型.方法 使用不同浓度的谷氨酸不同时间点作用于原代培养的大鼠结肠ICC,免疫荧光鉴定大鼠结肠ICC细胞;CCK-8法检测谷氨酸对ICCs活力的影响;Western印迹检测不同浓度谷氨酸在不同作用时点对大鼠结肠ICC自噬蛋白微管相关蛋白轻链(LC)...     

Loss of interstitial cells of Cajal network in severe idiopathic gastroparesis

INTRODUCTION Gastroparesis syndrome is a clinical entity characterized by chronic nausea, epigastric discomfort and recurrent vomiting, in the absence of mechanical obstruction[1]. Gastroparesis may be either primary (idiopathic) or secondary, i.e. associ…     

Interstitial cells of Cajal: a novel hypothesis for the pathophysiology of irritable bowel syndrome

Irritable bowl syndrome (IBS) affects a large proportion of the world's population, and accounts for a considerable number of visits to gastroenterologists and general practitioners. Despite its high prevalence, the precise mechanism of IBS has not been identified to date. The interstitial cells of Cajal (ICC) participate in the production of slow waves and the regulation of their propagation through the gastrointestinal system; thus, they are important components of gastrointestinal motility. The present review proposes that ICC play a central role in the pathophysiology of IBS. This hypothesis is based on many links between ICC and currently proposed mechanisms of IBS pathophysiology. It appears that ICC may be involved in almost all of the previously explained pathogenic mechanisms of IBS. If proven, this hypothesis may provide a key to solving the IBS mystery.     

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.     

干细胞因子对糖尿病小鼠小肠Cajal间质细胞的影响

目的探讨外源性干细胞因子（stem cell factor,SCF）对糖尿病（diabetes mellitus,DM）小鼠小肠Cajal间质细胞的影响。方法将雄性Balb/c小鼠随机分为正常对照组、糖尿病组（DM组）、糖尿病＋外源性SCF组（DM＋SCF组）,DM组和DM＋SCF组一次性腹腔注射（ip）链脲佐菌素（STZ,150 mg/kg）造模,选取成功模型;DM＋SCF组再ip SCF 0.2μg/（kg.d）;正常组和DM组每天ip等量的磷酸盐缓冲液（pH=7.4）。所有小鼠干预6周结束后,给予印度墨水灌胃测定小肠传输速率,免疫组化观察小肠c-kit的表达,Western blot观察c-kit蛋白的表达。结果饲养6周后,DM＋SCF组的体质量（g）、推进率、c-kit阳性细胞数（个/hpf）、c-kit蛋白的表达均比DM组明显增加（P均〈0.05）,仍低于正常组（P均〈0.05）;DM＋SCF组的血糖（mmol/L）比DM组明显降低（P〈0.01）,仍高于正常组（P〈0.05）。结论外源性SCF可以逆转Cajal间质细胞的异常改变,并对糖尿病小鼠的小肠动力障碍有一定的改善作用。     

Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation

Background and aims Slow transit constipation (STC) is a colonic motor disorder that is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the etiology of STC remains unclear. Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunctions, including idiopathic STC. This study aimed to determine the normal distribution of ICC within the colon of the Chinese and also to determine if ICC are decreased in Chinese STC patients.Patients and methods Twelve patients with STC and eight age-matched normal controls were studied. Specimens of sigmoid colon were obtained immediately after resection. ICC were identified with a monoclonal antibody to c-kit by an indirect immunofluorescence method. Immunostained tissues were examined with a laser scanning confocal microscope and the area occupied by ICC was calculated with an image analysis system.Results ICC were located in the external muscle layers including myenteric plexus (MP) and submucosal border (SMB). Two types of Kit-positive ICC were observed: bipolar cells characterized by one or two long processes and multipolar cells characterized by long stellate processes extending in various directions. A higher percentage of ICC was present in the MP regions and circular muscle (CM) layers compared with the SMB and longitudinal muscle (LM) layers. Tissues from STC patients showed a considerable decrease in the number of ICC located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), especially the ICC-SMB, in which ICC almost completely disappeared.Conclusions Similar distribution of ICC was observed in the normal sigmoid colon of the Chinese. Decreased area of c-kit+ ICC may play an important role in the pathophysiology of STC. It remains to be determined whether the loss of ICC is primary or secondary to another lesion.     

针刺对结肠吻合术后Cajal间质细胞修复与再生的影响

目的:探讨针刺促进术后肠道动力恢复的机制.方法:30只SD大鼠随机分为空白组、模型组(行结肠吻合术)、针刺组.针刺组予每日针刺双侧足三针(足三里、三阴交、太冲),连续3d.观察大鼠排便情况,测量小肠推进率,观察结肠组织Cajal间质细胞(interstitial cells of Cajal,ICC)超微结构和胆碱能神经-ICC-平滑肌细胞网络结构.结果:针刺组术后首次排便时间较模型组提前,小肠推进率提高(2.00d±0.47d vs 2.50d±0.53d,66.30%±4.21% vs 46.33%±5.56%,P<0.05).与空白组比较,模型组ICC超微结构损伤明显,胆碱能神经-ICC-平滑肌细胞网络结构紊乱,ICC和小泡乙酰胆碱转移体(VAChT)阳性神经纤维数量明显减少(18.67±6.11 vs 32.33±5.51,18.67±3.79 vs 20.67±3.21,P<0.05)荧光强度减弱(35.00±9.54 vs 58.67±10.21,20.33±5.13 vs 34.67±6.81,P<0.05).而针刺组ICC超微结构损伤较模型组轻,网络样结构维持,ICC和VAChT阳性神经纤维的...     

不同时程慢性束缚水浸应激对大鼠胃窦Cajal间质细胞超微结构的影响

目的:研究不同时程慢性束缚水浸应激大鼠胃窦Cajal间质细胞(ICC)的超微结构改变.方法:♂ SD大鼠48只随机分为6组、即实验3、7、28 d组和对照3、7、28 d组,每组8只.实验组每日束缚水浸1 h,对照组自由摄食饮水;于实验第4、8、29天晨禁食12 h后脱颈处死.取胃窦组织2块放入3%戊二醛中固定并电镜下观察ICC超微结构.结果:所有对照组ICC的超微结构均无异常改变,试验3、7、28 d各组ICC的超微结构与同期对照组比较均有明显的损害,主要表现为ICC的缝隙连接减少、细胞器减少等,以肌内ICC(ICC-MY)和肌间ICC(ICC-IM)为主:随着应激时间的延长,ICC的超微结构受损逐渐加重.结论:慢性束缚水浸应激可以损伤大鼠胃窦ICC的超微结构.     

Slow transit colon constipation is not related to the number of interstitial cells of Cajal

Background and aims Recent studies have demonstrated decreased numbers of interstitial cells of Cajal in patients suffering from severe chronic constipation as measured by c-Kit (CD117) and CD34 immunohistology. In this study, we wished to determine whether there were abnormalities in the number of neurons of the Auerbach's plexus, their CD117 and CD34 immunoreactivity, or the thickness of colon wall sections in patients with refractory slow transit colonic constipation as compared with control subjects.Patients and methods Specimens from 13 patients who had undergone subtotal colectomy for severe chronic constipation refractory to medical treatment were compared with normal controls. Enteric neurons of Auerbach's plexus were counted, and thickness of the circular and longitudinal layer of the muscularis externa as well as total muscularis externa was measured. Quantitative assessment of anti-CD117 and anti-CD34 immunoreactivity was performed using an Automated Cellular Imaging System and expressed as fractional scores.Results Except for a decreased circular muscle layer thickness in the constipated patients, no statistically significant differences were observed between the two groups. In particular, there was no relationship between CD117/CD34 fractional staining score and the duration or severity of disease, despite the selection of highly symptomatic individuals requiring colonic resection.Conclusion Using quantitative immunohistochemistry for CD117/CD34, we could not detect a relationship between fractional CD117/CD34 staining score and chronic constipation as compared to controls.