Cajal间质细胞与先天性巨结肠病的相关性研究
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摘要
无神经节细胞性巨结肠病(aganglionic megacolon)又称Hirschsprung氏病(Hirschsprung's disease,HD),是一种严重危害婴幼儿健康的先天性畸形,据统计,其发病率高达1/3000～1/5000,居消化道畸形第二位。先天性巨结肠的主要病理变化是一段结肠壁内缺乏神经节细胞,致使该段结肠持续性痉挛,肠腔狭窄,其近端肠腔扩张,内容物潴留。目前,有关先天性巨结肠的发病机制尚不完全清楚,多数观点认为该病属于多因子遗传性疾病,即由遗传和环境因素共同作用所致。先天性巨结肠的主要临床表现是腹胀、顽固性便秘、不完全性肠梗阻。目前临床上唯一有效的治疗方法是手术,但由于手术损伤比较大和结肠缩短,术后常出现小肠结肠炎、稀便、患儿心理障碍等后遗症。因此,有人尝试进行了神经干细胞移植治疗无神经节细胞性巨结肠病的实验研究,即用各种来源的神经干细胞移植入缺少神经节细胞的病变肠壁内。虽然移植进去的神经干细胞可以在肠壁的微环境中存活,并分化为功能性的肠神经细胞,但只是部分恢复了无神经节细胞肠段的运动功能。这说明在先天性巨结肠的发病机制中除了神经节细胞缺如之外,还有其他因素的参与,其中肠壁中的Cajal间质细胞引起了人们的关注。
Cajal间质细胞(interstitial cells of Cajal,ICCs)是分布在胃肠道内由中胚层间充质细胞分化而来的一类细胞,由西班牙神经解剖学家Cajal在1893年首次描述。它既不是神经元,也不是神经胶质细胞,更不是平滑肌细胞,而是具有独立功能的特殊间质细胞。目前Cajal间质细胞的生物学特性和特殊功能越来越受到国内外学者的关注。研究发现,这类细胞是胃肠道平滑肌的起搏细胞,能产生生理慢波,并参与电活动的扩布,从而调控胃肠道平滑肌的舒缩和蠕动,其数量减少或缺失与多种胃肠动力性疾病的发生密切相关。先天性巨结肠病属胃肠动力性疾病之一,Cajal间质细胞的异常与先天性巨结肠的发病也必然存在一定的相关性。揭示Cajal间质细胞的异常与先天性巨结肠病发病的相关关系,可进一步阐明无神经细胞性巨结肠病的发病机理,并为先天性巨结肠病的细胞替代治疗提供实验依据。
根据上述理念,我们对Cajal间质细胞与先天性巨结肠病的相关性进行了比较深入的研究,论文发表于Neuroscience Letters杂志,实验分为三个部分:
第一部分大鼠结肠Cajal间质细胞的分离与培养
为了更好地从形态上识别Cajal间质细胞和利用特异性表面抗原准确地鉴别Cajal间质细胞,从而为下一步的研究奠定基础,我们对大鼠结肠壁内的Cajal间质细胞进行了分离和体外培养。体外细胞培养,是研究细胞功能以及相应细胞信号转导机制的基础,也有利于更深入地掌握疾病本质,制定合理有效的防治措施。但是由于Cajal间质细胞的数量少,分散在结缔组织、神经纤维及平滑肌等组织之中,分离提取的难度较大。对于这类细胞的体外培养,由于无这样的经验可循,因而难度更大。我们采用胶原酶及胰酶酶解和密度梯度离心法,经过多次调配两种酶的浓度和作用时间,成功地对Cajal间质细胞进行了分离。分离出细胞后,将细胞悬液接种于含有干细胞因子(stem cell factor,SCF)的M199培养基中,进行培养。培养24小时后,ICCs即可贴壁生长,培养3天后,可分辨出ICCs形态,胞体呈三角形或梭形,有少量突起,核大。培养5天后,ICC形态更为清晰,自胞体发出的突起向周围伸长,相邻细胞胞体伸出的突起相互靠近。培养7天后,相邻ICC突起间有连接形成,细胞间彼此联系,突起相互交织,形成网络状结构。细胞培养稳定后,用Cajal间质细胞的特异性标志物c-kit进行免疫细胞化学染色,结果显示Cajal间质细胞呈阳性着色。
第二部分小儿结肠壁内Cajal间质细胞的正常分布模式的检测
研究表明,从小鼠的胚胎时期直至出生后,Cajal间质细胞在胃肠道内有一个不断发育成熟的过程,并形成不同的亚型。根据Cajal间质细胞在胃肠道内分布部位的不同,通常将其分为三个亚型,即位于肠壁粘膜下层的粘膜下ICCs,称为IC-SM;位于纵行肌与环形肌之间的肌间ICCs,称为IC-MY;位于肌层内的肌内ICCs,称为IC-IM。有关小儿结肠壁内的三种亚型的Cajal间质细胞的正常分布状况及其功能至今仍未见报道。基于此,我们收集临床手术切除的小儿正常结肠标本,用小鼠抗人c-kit单克隆抗体(CD117)作标记,免疫组织化学染色,荧光显微镜检测了结肠标本冰冻切片中ICCs的分布状况,观察了三种亚型的ICCs在小儿正常结肠壁中的分布状况,结果显示肌间ICCs(IC-MY)含量最为丰富,呈网络状包绕在肌间神经节细胞周围,肌层内ICCs(IC-IM)数量也较多,与平滑肌细胞平行分布,粘膜下ICCs(IC-SM)的数量最少。用兔抗人nNOS多克隆抗体标记NOS神经,免疫组织化学染色,荧光显微镜检测了标本冰冻切片及肠壁铺片中NOS神经元及神经纤维的分布情况,结果显示,在纵行肌和环形肌之间,NOS阳性神经元和神经纤维形成网络状结构,肌层内也有较多的NOS阳性神经纤维平行于平滑肌细胞分布。应用c-kit与nNOS双重染色检测了结肠壁中ICCs和NOS阳性神经元及神经纤维的位置关系,结果显示,NOS阳性神经纤维与ICCs之间密切接触,ICCs与平滑肌细胞的接触也很密切。上述实验结果提示,小儿结肠壁内的Cajal间质细胞已形成正常的规律性分布,并与肠神经及平滑肌之间存在着密切接触,可能由此形成肠壁规律性舒缩的结构基础。
第三部分Cajal间质细胞在先天性巨结肠病中的异常变化
选择确诊为先天性巨结肠的患儿21例,其中普通型巨结肠16例,全结肠型巨结肠(TCA)5例,取巨结肠根治术标本痉挛段全层肠壁作为实验组,另取7例正常结肠相应节段标本作为对照组,冰冻切片,片厚10μm。用小鼠抗人c-kit单克隆抗体(CD117)作标记,免疫组织化学染色,荧光显微镜检测了实验组及对照组结肠标本冰冻切片中ICCs的分布状况,结果显示,与正常组结肠相比,实验组各结肠标本中三种亚型的ICCs(IC-MY,IC-IM,IC-SM)数量均明显减少,有显著差异(P＜0.01),与普通型巨结肠相比,肌间ICCs(IC-MY)在全结肠型巨结肠中的数量减少更为明显,有统计学差异(P＜0.01),而粘膜下ICCs(IC-SM)和肌内ICCs(IC-IM)的分布则无明显差异性(P＞0.05)。用兔抗人nNOS多克隆抗体标记NOS神经,免疫组织化学染色,荧光显微镜检测了两组标本冰冻切片及肠壁铺片中NOS神经元及神经纤维的分布情况,结果显示,实验组NOS阳性神经元及神经纤维数量明显减少,甚至缺失,与对照组相比,差异显著(P＜0.01)。应用c-kit与nNOS免疫荧光双重染色,检测了两组标本冰冻切片中ICCs和NOS阳性神经的位置关系,结果显示NOS阳性神经元及神经纤维与ICCs位置疏远,无密切接触。同时,我们还将收集到的结肠标本的肌层,匀浆后提取总RNA和蛋白,应用逆转录多聚酶联反应(Reverse TranscriptionPolymerase Chain Reaction,RT-PCR),在mRNA水平上检测正常结肠和HD痉挛段结肠c-kit基因、nNOS基因与看家基因GAPDH的表达,用Western blot方法检测两组标本中c-kit蛋白及nNOS蛋白的表达,对实验结果条带进行半定量分析,结果证实,先天性巨结肠病变肠壁内c-kit蛋白和mRNA表达明显降低(P＜0.01),nNOS蛋白与mRNA表达也明显减少(P＜0.01)。以上实验结果提示,无神经节性巨结肠病患儿病变段结肠壁内三种亚型的Cajal间质细胞的数量明显减少甚至缺失,病变段肠管必然丧失产生电慢波的能力,同时NOS阳性神经元及神经纤维的数量也明显降低,导致或加剧了巨结肠病变段肠管的功能紊乱。
结论
1、本课题成功地对Wistar大鼠结肠Cajal间质细胞进行了分离和体外培养,体外培养的ICCs可以形成独立网络状结构,从而为这种细胞的深入研究提供技术支持。
2、小儿结肠壁内的Cajal间质细胞和NOS阳性神经网络状结构已经形成,具有了调控结肠运动的结构基础,肌间神经丛中NOS阳性神经元与肌间ICCs(IC-MY)关系密切,NO能神经可能对肌间ICCs(IC-MY)起调节作用。
3、无神经节性巨结肠病患儿病变段结肠壁内三种亚型的Cajal间质细胞(IC-IM,IC-SM和IC-MY)数量均明显减少甚至缺失,病变肠管丧失产生电慢波的能力,同时也有神经节细胞的减少共同导致了HD动力紊乱。
Aganglionic megacolon, i.e. Hirschsprung's disease (HD), is a functional intestinal obstruction with an incidence of 1/3000-1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. With regard to the treatment of aganglionic gut conditions, surgical intervention is frequently the only option, whereas surgical therapy does not always lead to a complete recovery and restoration of all bowel functions, which followed by some complications such as constipation, fecal incontinence and most serious enterocolitis, colonic rupture and psychic problem. Some researchers elucidated the possibility of intracolonic grafting of neural stem cells(NSCs) as a therapeutic methed for HD. Although the grafted NSCs can survive, differentiate into neuronal phenotypes and partly improve denervated colon motility recovery in the aganglionic colon wall after transplantation. The results were not so good, indicating that there might be some other factors participating in the development of gastrointestinal motility disorders in HD. Recently more and more attention has been paid to the studies associated with interstitial cells of Cajal.
Interstitial cells of Cajal (ICCs) was first described in 1893 by the great Spanish neuroanatomist, Santiago Ramon y Cajal. ICCs were located between gastrointestinal smooth muscle cells and nerve endings. After more than a century studies of morphology and physiology, investigators have been certain that ICCs are neither neurons nor glial cells, but not smooth muscle cells. They form cellular network in gastrointestinal tract and play a very important role in the regulation of the gastrointestinal motility by generating and propagating spontaneous electric slow-vave activity and mediating excitatory and inhibitory neurotransmission. Many studies show that decreased numbers or disrupted networks of ICCs were associated with gastrointestinal motility disorders, including Hirschsprung's disease. To study the relativity between abnormalities of ICCs and HD will help understanding the pathogenesis of HD, providing new base for cell transplantation research.
Based on the above concept, we addressed this issue to study the relationship between interstitial cells of Cajal and Hirschsprung's disease deeply. This study can be divided into three parts:
Part one: Isolation and culture of interstitial cells of Cajal from rat colon in vitro
Although there are many researches on ICC now, the procedure in isolation and culture of ICC still has many difficulties that should be studied and discussed. In order to identify ICCs more accurately from cell morphology, we make much efforts in studying the isolation, culture ICCs from rat colon in vitro. As we know, the quantity of ICCs in gastrointestinal tract is very small. They are located between smooth muscle cells and nerve fibers, embeded in dense connective tissue matrix. It is very difficult to isolate ICCs from rat colon. After trying so many times, using Collagenase and pancreatin digestion and ficoll density centrifugation, we successfully isolated and cultured ICCs from rat colon in vitro. The antibody of c-kit (CD117) was used to identify the cultured ICCs. The colon tissue of Wistar rat was dissected. Collagenase and pancreatin were used to isolate ICCs. The cell suspension was resuspended in M199 medium containing stem cell factor (stem cell factor, SCF). After 24 hours, we changed the medium for the first time. Then the medium was changed every other day. After 3 days, cultured ICCs exhibited a few axis-cylinders and longer axis-cylinders were observed to form synapse each other after 5 days. More widespread connections formed within 7 days in vitro. The change of its morphologic character were obvious within 7days. Flourescent staining with c-kit antibody confirmed that the cultured ICCs was successful.
Part two: The distribution pattern of interstitial cells of Cajal in children colon.
Normal colon specimens were obtained from seven children (age from 5 months to 26 months, mean age 11.6 months,5 boys and 2 girls) at the time of bladder augmentation. All specimens were obtained from the same part of a relevent region immediately after resection. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal was visualized by immunohistochemistrical method using monoclonal mouse anti-human c-kit (CD117) antibody. c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), in submucosal border (IC-SM) and smooth muscle layer (IC-IM). IC-MY formed a dense network encasing the myenteric plexus. IC-IM appeared as long, thin, bipolar cells with one or two processes connecting with each other. NOS positive neurons were evaluated by nNOS fluorescent staining on whole-mount preparations and frozen serial sections. A great number of NOS positive neurons and nerves were found within the circular muscle layer and myenteric plexus of the colon. Double labelling of nNOS and c-kit immunohistochemistry on frozen serial sections revealed a close distribution between NOS positive nerves and ICCs network surrounding myenteric plexus, which suggested that both NOS positive nerves and ICCs contribute to normal gastrointestinal tract motility.
Part three: Altered distribution of intersitial cells of Cajal in Hirschsprung's disease.
Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal and NOS positive neurons were visualized by c-kit (CD117) and nNOS fluorescent staining on frozen serial sections respectively. Morphometric analysis was performed to determine the distribution and density of c-Kit -positive ICCs in controls and patients. In the aganglionic colon of HD, ICCs located in the three regions (IC-SM, IC-IM, IC-MY) were markedly decreased in number. NOS positive nerves were aslo reduced in number. Expression of c-kit and nNOS mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), while expression of c-kit and nNOs protein were detected by Western blot analysis. The results show that c-kit mRNA and protein expression significantly declined in the HD group. Decreased expression of nNOS mRNA and protein were demonstrated in the HD group compared with the control group. The declined expression of c-kit gene and nNOS gene were associated with HD.
Conclusions
1. We have established the method to isolate and culture interstitial cells of Cajal from the Wistar rat colon in vitro. And our study offer the base for investigating the biologic property of ICCs.
2. The distribution pattern of interstitial Cajal cells, NOS-positive neurons and smooth muscle cells in the children colon is similar to that of adult. Each of them play an important role in conrolling gastrointestinal tract motility and physiological function. NOS-positve neurons in human intestinal myenteric plexus might have some effects on IC-MY.
3. We demonstrated the distribution and density of different subtypes of ICCs within the colon of recto-sigmoid HD and TCA patients and that each subtype of ICCs was decreased in number or was even lacking. The lack or reduction of ICCs in TCA and HD patients may lead to defective generation of pacemaker activity, thus contributing, together with the lack of neurons, to the motility dysfunction present in HD.
Cajal间质细胞(interstitial cells of Cajal,ICCs)是分布在胃肠道内由中胚层间充质细胞分化而来的一类细胞,由西班牙神经解剖学家Cajal在1893年首次描述。它既不是神经元,也不是神经胶质细胞,更不是平滑肌细胞,而是具有独立功能的特殊间质细胞。目前Cajal间质细胞的生物学特性和特殊功能越来越受到国内外学者的关注。研究发现,这类细胞是胃肠道平滑肌的起搏细胞,能产生生理慢波,并参与电活动的扩布,从而调控胃肠道平滑肌的舒缩和蠕动,其数量减少或缺失与多种胃肠动力性疾病的发生密切相关。先天性巨结肠病属胃肠动力性疾病之一,Cajal间质细胞的异常与先天性巨结肠的发病也必然存在一定的相关性。揭示Cajal间质细胞的异常与先天性巨结肠病发病的相关关系,可进一步阐明无神经细胞性巨结肠病的发病机理,并为先天性巨结肠病的细胞替代治疗提供实验依据。
根据上述理念,我们对Cajal间质细胞与先天性巨结肠病的相关性进行了比较深入的研究,论文发表于Neuroscience Letters杂志,实验分为三个部分:
第一部分大鼠结肠Cajal间质细胞的分离与培养
为了更好地从形态上识别Cajal间质细胞和利用特异性表面抗原准确地鉴别Cajal间质细胞,从而为下一步的研究奠定基础,我们对大鼠结肠壁内的Cajal间质细胞进行了分离和体外培养。体外细胞培养,是研究细胞功能以及相应细胞信号转导机制的基础,也有利于更深入地掌握疾病本质,制定合理有效的防治措施。但是由于Cajal间质细胞的数量少,分散在结缔组织、神经纤维及平滑肌等组织之中,分离提取的难度较大。对于这类细胞的体外培养,由于无这样的经验可循,因而难度更大。我们采用胶原酶及胰酶酶解和密度梯度离心法,经过多次调配两种酶的浓度和作用时间,成功地对Cajal间质细胞进行了分离。分离出细胞后,将细胞悬液接种于含有干细胞因子(stem cell factor,SCF)的M199培养基中,进行培养。培养24小时后,ICCs即可贴壁生长,培养3天后,可分辨出ICCs形态,胞体呈三角形或梭形,有少量突起,核大。培养5天后,ICC形态更为清晰,自胞体发出的突起向周围伸长,相邻细胞胞体伸出的突起相互靠近。培养7天后,相邻ICC突起间有连接形成,细胞间彼此联系,突起相互交织,形成网络状结构。细胞培养稳定后,用Cajal间质细胞的特异性标志物c-kit进行免疫细胞化学染色,结果显示Cajal间质细胞呈阳性着色。
第二部分小儿结肠壁内Cajal间质细胞的正常分布模式的检测
研究表明,从小鼠的胚胎时期直至出生后,Cajal间质细胞在胃肠道内有一个不断发育成熟的过程,并形成不同的亚型。根据Cajal间质细胞在胃肠道内分布部位的不同,通常将其分为三个亚型,即位于肠壁粘膜下层的粘膜下ICCs,称为IC-SM;位于纵行肌与环形肌之间的肌间ICCs,称为IC-MY;位于肌层内的肌内ICCs,称为IC-IM。有关小儿结肠壁内的三种亚型的Cajal间质细胞的正常分布状况及其功能至今仍未见报道。基于此,我们收集临床手术切除的小儿正常结肠标本,用小鼠抗人c-kit单克隆抗体(CD117)作标记,免疫组织化学染色,荧光显微镜检测了结肠标本冰冻切片中ICCs的分布状况,观察了三种亚型的ICCs在小儿正常结肠壁中的分布状况,结果显示肌间ICCs(IC-MY)含量最为丰富,呈网络状包绕在肌间神经节细胞周围,肌层内ICCs(IC-IM)数量也较多,与平滑肌细胞平行分布,粘膜下ICCs(IC-SM)的数量最少。用兔抗人nNOS多克隆抗体标记NOS神经,免疫组织化学染色,荧光显微镜检测了标本冰冻切片及肠壁铺片中NOS神经元及神经纤维的分布情况,结果显示,在纵行肌和环形肌之间,NOS阳性神经元和神经纤维形成网络状结构,肌层内也有较多的NOS阳性神经纤维平行于平滑肌细胞分布。应用c-kit与nNOS双重染色检测了结肠壁中ICCs和NOS阳性神经元及神经纤维的位置关系,结果显示,NOS阳性神经纤维与ICCs之间密切接触,ICCs与平滑肌细胞的接触也很密切。上述实验结果提示,小儿结肠壁内的Cajal间质细胞已形成正常的规律性分布,并与肠神经及平滑肌之间存在着密切接触,可能由此形成肠壁规律性舒缩的结构基础。
第三部分Cajal间质细胞在先天性巨结肠病中的异常变化
选择确诊为先天性巨结肠的患儿21例,其中普通型巨结肠16例,全结肠型巨结肠(TCA)5例,取巨结肠根治术标本痉挛段全层肠壁作为实验组,另取7例正常结肠相应节段标本作为对照组,冰冻切片,片厚10μm。用小鼠抗人c-kit单克隆抗体(CD117)作标记,免疫组织化学染色,荧光显微镜检测了实验组及对照组结肠标本冰冻切片中ICCs的分布状况,结果显示,与正常组结肠相比,实验组各结肠标本中三种亚型的ICCs(IC-MY,IC-IM,IC-SM)数量均明显减少,有显著差异(P＜0.01),与普通型巨结肠相比,肌间ICCs(IC-MY)在全结肠型巨结肠中的数量减少更为明显,有统计学差异(P＜0.01),而粘膜下ICCs(IC-SM)和肌内ICCs(IC-IM)的分布则无明显差异性(P＞0.05)。用兔抗人nNOS多克隆抗体标记NOS神经,免疫组织化学染色,荧光显微镜检测了两组标本冰冻切片及肠壁铺片中NOS神经元及神经纤维的分布情况,结果显示,实验组NOS阳性神经元及神经纤维数量明显减少,甚至缺失,与对照组相比,差异显著(P＜0.01)。应用c-kit与nNOS免疫荧光双重染色,检测了两组标本冰冻切片中ICCs和NOS阳性神经的位置关系,结果显示NOS阳性神经元及神经纤维与ICCs位置疏远,无密切接触。同时,我们还将收集到的结肠标本的肌层,匀浆后提取总RNA和蛋白,应用逆转录多聚酶联反应(Reverse TranscriptionPolymerase Chain Reaction,RT-PCR),在mRNA水平上检测正常结肠和HD痉挛段结肠c-kit基因、nNOS基因与看家基因GAPDH的表达,用Western blot方法检测两组标本中c-kit蛋白及nNOS蛋白的表达,对实验结果条带进行半定量分析,结果证实,先天性巨结肠病变肠壁内c-kit蛋白和mRNA表达明显降低(P＜0.01),nNOS蛋白与mRNA表达也明显减少(P＜0.01)。以上实验结果提示,无神经节性巨结肠病患儿病变段结肠壁内三种亚型的Cajal间质细胞的数量明显减少甚至缺失,病变段肠管必然丧失产生电慢波的能力,同时NOS阳性神经元及神经纤维的数量也明显降低,导致或加剧了巨结肠病变段肠管的功能紊乱。
结论
1、本课题成功地对Wistar大鼠结肠Cajal间质细胞进行了分离和体外培养,体外培养的ICCs可以形成独立网络状结构,从而为这种细胞的深入研究提供技术支持。
2、小儿结肠壁内的Cajal间质细胞和NOS阳性神经网络状结构已经形成,具有了调控结肠运动的结构基础,肌间神经丛中NOS阳性神经元与肌间ICCs(IC-MY)关系密切,NO能神经可能对肌间ICCs(IC-MY)起调节作用。
3、无神经节性巨结肠病患儿病变段结肠壁内三种亚型的Cajal间质细胞(IC-IM,IC-SM和IC-MY)数量均明显减少甚至缺失,病变肠管丧失产生电慢波的能力,同时也有神经节细胞的减少共同导致了HD动力紊乱。
Aganglionic megacolon, i.e. Hirschsprung's disease (HD), is a functional intestinal obstruction with an incidence of 1/3000-1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. With regard to the treatment of aganglionic gut conditions, surgical intervention is frequently the only option, whereas surgical therapy does not always lead to a complete recovery and restoration of all bowel functions, which followed by some complications such as constipation, fecal incontinence and most serious enterocolitis, colonic rupture and psychic problem. Some researchers elucidated the possibility of intracolonic grafting of neural stem cells(NSCs) as a therapeutic methed for HD. Although the grafted NSCs can survive, differentiate into neuronal phenotypes and partly improve denervated colon motility recovery in the aganglionic colon wall after transplantation. The results were not so good, indicating that there might be some other factors participating in the development of gastrointestinal motility disorders in HD. Recently more and more attention has been paid to the studies associated with interstitial cells of Cajal.
Interstitial cells of Cajal (ICCs) was first described in 1893 by the great Spanish neuroanatomist, Santiago Ramon y Cajal. ICCs were located between gastrointestinal smooth muscle cells and nerve endings. After more than a century studies of morphology and physiology, investigators have been certain that ICCs are neither neurons nor glial cells, but not smooth muscle cells. They form cellular network in gastrointestinal tract and play a very important role in the regulation of the gastrointestinal motility by generating and propagating spontaneous electric slow-vave activity and mediating excitatory and inhibitory neurotransmission. Many studies show that decreased numbers or disrupted networks of ICCs were associated with gastrointestinal motility disorders, including Hirschsprung's disease. To study the relativity between abnormalities of ICCs and HD will help understanding the pathogenesis of HD, providing new base for cell transplantation research.
Based on the above concept, we addressed this issue to study the relationship between interstitial cells of Cajal and Hirschsprung's disease deeply. This study can be divided into three parts:
Part one: Isolation and culture of interstitial cells of Cajal from rat colon in vitro
Although there are many researches on ICC now, the procedure in isolation and culture of ICC still has many difficulties that should be studied and discussed. In order to identify ICCs more accurately from cell morphology, we make much efforts in studying the isolation, culture ICCs from rat colon in vitro. As we know, the quantity of ICCs in gastrointestinal tract is very small. They are located between smooth muscle cells and nerve fibers, embeded in dense connective tissue matrix. It is very difficult to isolate ICCs from rat colon. After trying so many times, using Collagenase and pancreatin digestion and ficoll density centrifugation, we successfully isolated and cultured ICCs from rat colon in vitro. The antibody of c-kit (CD117) was used to identify the cultured ICCs. The colon tissue of Wistar rat was dissected. Collagenase and pancreatin were used to isolate ICCs. The cell suspension was resuspended in M199 medium containing stem cell factor (stem cell factor, SCF). After 24 hours, we changed the medium for the first time. Then the medium was changed every other day. After 3 days, cultured ICCs exhibited a few axis-cylinders and longer axis-cylinders were observed to form synapse each other after 5 days. More widespread connections formed within 7 days in vitro. The change of its morphologic character were obvious within 7days. Flourescent staining with c-kit antibody confirmed that the cultured ICCs was successful.
Part two: The distribution pattern of interstitial cells of Cajal in children colon.
Normal colon specimens were obtained from seven children (age from 5 months to 26 months, mean age 11.6 months,5 boys and 2 girls) at the time of bladder augmentation. All specimens were obtained from the same part of a relevent region immediately after resection. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal was visualized by immunohistochemistrical method using monoclonal mouse anti-human c-kit (CD117) antibody. c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), in submucosal border (IC-SM) and smooth muscle layer (IC-IM). IC-MY formed a dense network encasing the myenteric plexus. IC-IM appeared as long, thin, bipolar cells with one or two processes connecting with each other. NOS positive neurons were evaluated by nNOS fluorescent staining on whole-mount preparations and frozen serial sections. A great number of NOS positive neurons and nerves were found within the circular muscle layer and myenteric plexus of the colon. Double labelling of nNOS and c-kit immunohistochemistry on frozen serial sections revealed a close distribution between NOS positive nerves and ICCs network surrounding myenteric plexus, which suggested that both NOS positive nerves and ICCs contribute to normal gastrointestinal tract motility.
Part three: Altered distribution of intersitial cells of Cajal in Hirschsprung's disease.
Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal and NOS positive neurons were visualized by c-kit (CD117) and nNOS fluorescent staining on frozen serial sections respectively. Morphometric analysis was performed to determine the distribution and density of c-Kit -positive ICCs in controls and patients. In the aganglionic colon of HD, ICCs located in the three regions (IC-SM, IC-IM, IC-MY) were markedly decreased in number. NOS positive nerves were aslo reduced in number. Expression of c-kit and nNOS mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), while expression of c-kit and nNOs protein were detected by Western blot analysis. The results show that c-kit mRNA and protein expression significantly declined in the HD group. Decreased expression of nNOS mRNA and protein were demonstrated in the HD group compared with the control group. The declined expression of c-kit gene and nNOS gene were associated with HD.
Conclusions
1. We have established the method to isolate and culture interstitial cells of Cajal from the Wistar rat colon in vitro. And our study offer the base for investigating the biologic property of ICCs.
2. The distribution pattern of interstitial Cajal cells, NOS-positive neurons and smooth muscle cells in the children colon is similar to that of adult. Each of them play an important role in conrolling gastrointestinal tract motility and physiological function. NOS-positve neurons in human intestinal myenteric plexus might have some effects on IC-MY.
3. We demonstrated the distribution and density of different subtypes of ICCs within the colon of recto-sigmoid HD and TCA patients and that each subtype of ICCs was decreased in number or was even lacking. The lack or reduction of ICCs in TCA and HD patients may lead to defective generation of pacemaker activity, thus contributing, together with the lack of neurons, to the motility dysfunction present in HD.
引文
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13. Rich A, Miller SM, Gibbons SJ, et al. Local presentation of steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture. Am. J. Physiol. Gastrointest. Liver Physiol, 2003, 284:G313-G320.
14. Vanderwinden JM, Rumessen JJ, Liu H, et al. Interstitial cells of Cajal in human colon and in Hirschsprung's disease. Gastroenterology, 1996, 111: 901-910.
15. Yamataka A, Kato Y, Tibboel D, et al. A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung's disease. J Pediatr Surg, 1995, 30:441-444.
16. Gershon MD, Erde SM. The nervous system of the gut. Gastroenterology, 1981, 80: 1571-94.
17. Costa M, Brookes SJ. The enteric nervous system. AM J Gastroentero, 1994, 89: S129-137.
18. Bult H, Boeckxsanens GE, Pelckmans PA, et al. Nitric oxide as an inhibitory non-adrenerige non-cholinergic neurotransmitter. Nature, 1990, 345: 346—347.
19. Stark ME, Szurszewskl JH. Role of nitric oxide in gastrointestinal and hepatic functions and disease. Gastroenterology, 1992,103:1928-1949.
20. Nathaan C, Xie QW. Regulation of biosynthesis of nitric oxide. J biol Chem, 1994, 269: 13725-8.
21. Bredt DS, Hwang PM,Glatt CE,et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature, 1991, 351: 714-8.
22. Ekblad E, Mulder H, Uddman R, et al. NOS containing neurons in the rat gut and coeliac ganglia. Neuropharmology, 1994, 33: 1323-1331.
23. Jarvinen MK , Wollmann WJ, Powrozek TA, et al. Nitric oxide synthase-containing neuron in the myenteric plexus of the rat gastrointestinal tract: Distribution and regional density. Anal Embryol Bed, 1999,199: 99-112.
24. Ward SM, Beckett EAH, Wang XY, et al. Interstitial cells of Cajal mediated cholinergic neurotransmission from enteric motor neurons. J Neurosci, 2000,15: 1393-1403.
25. Ward SM, Morris G, Reese L, et al. Interstitial cells of Cajal mediated enteric inhibitory neurotransmission in the lower oesophagus and pyloric sphincters. Gastroenterology, 1998, 115 : 314-329.
26. Sanders KM. A case of interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology, 1996, 111: 429-515.
27. Der-Silaphet T, Malysz J, Hagel S, et al. Interstitial cells of cajal direct normal propulsive contractile activity in the mouse small intestine. Gastroenterology, 1998,114:724-736.
28. Boeckxstaens GE. Understanding and controlling the enteric nervous system, Best Practice & Research clinical. Gastroenterology, 2002, 16: 1013-1023.
29. Faussone-Pellegrini MS, Cortesini C. Ultrastructural features and localization of the interstitial cells of Cajal in the smooth muscle coat of human esophagus. J Submicrosc Cytol, 1985, 17 : 187-197.
30. Isozaki K, Hirota S, Miyagawa J, et al. Deficiency of c-Kit+ cells in patients with a myopathic form of chronic idiopathic intestinal pseudoobstruction. Am J Gastroenterol, 1997,92: 332-334.
31. Kenny SE, Vanderwinden JM, Rintala RJ, et al. Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. Report of two cases. J Pediatr Surg, 1998b, 33: 94-98.
32. He CL, Burgart L, Wang L, et al. Decreased interstitial cell of Cajal volume in patients with slow-transit constipation. Gastroenterology, 2000, 118: 14-21.
33. Kenny SE, Connell MG, Rintala RJ, et al. Abnormal colonic interstitial cells of Cajal in children with anorectal malformations. J Pediatr Surg, 1998a, 33: 130-132.
34. Burns AJ, Lomax AEJ, Torihashi S, et al. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci U S A, 1996, 93: 12008-12013.
35. Lyford GL, He CL, Soffer E, et al. Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation. Gut, 2002, 51: 496-501.
36. Torihashi S, Horisawa M, Watanabe Y. C-Kit immunoreactive interstitial cells in the human gastrointestinal tract. J Auton Nerv Syst, 1999, 75: 38-50.
37. Hagger R, Gharaie S, Finlayson C, et al. Distribution of the interstitial cells of Cajal in the human anorectum. J Auton Nerv Syst, 1998, 73 : 75-79.
38. Tong WD, Liu BH, Zhang LY, et al. Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation. Int J Colorectal Dis, 2004, 19:467-473.
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21. Sanders KM. A case of interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology, 1996, 111: 429-515.
22. Ward SM, Morris G, et al. Interstitial cells of Cajal mediated enteric inhibitory neurotransmission in the lower oesophagus and pyloric sphincters. Gastroenterology, 1998, 115:314-329.
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9. Vanderwinden JM, Rumessen JJ. Interstitial cells of Cajal in human gut and gastrointestinal disease. Microse Res Tech, 1999,47:344-60.
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26. Lyford GL, He CL, Soffer E,et al. Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation. Gut, 2002, 51: 496-501.
27. Torihashi S, Horisawa M, Watanabe Y. c-Kit immunoreactive interstitial cells in the human gastrointestinal tract. J Auton Nerv Syst, 1999, 75: 38-50.
28. Hagger R, Gharaie S, Finlayson C,et al. Distribution of the interstitial cells of Cajal in the human anorectum. J Auton Nerv Syst, 1998, 73: 75-79.
29. Tong WD, Liu BH, Zhang LY, et al. Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation. Int J Colorectal Dis, 2004,19:467-473.
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5. Liu w, Wu RD, Dong YL, et al. Neuroepithelial Stem Cells Differentiate into Neuronal Phenotypes and Improve Intestinal Motility Recovery after Transplantation in the Aganglionic Colon of the Rat. Neurogastroent Motil, 2007, 19: 1001-1009.
6. Dong YL, Liu W, Gao YM, et al. Neural stem cell transplantation rescues rectum function in the aganglionic rat. Transplant Proc, 2008 Dec, 40: 3646-52.
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10. Hirota S, Isozaki K, Nishida T, ea al. Effects of loss-offunction and gain-of-function mutations of c-kit on the gastrointestinal tract. J Gastroenterol, 2000, 35 [Suppl 12]: 75-79.
11. Kitamura Y, Hirota S, Nishida T. A loss-of-function mutation of c-kit results in depletion of mast cells and interstitial cells of Cajal, while its gainof-function mutation results in their oncogenesis. Mutat Res, 2001, 477:165-171.
12. Lecoin L, Gabella G, Le Douarin N. Origin of the c-kit-positive interstitial cells in the avian bowel. Development, 1996, 122: 725-733.
13. Rich A, Miller SM, Gibbons SJ, et al. Local presentation of steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture. Am. J. Physiol. Gastrointest. Liver Physiol, 2003, 284:G313-G320.
14. Vanderwinden JM, Rumessen JJ, Liu H, et al. Interstitial cells of Cajal in human colon and in Hirschsprung's disease. Gastroenterology, 1996, 111: 901-910.
15. Yamataka A, Kato Y, Tibboel D, et al. A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung's disease. J Pediatr Surg, 1995, 30:441-444.
16. Gershon MD, Erde SM. The nervous system of the gut. Gastroenterology, 1981, 80: 1571-94.
17. Costa M, Brookes SJ. The enteric nervous system. AM J Gastroentero, 1994, 89: S129-137.
18. Bult H, Boeckxsanens GE, Pelckmans PA, et al. Nitric oxide as an inhibitory non-adrenerige non-cholinergic neurotransmitter. Nature, 1990, 345: 346—347.
19. Stark ME, Szurszewskl JH. Role of nitric oxide in gastrointestinal and hepatic functions and disease. Gastroenterology, 1992,103:1928-1949.
20. Nathaan C, Xie QW. Regulation of biosynthesis of nitric oxide. J biol Chem, 1994, 269: 13725-8.
21. Bredt DS, Hwang PM,Glatt CE,et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature, 1991, 351: 714-8.
22. Ekblad E, Mulder H, Uddman R, et al. NOS containing neurons in the rat gut and coeliac ganglia. Neuropharmology, 1994, 33: 1323-1331.
23. Jarvinen MK , Wollmann WJ, Powrozek TA, et al. Nitric oxide synthase-containing neuron in the myenteric plexus of the rat gastrointestinal tract: Distribution and regional density. Anal Embryol Bed, 1999,199: 99-112.
24. Ward SM, Beckett EAH, Wang XY, et al. Interstitial cells of Cajal mediated cholinergic neurotransmission from enteric motor neurons. J Neurosci, 2000,15: 1393-1403.
25. Ward SM, Morris G, Reese L, et al. Interstitial cells of Cajal mediated enteric inhibitory neurotransmission in the lower oesophagus and pyloric sphincters. Gastroenterology, 1998, 115 : 314-329.
26. Sanders KM. A case of interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology, 1996, 111: 429-515.
27. Der-Silaphet T, Malysz J, Hagel S, et al. Interstitial cells of cajal direct normal propulsive contractile activity in the mouse small intestine. Gastroenterology, 1998,114:724-736.
28. Boeckxstaens GE. Understanding and controlling the enteric nervous system, Best Practice & Research clinical. Gastroenterology, 2002, 16: 1013-1023.
29. Faussone-Pellegrini MS, Cortesini C. Ultrastructural features and localization of the interstitial cells of Cajal in the smooth muscle coat of human esophagus. J Submicrosc Cytol, 1985, 17 : 187-197.
30. Isozaki K, Hirota S, Miyagawa J, et al. Deficiency of c-Kit+ cells in patients with a myopathic form of chronic idiopathic intestinal pseudoobstruction. Am J Gastroenterol, 1997,92: 332-334.
31. Kenny SE, Vanderwinden JM, Rintala RJ, et al. Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. Report of two cases. J Pediatr Surg, 1998b, 33: 94-98.
32. He CL, Burgart L, Wang L, et al. Decreased interstitial cell of Cajal volume in patients with slow-transit constipation. Gastroenterology, 2000, 118: 14-21.
33. Kenny SE, Connell MG, Rintala RJ, et al. Abnormal colonic interstitial cells of Cajal in children with anorectal malformations. J Pediatr Surg, 1998a, 33: 130-132.
34. Burns AJ, Lomax AEJ, Torihashi S, et al. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci U S A, 1996, 93: 12008-12013.
35. Lyford GL, He CL, Soffer E, et al. Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation. Gut, 2002, 51: 496-501.
36. Torihashi S, Horisawa M, Watanabe Y. C-Kit immunoreactive interstitial cells in the human gastrointestinal tract. J Auton Nerv Syst, 1999, 75: 38-50.
37. Hagger R, Gharaie S, Finlayson C, et al. Distribution of the interstitial cells of Cajal in the human anorectum. J Auton Nerv Syst, 1998, 73 : 75-79.
38. Tong WD, Liu BH, Zhang LY, et al. Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation. Int J Colorectal Dis, 2004, 19:467-473.