【作者】 侯豫；

【导师】 王宝西；

【作者基本信息】 第四军医大学 ， 儿科学， 2008， 硕士

【摘要】 实验背景及目的GDNF基因编码胶质细胞源性神经营养因子( glial cell derived neurotrophic factor,GDNF),它的编码产物GDNF参与细胞生长分化、增殖和凋亡有关的信号传导。近年研究表明,GDNF与众多疾病关系密切,其异常表达不仅导致某些中枢神经系统疾病,而且也是众多肠神经系统疾病的重要机制之一[1,2,3]。Cajal间质细胞(interstitial cells of Cajal,ICC)广泛分布于消化道,是胃肠慢波活动的起搏细胞及其基本电节律的主要传播细胞[4],并参与胃肠道神经信息的传递[5]。有关ICC的胚胎起源争论较多,多认为起源于间充质细胞,近年研究表明,ICC与众多胃肠道疾病关系密切,其分布异常、数目减少可能是这些疾病发病的重要病理生理机制之一。先天性巨结肠(Hirschsprung’s disease,HD)是一种肠神经系统先天性异常疾病,其具体致病因素及发病机制目前尚未完全阐明。一般认为与遗传、肠壁内环境改变、免疫及感染等因素有关。近年研究发现,HD患儿有多种基因突变,其基因的编码产物在肠道表达异常。ICC作为胃肠道基本电活动的起搏细胞,对其形态、生理功能已有较多的研究,在HD,一些基因编码产物(神经营养因子)表达异常,ICC在肠壁分布异常,HD成为深入研究ICC和某些因子关系的良好模型。但迄今为止,国内外文献尚未见有关神经营养因子和ICC比较的研究。本实验通过收集HD的标本,全部取材经组织病理学检测符合HD诊断。应用半定量RT-PCR及免疫组化技术检测HD患儿结肠手术标本组织中GDNFmRNA表达水平和ICC的分布,分析并统计其在不同肠段的表达及其两者间的关系,为进一步研究HD的发病机制和深入研究ICC提供必要的实验依据。试验方法收集42例经病理诊断为先天性巨结肠的标本,其中男33例,女9例,年龄2月-10岁,全部实验标本均为散发病例,取材经组织病理学检测符合HD诊断,5例肠套叠患儿(术前取得家属同意)结肠手术标本作为正常对照。分别收集HD患儿手术切除肠管狭窄段、移行段和扩张段全层标本,一部分液氮保存,对应部分多聚甲醛固定保存。第一部分:GDNFmRNA在HD不同肠段的表达研究。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GDNFmRNA水平,及5例肠套叠患儿结肠手术标本组织中GDNFmRNA水平,分析并统计其与临床病理特点之间的关系。第二部分:ICC在HD不同肠段的分布观察。分别取42例HD患儿不同肠段组织标本,多聚甲醛固定、脱水、包埋,常规超薄切片,采用特异性酪氨酸激酶受体(c-kit)抗体免疫组织化学法观察ICC分布。实验结果1. 5例正常对照肠管GDNFmRNA均表达阳性(100%);42例新鲜标本,狭窄段11例GDNFmRNA表达阳性(26.2%);移行段15例GDNFmRNA表达阳性(35.7%);扩张段40例GDNFmRNA表达阳性(95.2%)。与扩张段和正常对照结肠相比,狭窄段结肠组织中GDNFmRNA水平明显降低,差异具有显著性(P〈0.05);扩张段结肠和正常对照GDNFmRNA水平相当,差异无显著性(P >0.05)。2.免疫组化观察结果:(1)正常对照组结肠组织中ICC主要分布于粘膜下丛(IC-SM)和肌间丛(IC-MY),IC-MY在切面上几乎呈现连续性分布,相互连接形成网络状结构。(2)在HD患儿结肠,①狭窄段组结肠组织内ICC的分布较正常对照组和扩张段组显著减少(P<0.001),IC-MY的网络状结构完全破坏,残存ICC形态也出现异常;②移行段组结肠组织内ICC的分布较正常对照组和扩张段组减少(P<0.05),但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,未能形成正常的网络状结构;③扩张段组与正常对照组结肠组织内ICC分布相当,差异无显著性(P>0.05)。结论1. HD患儿结肠不同肠段GDNFmRNA表达有差异。GDNFmRNA在狭窄段表达量较扩张段和正常对照肠段明显减少,甚至无表达;在扩张段和正常对照肠段的表达无明显差异;在移行段的表达量与狭窄段和扩张段比较均有差异。2. HD患儿结肠不同肠段ICC的数量、形态、分布不同。在狭窄段肌间丛偶见残存的ICC,其形态变钝、突起减少或者消失;在扩张段和正常对照肠段粘膜下丛和肌间丛均可见连续分布的ICC,呈梭形有2-3个突起;在移行段ICC主要分布于肌间丛,数量居于狭窄段和扩张段之间。3. HD患儿结肠不同肠段,GDNF表达量和ICC数量存在正相关性。在狭窄段,GDNFmRNA表达减少,ICC的数量亦减少,结构破坏;两者在移行段与其在狭窄段和扩张段之间的表达量或分布均有差异;两者在扩张段的表达或分布与正常对照无明显差异。提示GDNF表达异常可能是HD发病的机制之一;ICC分布减少可能是HD胃肠动力障碍的原因之一。更多还原

【Abstract】 Background and ObjectiveThe GDNF gene encode glial cell derived neurotrophic factor, GDNF participate signal conduction associated with cell growth, differentiation, generation and apoptosis. Recent studies have indicated that the abnormal expression of GDNF have relations with many diseases, not only with central nervous system but also with enteric nervous system.Interstitial cells of Cajal (ICC),origin of the mesenchymal cells,are widely distributed throughout the gastrointestinal tract, which are believed to have a crucial role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recent studies have indicated that the abnormal distribution and the loss or decrease of ICC might play an important role in several disorders of human gastrointestinal motility.Hirschsprung’s disease(HD ) is a congenital disorder characterized by absence of enteric ganglia, its etiological facto and nosogenesis have not been completely elucidated. It is generally considered that the pathogenesis of HD have close relationship with heredity, the disorder of internal environment, immune function and infection. Recent studies have found several kinds of gene mutation, and the gene encoding productions have abnormal expression in intestinal tract. ICC, as the pacemaker cell in gastrointestinal tract, much studies focus on its morphology and physiologic function, its origin is still unknow .The abnormal expression of gene encoding production(neurotrophic factor) and the abnormal distribution of ICC in different segment of HD make it the perfect model to study the relationship between the neurotrophic factor and ICC. But until now, there are no correlative reports about the relationship between the distribution of ICC and neurotrophic factor in HD.In this study, we collected full thickness specimens from Hirschsprung’s disease patients which were proved by histopathologic diagnosis. The GDNF mRNA expression and the distribution of ICC in different parts of HD segments and non-HD segments were detected using semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR)and immunohistochemistry(IHC), then we analyzed the relationship between their expression and the clinical features, provide more experimental argument in studying pathogenesis of HD and ICCMethodsForty-two pairs of full thickness specimens(aganglionic segments and ganglionic segments)from Hirschsprung’s disease patients were collected from several hospitals soon after operation(male 33,female 9,aged from 2 months to 10 years). All the specimens were sporadic cases, and all the cases were proved by histopathologic diagnosis, including the common type 30 cases, short term type 12 cases. Another five colon specimens were obtained from intussusception patients as normal control(male 4,female1,aged from 30 days to 8 years).One part of the specimen was saved in liquid nitrogen(LN), the corresponding part was save in paraformaldehyde(PFA).Part one: The expression of GDNFmRNA in different segments of HD. The GDNF mRNA expression in different parts of HD segments of 42 cases were detected using semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR),then we analyzed the relationship between their expression and the clinical features, another five colon specimens were obtained from intussusception patients as normal control.Part two: Study of the distribution of ICC in different segments of HD.The samples from 42 HD cases were paraformaldehyde fixation, deaquation, embedment, normal extra thin section, using IHC by specific antibody(c-kit).Results1. In normal control, GDNF mRNA was positive in 5 out of 5(100%).Meanwhile, of all the 42 fresh samples of HD, GDNF mRNA was positive in 40(95.2%) in dilated segment.However, positive rate of GDNF mRNA was decreased to 15 in transitional zone(35.7%) and to 11 in spastic segment(26.2%) GDNF mRNA expression in the aganglionic segments were decreased,copared with the ganglionic segments and the control group(P<0.05〉.There were no statistical differences in GDNF mRNA expression between control group and the ganglionic segments group.2. IHC results: (1)ICC were mainly distributed in submucosal plexus (IC-SM) and myenteric plexus (IC-MY) of the distal colon, and also distributed in circular muscle layer and longitudinal muscle layer (IC-IM). IC-MY were distributed continuously between the circular and longitudinal muscle layers and formed a network;(2)In the segments of HD,①C ompared with the control group and the ganglionic segments group, ICC were decreased obviously in aganglionic segments group (P<0.001). The IC-MY network were disappeared, and the configuration of the residual ICC were abnormal.②In the transitional zone group, ICC were reduced than those of ganglionic segments group and the control group(P<0.05), but were more than those of the aganglionic segments group (P<0.001). Their configuration were close to normal, but the IC-MY did not form a normal network.③There were no difference of the distribution of ICC in ganglionic segments group and the control group (P>0.05).Conclusions1. The expression of GDNFmRNA is different in different segments of HD patients. It’s exprerssion is obvious less in aganglionic segments than that in the control and the ganglionic segments, even absence in some aganglionic segments. It’s exprerssion has no difference in control and the ganglionic segments.It has difference that the expression in transitional zone than in control and the ganglionic segments.2. The number,morphous and the distribution of ICC were different in each segments of HD. In most aganglionic segments,the ICC is absence, the relic ICC gets blunting, decreased or disappeared foot process. The ICC can be obviously found in control and the ganglionic segments,most of ICC in submucosal plexus and IC-MY form network. The number of ICC in transitional zone is small than it in ganglionic segments,and greater in aganglionic segments.3. In each segments of HD,the expression of GDNFmRNA and thenumber of ICC have positive correlation. In aganglionic segments, the number of ICC and are both decreased,and in transitional zone,their expression and distribution are between that in aganglionic segments and ganglionic segments. This sussest that the abnormal expression may be one of the pathogenesy of HD ,and the decreased of ICC in HD may be one of the cause to gastrointestinal motility disorder.更多还原