Expression Of Glial Cell Derived Neurotrophic Factor MRNA And Interstitial Cells Of Cajal In Hirschsprung's Disease

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.