The Study Of The Change Of Microenvironment In Colon And The Correlation Of Laminin Gene And Ret Gene In Hirschsprung's Disease

ObjectiveThe incidence of Hirschsprung's disease (HD ) is very high, and it is one hot spot of research work in pediatric surgery. Although there are marked absence of ganglion cells and proliferation of nerve fibers in the aganglionic colon of patients with Hirschsprung's disease, the cause of Hirschsprung's disease is still not clear. Controversy exists as to not only why the development of enteric nervous system (ENS) is abnormal but also the mechanism of the occurrence of constricted segment in Hirschsprung's disease. In order to explain the occurrence of HD, we discuss the role of microenvironment in the occurrence of HD through the immunohistochemistry study of the expression of laminin (LN), laminin receptor(LNR) and fibronectin(FN) in Hirschsprung's disease, and studying the expression of laminin gene and RET gene in the aganglionic segment, ganglionic segment and normal segment of colon in HD by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative PCR.MethodsSpecimens were attained from 27 patients with confirmed pathological diagnosis of HD, pre- or post-operatively, in the pediatric surgery department of Qilu Hospital of Shandong University in 2004. The narrow segment was taken as the aganglionic segment, the transitional segment or the distal end of the dilated segment with normal ganglion cells conformed by pathological diagnosis was taken as the ganglionic segment, and the proximate of the dilated segment was taken as the normal segment. 200 mg colonic tissue from each of the three segments was taken respectively and was stored in -84℃. Similar groups of tissue from the three segments were fixed in 10% neutral-formalin and embed with paraffin following.Specimen of the aganglionic segment, ganglionic segment and normal segment of colon in 27 cases of HD were stained by polmer detection system for immunohistological staining, photos were taken by RS Image system, and the staining area of every image of LN,LNR and FN were calculated by JD 801 Image analysis system.The qualitative expressions of laminin gene and RET gene of these three segments in 27 cases were detected by RT-PCR, and the difference of the expression was analyzed by alpha 9900 image analysis system, and the relative coefficient was calculated, which equals to the expressive intension of the goal gene divided by the expressive intension of b-actin. The relative coefficients were compared by One-way ANOVA of SPSS for windows 11.5 Software.The quantitative expressions of laminin gene and RET gene in the three segments were detected by real-time quantitative PCR, and the difference of the expression was concluded by SDS software and the 2-⊿⊿ CT method. The correlation between the expression of laminin alpha 1 and RET gene was calculated by Bivariate-pearson way of SPSS for windows 11.5 Software.The relation between the expression of LN,LNR,FN and the cause of HD and thecorrelation between the expression of two gene would be concluded.ResultsLN,LNR and FN were expressed in the three segment, the expression in aganglionicsegment was highly increased compared to the dilated segment(P<0.05), and it wasalso different compared between aganglionic segment and transitional segment(P<0.05) , no evident different between transitional segment and dilated segment except FN.The OD value of RNA which was calculated by A260/A280 was from 1.8 to 2.0. The laminin gene expressed in all the three segments. The relative coefficient of laminin gene in aganglionic segment, ganglionic segment and normal segment were 0.73±0.18,0.39±0.15,0.15±0.12.The expression in aganglionic segment showed highly increasing as compared to the dilated segment(P<0.05 ). It was showed that the expression of laminin alpha 1 in aganglionic segment was 2 times of that in normal segment by the real-time quantitative PCR, and the expression from aganglionic segment to normal segment showed decreased stepwise. While the expression of RET gene was just the opposite, the relative coefficient of RET gene in aganglionic segment, ganglionic segment and normal segment were 0.07±0.05, 0.33±0.14, 0.79±0.19 (P<0.01) . It was showed that the expression of RET in aganglionic segment was 0.07 times than that in normal segment by the real-time quantitative PCR, and the quantity of RET mRNA was increasing gradually from the aganglionic segment to the normal segment.The correlation between the expressions of two gene was inverse correlation, the coefficient correlation was -0.638, P<0.001. In aganglionic segment, the laminin alpha 1mRNA increased, while the RET mRNA reduced.ConclusionsThe expression of LN,LNR,FN in aganglionic segment were highly increased, and The expression of LNmRNA was also highly increased. So we caught conclude that the highly increased expression of LN,LNR,FN in aganglionic segment were intrinsic, and they may cause early differentiation, early maturation and premature ecesis of enteric nervous cells. The change of the microenvironment of colon wall may be the cause of Hirschsprung's disease. The expression of RETmRNA in aganglionic segment was evidently decreased. The inverse correlation between the expression of the two genes may have some close relation to the occurrence of HD.