The Study Of Differentially Expressed Gene And Expressed Changes Of Tight Junction Protein In Children With Pancreaticobiliary Maljunction

Background: The pancreaticobiliary maljunction is considered to be due to the early join of the common bile duct and the pancreatic duct outside the duodenal wall.The excessive common channel causes that the Oddi sphincter of the duodenal papilla cannot control and regulate the confluence,causing the fluid mechanical abnormality in the join of pancreatic duct and bile duct.Due to reflux of bile and pancreatic juice and long time of pancreatic juice and bile mixed,all kinds of pancreatin,such as phospholipase A2 and trypsin,are activated in the bile duct.It poses a serious challenge to the quality of life and health of the patients.An important link in the course of the occurrence of biliary tract diseases is considered to be the destruction of the bile duct barrier.The structural basis of the function of the mucosal barrier of the biliary tract derived from the tight-junction of the Interepithelial cell.Tight junctions of the bile duct epithelium maintain the polarity of the epithelial cells of the bile duct and the one-way flow of bile in the lumen.The loss of the integrity of the tight junction leads to the structural deformation of the bile duct.A large number of evidence suggests that many cholestatic diseases,such as cirrhosis and biliary atresia,are associated with tight junction destruction and biliary barrier dysfunction.We used gene chip technology to screen differentially expressed genes in children with pancreaticobiliary maljunction.qRT-PCR verification shows that tight junction signaling pathway may play an important role in the pathogenesis of PBM children.However,there are few domestic research reports on the tight junction of the biliary tract epithelia in children with PBM.Therefore,we studied the expression and distribution of tight junction proteins in biliary tract epithelium of PBM patients.This provides a basis for further study of the evolution of the tight junction damage of the biliary tract mucosa in children with PBM.Part One The Study of differentially expressed genes in children with pancreaticobiliary maljunction.Objective: Using human genome expression spectrum chip technology,we analyzed the difference of gene expression profiles between children with pancreaticobiliary maljunction and healthy children,and screened out abnormal genes associated with pancreaticobiliary maljunction.Methods: The total RNA of peripheral blood mononuclear cells from 5 children with pancreaticobiliary maljunction and 3 healthy controls was extracted and purified byTrizol method.RNA formed cDNA by reverse transcriptase.We used cDNA microarray to detect and compare gene expression in children with pancreaticobiliary maljunction and normal children.Then we analyzed the differentially expressed genes by GO and Pathway.At the same time,we made qRT-PCR verification for differentially expressed genes EPB41L2,GNAI3,MPP5,MRAS in Tight-junction signaling pathway and differentially expressed genes IL13?IL4 in Jak-STAT signaling pathway.Results: A total of 4949 differentially expressed genes(Cut off=2.0)were screened for children with pancreaticobiliary maljunction,of which 2628 were up and 2321 were down.The cluster map showed that there was a distinct difference in expression profiles between PBM and normal children.GO analysis showed there were a large difference in biological processes and molecular functions.The pathway analysis showed that the up-regulated pathways were mainly Tight-juntion signaling and Jak-STAT signaling.The results of qRT-PCR showed that the up-regulated multiple of genes EPB41L2,GNAI3,MPP5,MRAS,IL13?IL4 respectively was 1.684211±0.26885925,2.742857±0.833693,2.742857±0.833693,1.8125±0.269251,2±0.351104,1.818182±0.298205,1.761905±0.321678.the expression trend was consistent with the result of the chip.Conclusion: 1.In children with PBM,the expression of genes EPB41L2,GNAI3,MPP5,MRAS,IL13 and IL4 is up-regulated.2.Tight-junction signal pathway may be involved in the destruction process of epithelial barrier in PBM children.3.Jak-STAT signal pathway may participate in the chronic inflammatory process of biliary tract in children with PBM.Part Two The significance and change of the expression of tight junction and related protein in biliary duct epithelium of children with pancreaticobiliary maljunctionObjective: To investigate the expression of Occludin,Claudin-2,ZO-1 and myosin light chain kinase in biliary duct epithelium of children with pancreaticobiliar maljuntion.Methods: We collected 15 cases of bile duct tissue specimens of patients with PBM as experimental group,using Western-Blot and immunohistochemical staining technique for detecting the expression of Occludin,Claudin-2,ZO-1 and myosin light chain kinase.10 cases of normal bile duct tissue were taken as control.We used Spearman correlation analysis to analyze the relationship between myosin light chain kinase and expression of Claudin-2,Occludin,and ZO-1.Results: 1.Western-blot results showed that the expression of Occludin and Claudin-2 in biliary tract epithelium of children with pancreaticobiliary maljunction was decreased and the expression of ZO-1 and myosin light chain kinase was increased(P(27)0.01).2.Immunohistochemistry showed that Occludin,Claudin-2 and ZO-1 were mainly expressed on cell membrane and cytoplasm,MLCK was mainly expressed in cytoplasm,and the expression trend was consistent with Western-blot results.Spearman correlation analysis showed that the expression of MLCK was negatively correlated with the expression of Occludin(rs =-0.597,P = 0.002),and the expression of MLCK was negatively correlated with Claudin-2 expression(rs =-0.457,P = 0.022);and the expression of MLCK was positively correlated with the expression of ZO-1(rs = 0.843,P = 0.000).Conclusion: The destruction of biliary tract barrier in children with PBM may result in downregulation of tight junction protein Claudin-2 and Occludin and increased expression of ZO-1 in biliary epithelium.MLCK may be involved in the destruction of the biliary tract epithelial barrier in children with PBM.