Reaserch Of Differential Proteomics On Liver Of Patients With Biliary Atresia And Idiopathic Cholestasis

Biliary atresia and intrahepatic cholestasis are both belong to common liver and gallbladder diseases during neonatal stage, there are many similar characteristics of them, such as occurrence during neonatal stage, light yellow or white of stool, enlargement and hardening of liver; High serum cholestatic jaundice and abnormal enzyme including increasing of gamma-GT and AST; Besides, they have common pathological characteristics too, such as hepatic cells and capillaries cholestasis, portal area of inflammatory infiltration, degeneration of liver cell necrosis. These similar characteristics make clinical and differential diagnosis difficult. As the treatment of ailiary atresia and intrahepatic cholestasis are different, surgical treatment for ailiary atresia and internal medicine treatment for intrahepatic cholestasis respectively, there exists need for differential diagnosis between these two diseases. The pathogenesis of ailiary atresia is still unclear, with participation of virus infection, immunoreactions, heteroplasia and genetic factors, the complicated reactions between gene and protein or interactions between proteins and proteins would involve; intercellular activities and environment influence expression of gene and its post transcription modification. In this study, two-dimensional electrophoresis and iTRAQ were applied to detect liver tissues from patients with ailiary atresia and intrahepatic cholestasis, analyzing differential expression of proteins to select differential expressed proteins, testified differential expression of HSP90at the same time. This study was aimed to providing clues to differential diagnosis and pathogenesis of biliary atresia and intrahepatic cholestasis.Part1:Reaserch of Differential Proteomics on Liver of Patients with Biliary Atresia and Idiopathic Cholestasis by Two-Dimensional ElectrophoresisObjectiveCollected surgical specimens from patients with biliary atresia and intrahepatic cholestasis, two-dimensional electrophoresis were applied to detect differentia] protein expression at protein level; testified expression level of HSP90. The research was aimed to provide clues for discovering pathogenesis of biliary atresia and intrahepatic cholestasis and differential diagnosis between them.Materials and MethodsCollected patients with obstructive jaundice treated by cholangiography in laparoscopic surgery,20patients diagnosed as biliary atresia were set as case group,12patients without biliary atresia were set as control group. Total proteins in liver specimen were extracted and mixed, two-dimensional electrophoresis was operated on nonlinear PH3-1024cm long IPG gel with immobilized pH gradient and12%SDS-PAGE. Stained by Coomassie brilliant blue and then utilized ImageScanner software to scan the gel, PDquest8.0software was used to analyze differential expression of proteins. Filtered expression differences twice times or more in two groups. Extracted18different protein specimens analyzed peptides after enzymatic hydrolysis by MALDI-TOF mass spectrometry then searched in MASCOT search engine. Another13liver tissues of patients were analyzed by western-blot to detect expression of HSP90.Results1. MALDI-TOF mass-spectrometric analysis on60well-separated, different and clear displayed proteins,15proteins were indentified.9proteins were up-regulated in case group including Myosin, RhoGDI, MnSOD, albumin, calreticulin and vimentin;6proteins were up-regulated in control group including HSP, PDI, carbamoyl phosphate synthetase and Annexin.2. Western-Blot showed significant increasing expression of HSP90in case group, mean gray value of53279±12288for case group and276669±70025for control group in Quantity One software,p=0.03<0.05.Conclusions:These differential proteins of liver tissue detected by tow-dimensional electrophoresis are probably correlated with occurrences, development and clinical outcomes of biliary atresia and Idiopathic cholestasis; HSP90has the potential of becoming biomarker for differential diagnosis between biliary atresia and idiopathic cholestasis, providing theoretical basis for exploration of biliary atresia and Idiopathic cholestasis. PART II:Research of Protein Differential Expression in Liver of Patients with Biliary Atresia and Idiopathic Cholestasis by iTRAQObjectiveTo acquire a broader range of differential protein expression profiling, iTRAQ was applied to detect the same specimen in part I. To discuss application of iTRAQ in differential protein expression in biliary atresia and intrahepatic cholestasis, providing further information for research of pathogenesis, early diagnosis and differential diagnosis.Materials and MethodsSpecimens utilized in this part was the same as in part Ⅰ. Patients were divided into four groups which were case group1, case group2, control group1and control group2. iTRAQ with combination of Liguid chromatography-tandem mass spectrometry was applied to analyze specimen from liver. Protein Pilot3.0software was utilized to analyze MS/MS data, Identification of protein used by the confidence interval is greater than95%, when error factor<2, P<0.05, identification of proteins has statistical significance. Quantitative results of identified proteins were processed by the Pro GroupTM Algorithm. Expression difference between groups was greater than20, which was>1.2times or<0.8times, was considered different.Results:1.After analysis of software,593proteins were identified by iTRAQ. Relativ e expression values were obtained by setting ratio of the same protein expressi on in each group. Mean expression values were calculated,25protein with hig hest expression and lowest ration were selected, exclude points with inter grou p differences> intra group differences,38proteins were identified.21were upregulated in case group including SERPH,LV301,LV403,TBB2C,SSBP,DPYL2, RL7,PRDX5,ECHD2,THTM,KAD2,COMT,H2A2C,ACOT2,SRSF3,H15,IF4B,H2A Z,HNRPM,MYH9and THIM;17were up-regulated in control group including PRDX1,HMCS2,TCPD,HBD,G ATM,THIK,FUCM,COF1,ACBP,GSTA2,CK054,HC DH,EST1,PRDX6,TPIS, RLA2and ASSY.2.Compared with the result of2D,we found HSP90、Annexin A6、Myosin, light polypeptide6and Protein disulfide isomerase also be identified by iTRAQ,and their expression tendency was similar in two different methods. Conclusions:iTRAQ was applied to detect differential expression proteins in patients with biliary atresia and Idiopathic cholestasis. Expression profiling was broader than which found by2D electrophoresis. Differential expression proteins identified by iTRAQ provided further clues in researching pathogenesis, diagnosis and prognosis in biliary atresia and Idiopathic cholestasis.