Comparative Proteomic Investigation Of Proteins Of Gallbladder Bile Involved In The Formation Of Cholesterol Gallstone And Biological Function Analysis Of Retinol Binding Protein

Part oneComparative proteomic analysis of gallbladder bile proteins from cholesterol gallstone and control groupsObjective To establish and optimize a stable and standard proteomic technique platform and screen those proteins involved in the formation of cholesterol gallstone,this study is designed to comparative analysis of the proteome of gallbladder bile from cholesterol gallstone patients and control ones.Methods Total proteins of gallbladder bile from the two groups were extracted,and the protein profiles were established by using two-dimensional electrophoresis respectively. These maps were analyzed by ImageMaster two-dimensional electrophoresis analysis software.The differentially expressed proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Results The concentrations of bile proteins from cholesterol cholelithiasis patients and control ones were(6.4824±0.3403) mg/ml and(2.3156±0.3064) mg/ml respectively (P＜0.01).The average matched protein spots were 465±71 and 407±85 in the gels from the two groups respectively.There were 34 differentially expressed protein spots and 22 of them were identified successfully,15 expressed highly in cholesterol gallstone patients but lowly in the control ones,while 7 expressed just in the opposite way.Conclusions The differential protein profiles of gallbladder bile between cholesterol cholelithiasis patients and control ones were established and 22 differential protein spots were identified.The data may be a basis for further screening the key regulators of the formation of cholesterol gallstone.Part twoDifferential analysis of protein profiles of vesicular and micellar phases of gallbladder bile from cholesterol gallstone patientsObjective To identify the proteins which play key roles during the formation of cholesterol gallstone,differential analysis was carried out that the proteome of vesicular and micellar phases of gallbladder bile from cholesterol gallstone patients.Methods Vesicular and micellar phases were isolated by the density gradient ultracentrifugation method with Metrizamide.Total proteins from the two phases were extracted,and the protein expressional profiles were established by two-dimensional electrophoresis respectively.The differentially expressed protein spots were analyzed by ImageMaster two-dimensional electrophoresis analysis software and identified with MALDI-TOF-MS.Results The average matched protein spots were 120±24 and 198±37 in the two phases respectively.There were 19 differentially expressed proteins and 8 were identified from the two cholesterol-carrier phases.Among them,6 were up-regulated with 2 down-regulated in vesicular phase compared with micellar phase.Conclusions The differential protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established and 8 differential protein spots were identified successfully. The data may be a basis for further screening the key regulators of the formation of cholesterol gallstone.Part threeVerification of differentially expressed proteins between the cholesterol gallstone and control groups and also between the vesicular and micellar phasesObjective To further confirm the results of comparative proteomics and the next functional study,the differentially expressed proteins,sieved through comparative proteomics,were verified.Methods The expression of HSA, RBP,TPM,CRT and PFN in the gallbladder tissues from cholesterol gallstone group was compared with that of control group by immunohistochemistry.The expressional levels of 5 proteins in the gallbladder bile from cholesterol gallstone and control groups and also from the vesicular and micellar phases were compared by Western Blot.Results The results of immunohistochemistry showed that HSA,RBP and CRT were up-regulated with PFN and TPM down-regulated in gallbladder tissues from cholesterol gallstone group in comparison with control group.The expressional levels of HSA,RBP and CRT were higher in gallbladder bile from cholesterol gallstone group than control group,and HSA and RBP were higher in vesicular phase than in micellar phase by Western Blot.And PFN and TPM were down-regulated in the gallbladder bile from cholesterol gallstone patients.Conclusions The results of immunohistochemistry and Western Blot were consistent with those of comparative proteomic analysis.The results from our comparative proteomic platform with 2-DE combining with MS are accurate,stable and reliable.Part FourPreliminary investigation of nucleation-promoting potency of Retinoi Binding ProteinObjective To examine the nucleation-promotingpotency of RBP in the model biles in vitro.Methods Small model bile and Synthetic model bile were establish,the pro-nucleating effect of RBP were studied by a sensitive cholesterol crystal growth assay and the nucleation time detected by polarizing microscopy.Results The results of the crystal growth assay showed that the derived indices It,Ig and Ic presented as 0.73,1.02 and 0.95 respectively.RBP shortened the nucleation time of cholesterol monohydrate crystals when added to Small model bile(8.17d±1.47d ) or synthetic artificial model bile(7.5d±1.05d ). Conclusions These data suggest that RBP is pro-nucleation factor and provide some clues to further investigate the role of RBP in the formation of cholesterol gallstone.In conclusion,the differential protein profiles of gallbladder bile between cholesterol cholelithiasis patients and control ones,and between vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established. There were 22 and 8 differential protein spots were identified respectively.The results of immunohistochemistry and Western Blot were consistent with those of comparative proteomic analysis.Among the differentially expressed proteins,we preliminarily investigated on the nucleation-promoting function of RBP involved in the formation of cholesterol gallstone.