| Part one Establishment and differential analysis of protein profiles of human gallbladder tissueObjective To establish and optimize a stable and standard proteomic technique platform and differential analysis those proteins from cholesterol gallstone patients and control ones, this study is designed to comparative analysis of the proteome of gallbladder tissue from two groups, to provide experiment proof for elucidate mechanism of formation of cholelithiasis. Methods Total proteins of gallbladder tissue 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. Results The concentrations of bile proteins from cholesterol cholelithiasis patients and control ones were 7.45 mg/mL and 4.36 mg/ml(P<0.01)respectively (P<0.01). 2-DE patterns with high resolution and reproducibility were obtained.The average matched protein spots were 632±61和606±64 in the two groups, respectively. The matched rates were 73.46%和71.49% correspondingly. There were 474 matched spots in the two reference gels maps and the matched rate was 70.64%. There was a marked difference between two groups (P<0.05),in which 27 expressed highly in cholesterol cholelithiasis patients but lowly in the control ones, while 8 expressed just in the opposite way. Conclusions The well-resolved and reproducible differential protein profiles have been primarily established from patients with cholesterol gallstones and normals. The differential protein spots were analyzed by software. The data may provide experimental foundation for further study source of formation of cholesterol cholelithiasis.Part two Comparative proteomic analysis of sub-serosa of human gallbladder tissueObjective To establish and optimize a stable and standard proteomic technique platform of sub-serosa of human gallbladder tissue and differential analysis those proteins from cholesterol gallstone patients and control ones, to screen proteins for elucidate mechanism of formation of cholelithiasis. Methods Total proteins of sub-serosa of gallbladder tissue 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 and identified with MALDI-TOF/TOF MS. Results The well-resolved and reproducible differential protein profiles have been primarily established from sub-serosa tissue of gallbladder from patients with cholesterol gallstones and normals. There were 19 differentially expressed proteins and 11 were identified from the two cholesterol-carrier phases. Among them, Tropomyosin 4, Transgelin, Transthyretin, Cyanomet Rhb1.1, Haptoglobin-related protein precursor were up-regulated with Peroxiredoxin 3, Hypothetical protein, T cell receptor alpha chain were down-regulated in gallstone groups compared with normal groups. Conclusions The well-resolved and reproducible differential protein profiles have been primarily established from patients with cholesterol gallstones and normals. The differential protein spots were analyzed by software and were identified through MALDI-TOF-MS. The data may be a basis for further screening the key regulators of the formation of cholesterol gallstone.Part three Verification of differentially expressed proteins between the cholesterol gallstone and control groups Objective 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 TTR,SM22-alpha,TPM4,Prx3 in the gallbladder tissues from cholesterol gallstone group was compared with that of control group by Western Blotting and RT-PCR. Results The results of Western Blotting showed that TTR,SM22-alpha and TPM were up-regulated in gallbladder tissues from cholesterol gallstone group in comparison with control group. The expressional levels of SM22-alpha were higher in gallbladder tissues from cholesterol gallstone group than control group, and Prx3 were lower in cholesterol gallstone group by RT-PCR. Conclusions The results of Western Blotting and RT-PCR 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 Four Preliminary investigation of nucleation-promoting potency of TransthyretinObjective To examine the nucleation-promoting potency of TTR in the model biles in vitro. Methods Small model bile and Synthetic model bile were establish, the pro-nucleating effect of TTR were studied by a sensitive cholesterol crystal growth assay and the nucleation time detected by polarizing microscopy. Results TTR shortened the nucleation time of cholesterol monohydrate crystals when added to Small model bile (14.5±1.29)d or synthetic artificial model bile (13.5±0.58)d. Conclusions These data suggest that TTR is pro-nucleation factor and provide some clues to further investigate the role of TTR in the formation of cholesterol gallstone.In conclusion, the differential protein profiles of gallbladder tissue, seroa tissue of gallbladder, sub-seroa tissue of gallbladder between cholesterol cholelithiasis patients and control ones. There were 11 differential protein spots were identified respectively from sub-seroa tissue of gallbladder, one repeat. The results of Western Blotting and RT-PCR were consistent with those of comparative proteomic analysis. Among the differentially expressed proteins, we preliminarily investigated on the nucleation-promoting function of TTR involved in the formation of cholesterol gallstone.
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