| [Objective]:1.Select trauma and surgical patients serum sample is divided into groups of thrombosis, blood clots do not form a group and the normal control group, the use of two-way point of swimming, mass spectrometry screening differences between the three groups of samples of Proteins.2.Select some protein spots, using Elisa technique quantitative detection of protein expression in serum samples.[Materials and methods]:Part1:Human serum protein markers predict DVT Screening and analysis of protein function associated1. Select trauma and surgical patients, according to the diagnostic criteria screened DVT thrombosis group, thrombus formation group did not. Select healthy persons included in the normal control group. Eventually serum samples collected90cases divided into three groups: normal control group (30cases), thrombosis has not formed group (30cases), thrombosis group (30cases). Do not form a group and thrombosis thrombosis group are provided "venous thromboembolism prophylaxis NICE guidelines" standard treatment. Three groups were collected fasting venous blood samples, including blood clots do not form a group of dynamic fracture surgery on blood and observed two weeks without thrombosis in patients after diagnosis of thrombosis group gathering, in strict accordance with the time point of each group blood collecting blood specimens.2. The collected blood samples for routine blood testing, while the soluble protein samples were extracted, processed samples at-80℃good stand.3. The blood samples will stand isoelectric focusing, focusing underwent Immobiline DryStrip gel strip balanced10minutes, and immediately transferred to a second perpendicular to SDS-PAGE electrophoresis. After the end of the gel electrophoresis dye vat dye transfer to the stained gel using precision ImageScanner scanned into the two-dimensional electrophoresis pattern analysis than the right, to get protein spots.4. Test on blood samples obtained in different points protein MALDI-TOF-MS analysis to determine the source or nature. Observe them in a deep vein thrombosis in the expression changes.Part2:TpP in human serum by ELISA and expressed deep vein thrombosis 1. In group A randomly selected sample of9cases, B, C group randomly selected10cases of serum samples;2. Analysis of the expression of TpP in different groups;3. Application of SWISS-PORT and other related to qualitative analysis of TpP protein, role in thrombosis in.[Results]:Part1:analysis and prediction of protein markers in serum screening of DVT and related proteins.1. Three groups of samples identified a total of10protein spots, including thrombosis group (A) than not form clots (B group) and normal control group (C) There are four points of difference were successfully identified proteins. MS sequencing showed two spots in the thrombus group were up-regulated, they are:TpP, C4binding protein;2.The NCBI database query, only in the group appear in thrombosis or upregulated protein and inhibits fibrinolysis, procoagulant, inhibition of platelet aggregation and related.Part2:TpP in human serum by ELISA and expressed deep vein thrombosis1. The ELISA TpP test results as follows:In the thrombosis group (A) increased expression, and that, before the formation of thrombus group (B group) and normal group (C group) no significant changes in expression, TpP expression trends and thrombosis consistent with the formation of biological processes.[Conclusion]:1. Two-dimensional gel electrophoresis-mass spectrometry technology can effectively isolated and identified serum proteins, blood clots do not form a group with respect to thrombus differences in serum proteins, this technology is looking for deep venous thrombosis after trauma-related serum markers provide a methodological foundation.2. MALDI-TOF-MS analysis revealed:Thrombus Precursor Protein (TpP) group than in thrombosis thrombus formation group and the normal group, not the expression were increased, statistical difference. |
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