| [Objective] Harvest blood plasma sample corresponding the states of pre and post deep venous thrombosis of rat, to choose differential expression proteins of thrombogenesis and unthrombogenesis states, and to select predict diagnosis DVT molecular marker. On this basic, we focus on anti-fibrolysis associated proteins, investigating their expression changes, to further determine fibrolysis associated marker that can be used as predict diagnosis marker in rat blood, and to analysis the role of determined marker in thrombogenesis.[Materials and Methods] Part 1 The determination of DVT predict diagnosis differential protein in rat plasma and the analysis of protein associated to fibrolysis1.100 SD rats enrolled in this experiment were assigned into groups according to observe time phases and/or thrombogenesis state. Twice experiments were both divided to 4 groups, blank controlled group (Group A), pre-thrombogenesis group (Group B), peak state of thrombogenesis (Group C) and peak state of non-thrombogenesis (Group D);2. animal models were built by our team self invented method of bilateral thigh quantitative strike associated with plaster external fixation. The experiment rats were without anaesthetized, stroked both thighs on proximal end and lateral side once each, using trauma quantitative strike equipment with a strike energy of 5 joule, then fixed in hip spica cast, observing the color and edema state of rats both feet.3. The materials harvest:on corresponding time phase, initially determine thrombus state by macroscopic observation,10 rats were enrolled in each group,1.5ml peripheral blood were drawed individually, centrifuged on 3,500 r/m for 10 minutes, collecting upper layer plasma;4.Mix the plasma in each group, extract total protein in each group, plasma protein were treated by affinity chromatograph to removal high abundance protein interference.5.Plasma total proteins were detected by amphitropic protein electrophoresis in group C, D, diverse protein were analyzed and searched by ImageMaster 2D Elite3.0 software, take Volume value ratio increase and decrease 50% as standard to search up and down-regulate protein;6.Diverse protein sites were decolorized, enzyme cut in gum and peptide segments extraction, using MOLDI-TOF/MS to make PMF analysis.7.Making bioinformatics analysis of the determined proteins using NCBI, Pathway and etc. databases.Part 2 Serpinel,Serpinal in rats predict the diagnosis of traumatic deep vein thrombosis Experimental Study.1,In rats blood, using ELISA to detecte Serpinel and Serpinal expression of A, B, C, D group;2,Comparing the Serpinel and Serpinal expression of different change by the blood.[Results]Part 1 Determination of diverse proteins predict diagnosis DVT of rats and analysis the role of protein associated with fibrolysis.1.In this model, femoral vein thrombus occurred 25h after model built,64.63% cases in group C have femoral vein thrombogenesis;2.The observation result of vascular tissues by naked eye and light microscope enrolled in each group were coincidence in whole.3.Group D compared with C, the proteins distribution has no difference, protein site mostly concentrate 30-70KD and PI value rang 4-8;4.Group D compared with group G, there 13 up-regulation protein sites and 8 down-regulation sites after thrombogenesis;5.Through image analysis,16 of 21 diverse proteins had been determined, of which Serpins1 and Serpins3 were proteins associated fibrolysis. Serpins1 inhibit outer activation pathway through inhibiting tissue-type plasminogen activator, so as to effect thrombogenesis; Part 2 Serpinel,Serpinal in rats predict the diagnosis of traumatic deep vein thrombosis Experimental Study.1. The result of ELISA detection for Serpinel is appeared up-regulation in Group B, and continued up-regulation in group C, while unchanged in group D.2. The result of ELISA detection for Serpinal is appeared up-regulation in Group C, while unchanged in group B and D.[Conclusion] 1. Thrombosis and inhibition of fibrinolysis, apoptosis, transcriptional regulation of vascular activity and other functions related to changes in protein expression, this study identified some differences in proteins involved in MAPK Pathway affected by thrombosis.2. Serpinel for the fibrinolytic proteins, function as direct inhibition of plasminogen activator role; its expression in blood trend consistent with the biological processes of thrombosis, and thrombosis before the expression that is higher than the predicted diagnosis Deep vein thrombosis protein markers;3 Serpinal function can be divided into:(1) direct inhibition of plasmin role in promoting thrombosis; (2) the impact of the initial steps of endogenous coagulation and inhibit thrombosis. In thrombosis there was no significant change before and can not predict the diagnosis of deep venous thrombosis protein markers could be used as protein markers of thrombosis and further studies. |
