The Research Of Detecting Key Genes Influencing Coagulation Factors And Plasminogen In Blood For Molecular Diagnosis Of DVT

1. By testing genes in venous tissue of DVT rats and using Bioinformatics, lock the upstream genes highly relevant with coagulation factor Ⅴ, Ⅷ, Ⅹ and tissue-type plasminogen activator inhibitor. Then test the gene expression changes in blood of DVT rats, patients.2. Through genetic pathway, analyze the relationship between key genes with the injury of endothelial cell and enhancing of coagulation, discovering that the permeability of mitochondrial membrane in endothelial cells increase, leading to the decreasing of anticoagulant protein C secretion, and increasing of Ⅴ, Ⅷ, Ⅹ coagulation factor activity; analyze the relationship between key genes with platelet aggregation and Inhibition of fibrinolytic activity, discovering that the activity of glycoprotein receptor in platelet membrane increase, leading to the increasing of plasminogen activator inhibitor release and decreasing of plasminogen.3. The related results show that gene expression of TGF-β1and other genes are up-regulated, indicating that in traumatic DVT, focusing on these genes and combining with genetic information analysis, roles influencing the complicated issues of DVT are quickly locked, and is beneficial to the relevant research works on DVT. Meanwhile, diagnostic molecular markers are finally fixed.MATERIALS AND METHODS1. Sixty SD rats model of DVT were established and randomly divided into four groups. Group A (control group, n=10), Group B (pre-thrombosis group,2.5hours after trauma, n=10), Group C (thrombosis group,25hours after trauma, n=10), Group D (no thrombosis group,25hours after trauma, n=10). Dissect femoral vein tissues at different time points, observe the incidence and severity of thrombosis. Acquire femoral vein tissues, test differentially expressed genes by Genechip Rat Genome2302.0; select significantly expressed genes by Fold Change (FC) method; combining with research hotspots and databases of Ncbi Gene, Gene Card, Pathway analysis, lock anti-fibrinolysis genes like Serpine1and regulating genes (TGF-β1etc.) as research emphasis.2. Sixty SD rats model of DVT were established and randomly divided into four groups. Group A (control group, n=10), Group B (pre-thrombosis group,2.5hours after trauma, n=10), Group C (thrombosis group,25hours after trauma, n=10), Group D (no thrombosis group,25hours after trauma, n=10). Acquire venous blood samples in different time points, test previously locked TGF-β1and other genes expression quantitively and qualitatively.3. Acquire the annotation informations of TGF-β1, Serpine1, vWF and PF4in rats and human by databases of Ncbi Gene and Gene Card (2013). Compare the sequences of two genes in BLAST, and ascertain their homology in rats and human.4. Establish the risk factors and diagnostic criteria of DVT in the research based on "The NICE guidelines of prophylaxis for venous thromboembolism", and "Methodology for the development of anti-thrombotic therapy and prevention of thrombosis guidelines". From2009.1～2011.1, in the department of orthopedics of the first affiliated hospital of Kumming medical university, collect300blood samples in human with risk factors of DVT without limitations for gender and age. Select10samples in healthy human as Group A,12samples with thrombosis as Group B,15samples without thrombosis after trauma as Group C. Venous blood samples were collected, detect gene expression changes of TGF-β1, Serpine1, vWF and PF4using PCR and real-time PCR. Explore the gene expression changes and their effects in endothelial cell injury, platelet aggregation, coagulation and anti-fibrinolysis.RESULTS1. The results of testing femoral vein tissues in DVT rats by gene chip display that:2458genes differential expressed, of which1146up regulated,1312down regulated. anti-fibrinolysis genes of TGF-β1、Serpine1、vWF and PF4were locked as objects in next research. 2.2.5hours after trauma, TGF-β1, Serpine1, vWF, PF4were up regulated in rats blood. After thrombosis, these gene were up regulated significantly. The gene expression in group C (thrombosis) were higher than group B (pre-thrombosis) and Group D (no thrombosis).3. The results of BLAST comparison between rats and human show that TGF-β1was89.4%, Serpinel was91.2%, vWF was86.9%and PF4was92.6%. Homology comparison rate was all higher than85%, indicating that TGF-β1and other genes have high homology between rats and human.4. The real-time PCR results in clinical patients’blood display that the expression level of TGF-β1, Serpine1, vWF and PF4in Group B (thrombosis) were the highest, while the expression level of those in Group C (trauma) were also higher, both groups were higher than Group A (control group). The conclusion of Pathway analysis demonstrating that TGF-β1and Serpinel was upper stream regulating gene of vWF, while vWF was a key gene to regulate the secretion of PF4activating platelet.CONCLUSION1. The expressions of TGF-β1, Serpine1, vWF and PF4are possibly have relationship with DVT in rats and human.2. TGF-β1, Serpine1, vWF and PF4affect the injury of endothelial cell and activation of platelet, which are crucial states of coagulation system and anti-fibrinolysis system, prompting the generation of deep vein thrombosis.3. The expressions of TGF-β1, Serpine1, vWF and PF4were up regulated before thrombosis, and this trend was more significant after thrombosis, indicating that these genes may probably have effects to prompt thrombosis and was able to be the molecular markers for the diagnosis of DVT.