The Experimental And Clinical Study Of IL-1β, A2M, Cathepsin D Affecting Deep Venous Thrombosis

OBJECTIVE:(1)To screen the distinguishing patterns of protein and gene expression of femoral vein vascular tissue at different status (status of pre-thromgenesis, thrombogenesis peak, and non-thrombogenesis), by establishing a rat traumatic deep vein thrombosis model. (2) To formulate uniform clinical standards for diagnosis,inclusion and exclusion in patients, collect DVT patient's venous blood, and screen differentially proteins expression in 2 groups (the human thrombosis group/non-thrombosis group). (3) Comparing the expression of the protein and gene in rats'venous vascular tissue and in human's venous blood, in order to find out characteristic protein expression (including IL-1β, IL-6, A2M, Cathepsin D and Cathepsin G) in rats'vein vascular tissue, which were highly relevant to the endothelium and platelet, (4) Using ELISA to further identify the different changes of expression about these important proteins in human blood, and explore the potential role of these proteins in the process of endothelial cell injury, platelet activation, inflammatory factor activation, regulation of the interaction mechanism, and the function of promoting thrombosis.METHODS:The first part:150 SD TDVT (traumatic deep vein thrombosis) rats model were established by combination with clamping both femoral vein and fixing with cast. The rats were randomly divided into control group (group A,n=10);Post-traumatic instant group (group B,0.5 hour after trauma,n=10);pre-thrombogenesis group (group C,2.5 hour after trauma,n=10);Thrombotic crest-time group (group D,24 hour after trauma n =10);Thrombus resolution group (group E,72 hour after trauma, n=10);Thrombus insolution group (group F,72 hour after trauma, n=10);Nonthrombogenesis group (group G, 168 hour after trauma, n=10); No thrombus at crest-time group (group H,24 hour after trauma, n=10). Rats femoral venous were dissected and observed the incidence and serious degree of thrombus in different time point. Then total RNA were extracted from the localized venous endothelial. Using the gene chip-based screening technology to detect difference expression of genes.The second part:DVT rats models were established again by same method. The 100 SD rats were randomly divided into control group (group A, n=10), pre-thrombogenesis group (group B,2.5 hour after trauma), thrombogenesis group (group C,24 hour after trauma) and non-thrombogenesis group (group D,24 hour after trauma). Rats' femoral venous were dissected for observing the incidence and serious degree of thrombus in different time point. Then total protein was extracted from the localized venous vascular tissue. The difference of protein expression in B and C groups were detected by a Two-dimensional gel electrophoresis (2D DIGE) in B and C groups.The third part:To formulate uniform diagnosis,inclusion and exclusion criteria for monitoring thrombosis in clinical patients, and collect DVT patient's venous blood in order to detect. Clinical cases were divided into normal control group (A group, n=10), thrombogenesis group (B group, n=10), non-thrombogenesis group (C group, n= 10). Collecting patients'blood samples, using 2D-DIGE technology to detect different proteins expression in B and C groups. Comparing these key proteins (including IL-1β, IL-6, A2M, Cathepsin D and Cathepsin G) with the potential thrombogenesis proteins and genes in rats' venous endothelium, combined with information of literatures, to find out the potential indicated thrombogenesis proteins which were highly relevant to the endothelium and platelet.The fourth part:Detecting the expression changes of these potential indicated proteins (IL-1β,A2M,Cathepsin G) in human blood at different time points by ELISA.RESULTS:(1) The micro-array results of Rats' femoral venous wall tissue was:There were 2458 differentially expressed genes in total genes (15,864). Of there genes, there are 1146 (46.6%) genes'(such as IL-1B) expression increased, and the expression of 1312 (53.4%) genes depressed.(2) The of 2D DIGE's results of Rat femoral venous wall suggest that:Selected 21 different potential thrombogenesis protein spots in gel, and confirm 16 different potential thrombogenesis protein (such as A2M) by mass spectrometry analysis.(3) The 2D DIGE's results of human blood indicated that:Selected 25 different potential thrombogenesis protein spots in gel, and confirm 20 potential thrombogenesis protein (such as Cathepsin D) by mass spectrometry analysis.(4) Analyzed and compared of the different potential thrombogenesis relative proteins and genes between in human blood and rats'vascular tissue showed that:IL-1β, IL-6, A2M, Cathepsin D, Cathepsin G gene and relative proteins'expression increased and may be the potential thrombogenesis protein.(5) The results of ELISA in human blood showed that:at the thrombogenesis instant group (group A)and non-thrombogenesis group (group C), the expression of IL-1β, IL-6, A2M, Cathepsin D increased slightly, in thrombogenesis crest time group (group B),the expression increased significantly compared to group A and C.CONCLUSUON:(1) An acute TDVT animal model can be established successfully through vein direct clamping combined with hip spica cast fixation:the method is convenient and the successful rate is high.(2) TDVT is associated highly with an increased expression of IL-1β, IL-6, A2M, Cathepsin D, Cathepsin G in thrombogenesis venous vascular wall and blood.(3) IL-1β, IL-6, A2M, Cathepsin D and Cathepsin G may play an important role in the process of DVT generating, and may be candidate molecular markers for early diagnosis of deep vein thrombosis.