Research Of Integrin αV And β3Expression Changes In Deep Venous Thrombosis

Through experiments on DVT rat model, DVT rabbit model and human blood, initial research was made in ITG family members who have close relationship with DVT.Objective:1. Establish DVT rat model, gather rat femoral vein tissue in different thrombosis states, then extract mRNA and screen differencial expressed key genes that have close relationship with DVT by cDNA gene chips.2. Explor the DVT rabbit model built method and try to detect model blood vessels with color Dopplor ultrasonography; gather blood samples in different thrombosis states comfirmed with ultrasound, then extract mRNA and detect the ITG genes Integrin aV, Integrin β1and etc. expression changes on different thrombosis status, which screened in rat DVT model.3. Gather blood samples and detect the expression changes of ITG members like Integrin aV and Integrin P3and etc. in DVT group, non-thrombosis group and healthy control, then explor the relationship of gene expression changes and thrombosis states.Method:1.60SD rats were randemely divided into control group (n=10) and model group (n=50). Model was built by clamp both femoral veins with masquito clamp in combination with plaster. Kill model rats in different time (2.5h and25h after model built), dissect and make femoral vein free, observing thrombosis incidence, then divided the model group into pre-thrombosis group (2.5h after model built), thrombosis group (25h after model built) and non-thrombosis group(25h after model built). Gather rat vein tissue and extract mRNA with Trizol one-step method, then search data in PubMed and GeneCard and detect the expression changes of genes that have close relationship with DVT by cDNA gene chips screen, screening significant differential expressed genes by FC mulitiple analysis method.2. Establish DVT rabbit model by small incision and ligation in proximal part method, then comfirm thrombosis state by color Dopplor ultrasonography and gather blood sample with coincident state. Extract mRNA from rabbit blood with RNeasy Protect Animal Blood Kit, then amplificate object genes and the expression changes were detected by agarose gel electrophoresis, semiquantitative analyzing electrophoresis result by Bio-Rad software Quantity one4.6and making conclusion through statistical analysis.3. Gather human blood samples and divided into DVT group, non-thrombosis group and healthy control group according to diagnosis. Extracte mRNA with Paxgene blood RNA kit, validate Integrin αv and Integrin P3genes that differential expressioned in previous animal models, verify PCR result by sequencing, and quantitatively detected by real-time PCR, then analyze the differences among the groups. Gather clinical data including the results of complete blood count, blood biochemistry, blood coagulation, color Doppler ultrasonography and etc, and then make statistical analysis.Result:1. The expressions of gene Integrin aV and Integrin β3were remarkablely higher in thrombosis group than those in pre-thrombosis, non-thrombosis and control group (>3.2folds) in rat model.2. Small incision expose and ligation of femoral vein in proximal part has a high DVT incidence, and rabbit blood flow, thrombosis formation, regression and thrombosis recanalization can be monitored by color Doppler ultrasonography.3. There is blood flow signals illuminate thrombosis reanalization2days after model built.4. The expression of Integrin aV mRNA was highly elevated after thrombosis than that before model built and sham operation group in rabbit model. 5. Gene expression changes of Integrin aV and Integrin β33were detected by PCR amplificate and electrophoresis, as well as real-time PCR. The gene expressions in DVT group were higher than non-thrombosis group and control group, and there is no difference between non-thrombosis and control group. The rest ITG family members have no expression difference among three groups.Conclusion:1. ITG genes Integrin aV and Integrin β33have a high expression in femoral vein tissues when rat DVT formation.2. Extracting mRNA from rabbit and human blood has maneuverability and accuracy.3. DVT rabbit model can be successfully built by small incision and femoral vein ligation in proximal part, and color Doppler ultrasonography can be used for real time monitoring femoral vein thrombosis states in rabbit model.4. Recanalization has been stared from early period (<3d) of thrombosis in DVT rabbit model.5. αVβ3may play role in recruit inflammatory cells and thrombosis recanalization.