| Object In rat traumatic deep vein thrombosis before the formation of the peak of the femoral vein of rats TDVT tissue microarray analysis of test results, focusing on the peak of procoagulant thrombosis-endothelial cell expression of genes related to LOX-1, in Blood cells in rats with its Real-time PCR detection, and study its expression in blood cells.MethodsThe first part Traumatic deep vein thrombosis in animal data analysis and BLAST homology comparison1.Establish a rat model of traumatic deep vein thrombosis, based on visual observation modeling time and thrombosis in the state of 150 rats were divided into five groups, A group (normal control group, Oh), B group (trauma immediate group, Oh), C group (thrombosis in the former group,2.5h), D group (peak thrombosis group, 24h), E group (blood clots do not form groups,168h).2.10 in each group were selected stocks veins removed, cut about 4-5cm of the femoral vein and main branches of, the use of Trizol extraction of total RNA, and the RNA quality control.3.Microarray gene expression profiling using Affymetrix Rat230, the results of microarray data analysis using fold change (between the two groups of the same gene Signal log2 ratio comparing up-regulated genes:Log2 Ratio≥1, down-regulated genes:Log2 Ratio≤-1,) differential expression of selected genes;4.To "endothelial cell" as the search word in the chip test data Excel table Gene Symbol (gene symbol) query, and apply the fold change analysis (adjusted parameters for Signal log2 ratio≥3 or compare≤-3); with homologous of comparison to determine the expression of endothelial cell-related genes LOX-1 for the main object of attention5.CNKI database through the NCBI GenBank database, on behalf of the two gene sequences into the BLAST in the comparison, clearly LOX-1 in rat and human homology between the (sequence alignment using the MEGA 4.0 software) The second part Real-time PCR detection of blood cells in the rat LOX-1 gene expression1.Again, the rat model of traumatic deep vein thrombosis, the 100 SD rats were observed to be time and thrombosis grouped as follows:control group (A group), thrombosis former group (B group 2.5h), thrombosis the formation of the peak of thrombosis group (C group 24h) and the peak of thrombosis thrombosis group (D group).2.For the corresponding point in time, every time blood samples were collected each rat 5ml, centrifuged to obtain serum samples and blood cells, blood cells using Trizol extraction of total RNA, and the femoral vein cut.3.Of LOX-1 for Real-time PCR detection; of the femoral vein was cut for histological observation of vascular thrombosis level.Result1.Microarray LOX-1 gene in the rat femoral vein tissue TDVT Show:In the B group (trauma instant group) LOX-1 significantly upregulated (BvsA:10.04), and gradually increased, C (thrombosis former group), D group (peak thrombosis group) and A group (CvsA:14.78, DvsA:17.13), blood clots do not form a group (E group) was slightly upregulated (EvsA:5.45).2.BLAST comparison, LOX-1 in rat and human homology between the 92.6%, rat and human LOX-1 gene was highly homologous.3.Real-time PCR detection of blood cells in the rat TDVT model of the expression of LOX-1 showed:B group (early thrombosis group), C group (peak thrombosis thrombosis group) than in group A (control group) occurred significantly upregulated (BvsA:8.49, CvsA:14.76), while the D group (thrombosis of blood clots do not form a peak group) compared with A group (control group) slightly up-regulated expression (DvsA:3.21). Real-time PCR test results with the chip test results are basically the same on the trend.ConclusionLOX-1 in rat TDVT expression during the formation of a dynamic upward trend (BvsA:8.49, CvsA:14.76, DvsA:3.21), possibly through regulation of platelet adhesion and aggregation to influence thrombosis. This will be our future research focus of the center.
|
PDF Full Text Request