A Study Of IL-1β,IL-6 In Rats Predicts And Diagnoses The Traumatic Deep Vein Thrombosis

[Objective]In rat traumatic deep vein thrombosis based on animal models,before deep vein thrombosis and the peak period of the DVT of two key time points,femoral vein of rats results of the application of gene chip detection method for a wide range of information analysis,we focus on changes in inflammation-related genes and levels in vascular tissue and blood levels to verify RT-PCR detection.Investigating the IL-1β,IL-6 as a forecast of rat traumatic diagnosis of deep vein thrombosis of new molecular markers of the feasibility and reliability, the development of gene chips can be used to predict the clinical diagnostic kits to lay the foundation.[Materials and Methods]The first part:Rat traumatic deep vein thrombosis before the peak formation of the formation analysis of differentially expressed genes.1.In the two tests,the first experiment 150 SD rats and the second test 250 SD rats,model based on observed time points and / or thrombosis of the existence of the state of division,two experiments were divided into 4 groups,namely normal control group(A group),before thrombosis(C group),thrombogenesis at thrombotic crest-time(D group) and nonthrombosis at the thrombosis crest-time(G group).2.Two experiments were created beating on the bilateral thigh quantitative and piaster fixation model law.Non-narcotic rats use quantitative beating trauma devices,the moment to beat the energy of 5 J,respectively,in rats with bilateral thigh hit the 1 proximal lateral,hip cast immobilization word,observing the changes of color and swelling of both feet case.3.Based on the corresponding time points, the first tentatively determined through visual observation of the state of thrombosis in each group into 10 rats,respectively,both cut about 4-5cm of the femoral vein,femoral vein of each re-cut about the organization of 0.5cm to HE staining,light microscope to determine the status of thrombosis.4.Choosing the sub-conditions of each group shares the same vein mixed, one-step method using Trizol extraction of total RNA,using 2%agarose gel electrophoresis detection of its quality.5.Each group of qualified quality RNA samples were biotin-labeled using Genechip Rat Genome 230 2.0 chip hybridization,chip scanning and data processing.6.Using a multiple analysis of selected changes in each time point compared with a group of differentially expressed genes(gene increases:Log₂ Ratio≥1,and Change marked asâ… ;gene discreases:Log₂ Ratio≤-1,and Change marked as D) and GO classifications. 7.The C group,D group,G group and the differences in the expression of inflammation-related genetic data in order to probe set ID for the sort of uniform standards, combined.8.Using cluster analysis Cluster analysis of the differential expression of inflammation-related genes,more intuitive display C group,D and G group of differentially expressed genes between the groups change.9.Further analysis of the use of FC method,by adjusting the relevant parameters,to identify significant differentially expressed genes, parameter adjustment for the Single Log2 Ratio≥2.3 or Single Log2 Ratio≤-2.3,D group and G group gene expression levels of≥100.10.BLAST with than endogenous:The FC analysis method selected the significant differentially expressed genes in the NCBI website GENE query its human and rat gene sequences,the two gene sequences into the BLAST than in a clear and homology of the human genome..The second part:IL-1β,IL-6 in rats predict the diagnosis of traumatic deep vein thrombosis Experimental Study.1,In rats blood,using RT-PCR to detecte IL-1βand IL-6 mRNA expression of A,C,D,G group;2,In rats' vascular tissue using RT-PCR to detecte IL-1βand IL-6 mRNA expression of A,C,D,G group;3,Comparing the IL-1βand IL-6 mRNA expression of different change by the blood cell RT - PCR detection,vascular tissue RT - PCR detection,vascular tissue microarray testing.[Results]The first part of the trauma of physical model of deep vein thrombosis and femoral vein of the establishment of gene expression in vascular tissue.1.In the model, femoral vein thrombosis occurred in rats after 72h,Group D first experiment 50.0%of femoral vein thrombosis inside the second test D group 48.2%of femoral vein thrombosis inside.2.In the corresponding sub-groups of vascular tissue of the eye and light microscope observation of the results are basically the same aspects of thrombosis.3.Each group based on the total RNA and gene chip detection of successful completion of two approved the quality of monitoring and evaluation testing are qualified.4.In the pathological process of a total of 2458 differentially expressed genes,which increase gene 1146(Log2 Ratio≥1,and Change marked asâ… ),reduced gene 1312(Log2 Ratio≤-1,labeled as D and Change ).The majority of differentially expressed genes function is not clear,known mainly for functional genes involved in blood coagulation -Anti-coagulation and fibrinolysis-fibrinolytic resistance,inflammation,immune-related genes such as genes.5.In the gene chip to identify 55 inflammation-related genes,of which 19 inflammation-related genes in the process of expression of a difference.6.Cluster analysis showed that 55 inflammation-related genes,IL-1, IL-6 and Cinc2 clusters,such as performance,the level of gene expression in cluster formation and evolution of thrombosis was changed in the same direction.7.FC analysis of the inflammation-related genes significantly differentially expressed a total of 4 for:IL-1β, IL-6,CXCL2,and CCL3.5.D group,G group significant differences in the expression of inflammatory gefies and the human genome BLAST Homology comparison:IL-6 was 87.7%; IL-1βwas 86.7%;CCL3 was 78.5%;CXCL2 for 58.6 percent.The second part:IL-1β,IL-6 in rats predict the diagnosis of traumatic deep vein thrombosis Experimental Study 1.The result of RT-PCR detection is confirmed efficient by augmentation curve.2.The result of RT-PCR detection is confmned effective and high sensitivity by dissolve curve.3.IL-1β,IL-6 for RT-PCR test results with the gene chip on the test results are basically the same trend.[Conclusion]1.The application of gene microarray technology detecting rats deep vein thrombosis traumatic vascular tissue,locking the molecular markerlL6and IL1βwhich can forecast diagnosis rats'TDVT in the aspect of tissue.2.The application of Blast by comparing rats with human proved height homology while speculate the IL6 and IL1βcan be used for predicting the deep venous thrombosis.3 In blood,IL6,IL1βthe changing trends the same as in vascular tissues,which can pass in the blood test to predict the diagnosis of deep vein thrombosis in rats.