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ObjectTDVT rat femoral venous endothelial tissue,blood cells, focus on the key states of prethrombosis, peak of thrombosis, deteced the expression change of Argl、eNOS、vWF; identify the expressional change of Argl、eNOS in human blood at states of thrombosis, non-thrombosis and analysis the possible mechanism of DVT process.MethodsPart11.Esteblishment rat acute traumatic DVT model:clamping the femoral vein and immoblize the bilateral hind limbs(hip spica cast fixation).According to different observation phases and thrombosis observation results,150 TDVT model SD rats were divided into 5 groups:the control group (group A), traumatic instant group(group B) prethrombotic group (group C), thrombosis crest-time group (group D), non-thrombosis group (group E). In the corresponding time,4cm rat femoral venous tissue was collected and dissected endothelial tissue,which used to gene-chip detected.2.Summarized and analyzed the microarray data of Affymetrix 230A, applied fold change method to scan and analysis differential expression genes involved in DVT. (Comparison Signal log2 ratio between two groups with one gene,Set upregulation gene:Single Log2 ratio≥1,downregulation gene:Single Log2 ratio≤—1).3.The screened differential expression genes applied GO classification and bioinformatics research,focus on endothelial cell related genes.finally locked upexpression gene-Arg 1 and endothelial cell injury related factor vWF and downexpression gene-eNOS.4.Queried both rat and human gene sequences of Arg1、eNOS和vWF in the NCBI GENE website, than used BLAST to determine the homology.Part21.Esteblishment rat acute traumatic DVT model:clamping the femoral vein and immoblize the bilateral hind limbs(hip spica cast fixation).According to different observation phases and thrombosis observation results,100 TDVT model SD rats were divided into 4 groups:the control group (group A), prethrombotic group (group B), thrombosis crest-time group (group C), non-thrombosis group (group D).2.In the corresponding time,5ml blood samples were hemospasia from heart of each model.Used TRIzoI method to extracte total RNAs of blood, the qualities of RNA samples were evaluated by 3% agarose gel electrophoresis and spectrophotometer detection.3.Applied Oligo 6.69 software was to design Arg1、eNOS、vWF PCR primers, with GAPDH as an inner-reference. Each group blood cell RNA was reversed transcription into cDNA, and then PCR reaction was performed (force -- denaturalization degeneration--annealing -- extension -- end-extension). The PCR production was analyzed by fluorescent quantitative method. Identified Arg1、eNOS、vWF specific amplification products by melting curve and detected the amount of products by amplification curve.4.Data processing method of Real Time PCR:2-ΔΔCT method, the 2-ΔΔCT result is a ratio experimental group (group B, C, D) versus control group (group A) of Argl、eNOS、vWF. Then, analysis them expression tendency in rat blood of DVT.Part3To formulate uniform diagnosis、inclusion and exclusion criteria for monitoring thrombosis in clinical patients, and collect DVT patient’s venous blood. Clinical cases were divided into normal control group (A1group, n= 5), traumatic control group (A 1 group, n= 5),thrombogenesis group (B group, n= 6), non-thrombogenesis group (C group, n= 6).Collection of blood samples for RT-PCR detection of Arg1, eNOS expression.Results:1. Analyzed of microarray gene data in rat femoral venous endothelium show:Arg1 in the prethrombotic group (group C) was slightly higher expression (CvsA:3.1),and in the thrombosis crest-time group (group D)was significantly higher expression (DvsA:3.6). vWF in the prethrombotic group (group C) was slightly higher expression (CvsA:2.2),and in the thrombosis crest-time group (group D)was significantly higher expression (DvsA2.8).eNOS in the prethrombotic group (group C) was slightly lower expression (CvsA:-1.6),and in the thrombosis crest-time group (group D)was significantly lower expression (DvsA:-2.1)2.Real-time PCR deceted Arg1、eNOS expression changes in rat blood cells:Arg1 in the prethrombotic group (group B) was slightly higher expression, in the thrombosis peak-time group (group C)was significantly higher expression, (CvsA:9.673) and in the non-thrombosis group (group D)was slightly higher expression. vWF in the prethrombotic group (group B) was slightly higher expression, in the thrombosis peak-time group (group C)was significantly higher expression (CvsA:11.973),and in the non-thrombosis group (group D)was slightly higher expression.eNOS in the prethrombotic group (group B) was slightly lower expression,and in the thrombosis crest-time group (group C)was significantly lower expression (CvsA:-9.132), and in the non-thrombosis group (group D)was slightly higher expression.3.Rat and human Arg1、eNOS gene is highly homologous.4.RT-PCR deteced human blood cells Arg1、eNOS,the results show that Arg1 was high expression in B,C,D group and eNOS was no significant changes in all the four groups.Conclusion1. Arg1 is closely related to human traumatic deep vein thrombosis.2.The expresion changes of Arg1 and eNOS have the same tendency in rat femoral venous tissue and blood cells, which can reflected the throbosis states.
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