Post-traumatic Instant Gene Expression In A Rat Model Of Traumatic Femoral Vein Thrombosis

Objective 1．In a rat model of traumaic deep vein thrombosis (TDVT), to overall detect femoral vein genes expressions through genechip technique. 2．To screen genes not only presenting differential expression at post-traumatic instant, but also in thrombogenesis-nonthrombogenesis at thrombotic crest-time, and these genes might be significant to earlier dignosis of TDVT. The purpose of this study is to provide essential experimental data for further studies about the molecular mechanism and biomarker determination for earlier dignosis of deep vein thrombosis (DVT).Methods 1．Modeling. 150 SD rats were randomly divided into the control (group A, 10 rats) and model groups (140 rats). In model rats, nonanesthetized and beating on bilateral posterior limbs combined with hip spica cast fixation were performed. In which, fixation was not performed in post-traumatic instant group (group B) because of their femoral veins would be cut as soon as modeling. The beating location was at lcm below greater trochanter of femur (traumatic energy was 5J, once a limb).2．Grouping and sampling. According to different sampling times and thrombotic states, the model rats were divided into 7 groups: post-traumatic instant (group B, post-traumatic 0．5h), initial period of thrombogenesis (group C, post-traumatic 72h), thrombogenesis at thrombotic crest-time (group D, post-traumatic 120h), nonthrombogenesis at thrombotic crest-time (group H, post-traumatic 120h), thrombus resolution (group E, post-traumatic 168h), thrombus insolution (group F, post-traumatic 168h) and nonthrombogenesis in post-traumatic 168h (group G).â‘ Thrombotic states were determined initially through macroscopic observation.â‘¡More than 10 rats meeting to pathological features of corresponding group were selected. Afetr anesthesia through intraperitoneal injection, bilateral femoral veins and great saphenous veins were cut (about 3～4cm long), in which, 0．5 cm was used for pathological slice HE staining and microscopic observation in order to ensure thrombotic states and thronbus classification. Finally, according to macroscopic and microscopic observation results, each 10 rats were selected into corresponding group.3．Femoral vein tissue total RNA extraction and genechip detection. Femoral veins were mixed together in the same groups. And Trizol one-step method was applied for total RNA extraction. The qualities of the 8 extracted RNA samples were detected through 3% agarose gel electrophoresis and spectrophotometer. Valid RNA samples were stored in a temperature of-80(?).4．Genechip inspection: The extracted 8 RNA samples were sent to Shanghai Biochip CO. Ltd.. The qualities of the RNA samples were detected again by Lab-on quality determination system. According to the manipulation protocal of Genechip Rat Genome 230 2．0, 8 genechips detection were detected respectively through cDNA probe preparation, biotin labeling, hybridization, staining and scanning in order.5．Genechips data processing and analysis:â‘ Fold Change (FC) analysis: through comparing the genes expression differences, to screen differental expression genes of each group compared with the control (up-regulation: Signal Log₂ Ratio≥1 and Change was marked as I; down-regulation: Signal Log₂ Ratio≤-1 and Change was marked as D). And these genes were classified according to Gene Ontology (GO) classification.â‘¡To screen genes keeping differential expression in the post-traumatic instant group and control comparison (BvsA) and thrombotic crest-time thrombogenesis and control comparison (DvsA), genes keeping differential expression in the post-traumatic instant group and control comparison (BvsA) and thrombotic crest-time nonthrombogenesis and control comparison (HvsA). Based on this, to further screen genes presenting significantly differential expression in these genes (Signal Log₂Ratio≥2 and Signal Log₂Ratio≤-2) , and using Pubmed database to query information about them.â‘¢To Screen the genes keeping differential expression in BvsA and thrombotic crest-time thrombogenesis/nonthrombogenesis comparison (DvsH), and to analyze them through Gene Cluster 3．0 software.â‘£Differential expression genes Pathway analysis in BvsA and DvsH: the pathways involved in these genes were looked up in KEGG PATHWAY Database. To mainly analyzed varation of MAPK signaling pathway in BvsA, DvsA, HvsA.Results 1．Eighteen rats died in the 150 and the mortality was 12%. At the thrombotic crest-time (post-traumatic 120h), thrombotic rate was 50．6%. At post-traumatic 168h, thrombus resolution rate was 56．7% and thrombus insolution rate was 43．3%; nonthrombogenic rate was 44．6%.2．The microscopic observation results of fomoral vein thrombsis were basically conincidence with the macroscopic observation, which met to the requests of grouping.3．The quality determination results of the 8 RNA samples: the results of 3% agarose gel electrophoresis showed that 28S and 18S straps were clear, 28SRNA:18SRNA>2:1; the weight of RNA of each group >15μg which met to the requisitions of genechip detection.4．In the 31 042 genes which can be detected by Genechip Rat Genome 230 2．0:In B/A, 349 genes presented differential expression, in which, 214 were upregulated and 135 downregulated;In C/A, 2 393 genes presented differential expression, in which, 1386 genes were upregulated and 1 007 downregulated;In D/A, 1 743 genes presented differential expression, in which, 945 were upregulated and 798 downregulated;In H/A, 2 790 genes presented differential expression, in which, 1685 genes were upregulated and 1 105 downregulated;In D/H, 805 genes presented differential expression, in which, 51 genes were upregulated and 755 downregulated;In E/A, 1 913 genes presented differential expression, in which, 1 222 genes were upregulated and 691 downregulated;In F/A, 2564 genes presented differential expression, in which, 1535 genes were upregulated and 1029 downregulated;In G/A, 1849 genes presented differential expression, in which, 1235 genes were upregulated and 614 downregulated.5．GO classification results differential expression genes: most of them were unknown genes; the functions of the known genes involved cell apoptosis, binding, metablism, cell cycle, transcription regulator activity, etc..6．In total, 79 genes kept differential expression in BvsA and DvsA and 112 genes kept differential expression in BvsA and HvsA. In which, 19 genes kept differential expression in BvsA and DvsA, the functions of them included inflammatory response, immune response, acute phase reaction, chemotactic factor biosynthesis, DNA-dependent transcriptional control, etc.; 18 genes kept differential expression in BvsA and HvsA, the function of them included fibrinolytic regulation, inflammatory response, immune response, striated muscle contraction, calcium ion binding, DNA-dependent transcriptional control, etc..7．In total, 30 genes kept differential expression in BvsA and DvsH, which included 15 known genes and 15 unkown genes. The functions of the known genes involved cell apoptosis, binding, cell cycle, transcription regulator activity, etc..8．In the Cluster analysis result of above-mentioned 30 genes, 21 were in a similar temporary expression mode: these genes kept upregulation in B and H groups but no diferential expression in group D. The 21 genes included 14 known genes, such as Copeb, Plaur, Serpine1, Ptgs2, Dusp1, Nr4a1, Nr4a3, Junb, etc., and 7 unknown genes(RGD:1359581, LOC301105///Syncrip_predicted, Dnajb1_predicted, RGD1307124_predicted, 1374429_at, 1375422_at, 1383052_aat). Thse genes were involved in fibrolysis-antifibrolysis system, transcriptional control, inflammatory response, intra-cellular signaling cascade, etc.. In which, Serpinel and Plaur were key genes in fibrolysis-antifibrolysis system, Copeb was a trascriptional control gene of uPA, Nr4a1 was a trascriptional control gene of Serpine1, and Ptgs2 was a controlling gene in PGI2 biosynthesis. According their coexpression trend and functions, it is presumed that the 7 unknown genes could be related to TDVT.8．The Pathway analysis showed that the above-mentioned 30 genes were mainly involved in 12 signaling pathways, such as MAPK, complement and coagulation cascades, focal adhesion, Toll-like receptor signaling pathways, etc.. In which, MAPK pathway included several immediate early genes (IEGs), such as c-fos, c-jun, nur77, etc., which were related to proliferation, differentiation and inflammation response. These genes upregulated at the post-traumatic instant.Conclusions 1．Serpine 1 with antifibroplastic activity, which presents differential expression at post-traumatic instant, are related to thrombogenesis at thrombotic crest-time; Nr4a1 is through regulating Serpine1 on transcriptional level so as to play antifibroplastic effects.2．Plaur with fibroplastic activity, which presents differential expression at post-traumatic instant, are related to thrombogenesis at thrombotic crest-time; Copeb is through regulating uPA on transcriptional level so as to play fibroplastic effects.3．Ptgs2, Dusp1, Junb, etc, presents coexpression with Serpine 1, Plaur, Nr4a1, Copeb; they could have common regulatory elements or same cellular resouce and perform similar biological effects in TDVT.4．After MAPK pathway started by traumatic stimuli, immediate-early genes such as c-Jun, c-fos, etc. are upregulated, successively induce the upregulation of Ptgs2 expression level and increase synthesis and release of PGI2 by vascular endothelium so as to play vasodilatation and platelet aggregation inhibition effects to inhibit thrombogesis.5．Unknown genes RGD:1359581, LOC301105 /// Syncrip_predicted, Dnajb1 predicted, RGD1307124_predicted, 1374429_at, 1375422_at, 1383052_aat presents coexpression with Plaur, Serpine1, Nr4a1, Copeb, Ptgs2, Dusp1, Junb. They could be related to thrombogenesis at thrombotic crest-time in TDVT. And it is essential to do further studies on them. 6．Many genes presenting differential expression at post-traumatic instant are involved in MAPK signaling pathway. This indicated that after trauma, injuried tissue reparative process is through transcriptional control to regulate cells proliferation and differentiation.