| Objectives: Based on establishing a rat model of acute traumatic limb deep veinthrombosis. Through the Affymetrix 230A genechip, to study the differentialexpressed genes between the thrombi resolution group and thrombi insolution groupafter thrombosis. To explore the partial influence factors of resolution in traumaticdeep vein thrombosis on the level of gene.Methods: 1. Grouping. 20 SD rats from the total 250 (non-restriction female andmale) were divided randomly into the control group; the remained 230 Were dividedinto 6 groups: trauma instant group (B, at 0.5h after modeling), thrombosis prophasegroup (C, at 2.5h after modeling), thrombosis crest-time group (D, at 25h aftermodeling), thrombi resolution group (E, at 72h after modeling), thrombi insolutiongroup (F, at 72h after modeling), non-thrombosis group (G, at 72h after modeling)according to different phases after model being produced and the results ofpreliminary experiment.2. Modeling. Rats were not anesthetized in model producing process. After inguinalregions sterilized by Iodophors, 1cm long medial inguinal groove incision wasadopted to expose proximal femoral vein, artery and nerve. After blunt dissection,1.5cm exposed femoral vein was clamped at three points separately with 12.5mmmosquito-hemostatic forceps, once each point; the clamping strength was fasteningone barb of hemostatic forceps, lasting 3 seconds each time, then the incision wassutured, no drainage was set. Rat hibateral posterior limbs were fixed with hip spicacasts except for group A and B. At different phases after model being produced, colorand swelling extent of both feet were observed by gross observation. 3. Methods for obtaining femoral veins. Rats were anesthetized with 3％pentobarbital sodium (1ml/kg, intraperitoneal injection), supine position fixation,sterilizing anteromedial skin of hibateral posterior limbs through iodophors,exposing hibateral femoral veins. In group A, 4.5cm femoral vein and related maintributaries were resected. In group B, at 0.5h; group C, at 2.5h; group D, at 25h; groupE, F, G, at 72h after model being produced, the same region vascular tissue was alsoresected separately. Part of the 0.5cm vessel separated for HE staining histologicalanalysis, the rest were rinsed by 0.9％physiological saline to dean up blood andthrombio The vessel specimens were put into nitrogen canister in less than 30 secondsafter ex vivo, which would be used for total RNA extraction.4. Extraction of total RNA. After affirming the status of thrombosis by histologicalanalysis, total mRNAs of femoral vein specimens from the 7 phases were extractedseparately through TRIzol method. All total RNA samples were checked by agarosegel electrophoresis and devided into two portions for being detected through genechipand RT-PCR.5. RNA detection through genechip and RT-PCR. Through cRNA probespreparation, hybridization, washing, staining and scanning were performed orderly tofinish array detecting according to the operation flowsheet. To validate theaccuratissime of genechip through RT-PCR.6. Data screening and analysis. After performing the restrictive conditions:①thedata of experimental group in EvsA and FvsA should not be marked "absent"(that isto say, the signal intensity is weak);②Signal log2 ratios of the same gene comparedbetween the resolution group and insolution group must be≥1 or≤-1(representingvariability routinely), to search out the differential expression genes between theresolution group and insolution group. The screened genes were clustered throughsoftware of Gene Cluster 3.0 and drawn visual picture. These genes functions wereinquested from web "NCBI", "CNKI", etc. and aggregate analysis was done. Results: 1. 3 and 2 rats died because of femoral artery rupture and pulmonaryembolism respectively. In group A, B, C, no rat presented thrombosis. In the 190rats of group D, E, F and G, total 5 rats died. From 2.5h to 72h after model being built,149 rats presented thrombosis, 36 were no thrombosis, 64 presented thrombiresolution and 37 presented thrombi insolution. The mortality of rats is 2％, incidencerates of thrombosis, non-thrombosis and resolution are 80.54％, 19.46％and 63.37％respectively.2. The total RNA samples of 7 groups were proved to be high qualities withoutdegradation.3. The hybridization signal intensity of arrays was satisfactory. The change tendencyof IL-1βand Cinc2 detected by genechip and RT-PCR were in good coincidence.4. In the 15866 rat genes which can be detected by Affymetrix RAT 230A microarray,7389 presented differential expression, were involved in MAPK, Calcium, Focaladhesion, etc. signaling pathways; 8477 did not present differential expression in allphases.5. After comparison between group resolution and group control, 24 genesupregulated(Log2 Ratio≥4.0). Among them, 14 genes have no functional description,10 genes(Top2a,Stk6,Slpi,Olr1,Kdap,Itgam,I16,Cd8a,Brca1,A2m)withpartial GO functional description refer to DNA metabolism, protein metabolism,inflammatory reaction,etc.. 9 genes downregulated(Log2 Ratio≤-4.0). Among them,4 genes have no functional description, 5 genes(Lepr,Gpd3,Atp1b4,Af6,Adh7) withpartial GO functional description refer to cell-cell signaling, energy metabolism, etc..6. After comparison between group insolution and group control, 26 genesupregulated(Log2 Ratio≥4.0). Among them, 16 genes have no functional description,10 genes(Slpi,Olr1,Nos2,Mmp9,Kdap,Itgam,I16,Cxc12,Cinc2,Cd8a)withpartial GO functional description refer to DNA inflammatory reaction, cell-cell actionetc.; 8 genes downregulated(Log2 Ratio≤-4.0). Among them, 6 genes have nofunctional description, 2 genes(Gpd3, Cyp1a1) have partial GO functional description.7. The 118 differential expression genes between the resolution and insolution hadbeen searched out. Among them, 48 genes, which with symbols were assigned GOfunctions by Genebank and were related to the functions of apoptosis, tumor, binding,metabolism, cell cycling, signaling, construction molecular activity and transporter.8. Compared the resolution to insolution, the expressions of Mmp-9, Mmp-12,Mmp-13, IL-1, Cxc12, Cinc2, Cc13, Ptges and Arginase were conspicuous whichexpressed higher in groups of D and F and lower in groups E and G inversely.Conclusions: 1. The femoral vein express Mmp-9, Mmp-12, Mmp-13 related totissue repair, angiopoiesis, and IL-1, Cxc12, Cinc2, Cc13 related to inflammation topromote vascular repair and enhance anticoagulation. It express Ptges, Arginaserelated to vasodilatation and inhibition of platelet aggregation to regulate in commonthe thrombi resolution through changing the status of local vessel. Whether theprocess of thrombi resolution is promoted or prevented by them should be provedthrough more functional experiments at gene or/and protein level.2. The three unknown function genes(1391505xat,1379497at,1377365at)coexpress conspicuously with the above genes. It is presumed that the function ofthem are related to tissue repair, angiopoiesis, inflammation, vasodilatation andinhibition of platelet aggregation.3. The rat model of acute traumatic deep vein thrombosis can be established throughclamping femoral vein with hip spica cast fixation.4. The rat acute traumatic deep vein thrombosis is a complicated process whichinvolves in MAPK, calcium, focal adhesion, cytokine-cytokine receptor interaction,etc. signaling pathways.5. In the groups E and F, the differential expression genes (Top2a,Stk6,Slpi,Olr1,Kdap,Itgam,I16,Cd8a,Brca1,A2m,Lepr,Gpd3,Atp1b4,Af6,Adh7)refer to DNA metabolism, protein metabolism, inflammatory reaction, cell-cell signaling,energy metabolism, etc..6. Compared the resolution to insolutin, the functions of differential expression genesin the traumatic deep vein thrombosis are related to apoptosis or tumor (Itga6, Robo1,Alox12, Il1b, Cc13, Cxc12, Ptges, Il1a, Mmp-9), binding (Alox12, Igfbp3, Opr1, Mx2,Mmp12, Il1b, Cc13, Cinc2, Cxc12, Il1a, Arg1, Stxbp5, Il13ra2, Mmp-9), metabolism(Alox12, Mx2, Mmp12, Ptges, Arg1, Mmp-9), cell cycling (Il1b, Il1a), signaling(Robo1, Opr1, Il1b, Cc13, Cinc2, Cxc12, Il1a, Il13ra2), construction molecular activity(Col11a1) and transporter (Kcnj5).7. The results of Affymetrix are further verified through detection IL1 and Cinc2 byRT-PCR.
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