A Study Of Genes Expressions And Low Molecular Weight Heparin Intervention In Traumatic Femoral Vein Thrombosis

Objective Based on establishing a traumatic limb deep vein thrombosis (DVT) rat model, to dynamically detect femoral vein gene expression changes and screen differential expression genes at different phases in this pathological process. To focus on the differential expression genes between thrombus resolution and insolution groups. Meanwhile, to perform Low molecular weight heparin (LMWH) intervention in this model in order to select genes which could have a close relationship with DVT prognosis and could serve as drug action targets. This will be a basis of further studies about gene network in this pathological process and novel drug development for DVT.Methods:1. Establishing a traumatic limb DVT rat model and studying femoral vein gene expression.(1) 150 SD rats were randomly divided into control (group A, 10 rats) and model rats (140 rats). In the model rats, beating on bilateral posterior limbs without fixation was performed only in post-traumatic group (group B, 10 rats); beating on bilateral posterior limbs combined with hip spica cast fixation was performed in the others. According to different observation phases and/or pathological states in the process of TDVT, the model rats was subdivided into 7 groups: the control, post-traumatic instant (group B, post-traumatic 0.5h), initial period of thrombosis (group C, post-traumatic 72h), thrombogenesis at thrombotic crest-time (group D, post-traumatic 120h), nonthrombogenesis at the thrombotic crest-time (group H, post-traumatic 120h), thrombus resolution (group E, post-traumatic 168h), thrombus insolution (group F, post-traumatic 168h) and nonthrombosis at post-traumatic 168h (group G). (2) At the corresponding phases, according to gross observation, each 10 rats meeting to different pathological features were seleted into corresponding groups. Bilateral femoral veins (around 4～5cm long ) were cut respectively, in whch, 0.5 cm was cut for HE staining and histological observation in order to ensure the reliability of grouping and the rest was stored in nitrogen canister. (3) Femoral veins were mixed in the same groups. And Trizol one-step method was applied for total RNA extraction. And 1μl RNA sample of each group was taken for RNA content determination by spectrophotometer; other 1μ1 was for RNA quality determination by 2% agarose gel electrophoresis (AGE). (4) Valid RNA samples were sent to Shanghai Biochip CO. Ltd and the quality of RNA samples were detected again by Lab-on quality determination system. Then RNA samples were labeled with biotin and for hybridization. Then according to the manipulation process of Genechip Rat Genome 230 2.0, genechips were detected through cDNA probe preparation, hybridization, staining, scanning in order. (5)Combined with Fold Change (FC) analysis, to screen differental expression genes of each group compared with the control (up-regulation: Log₂ Ratio≥1 and Change was marked as I; down-regulation: Log₂ Ratio≤-1 and Change was marked as D). And the differential expression genes were classified according to GO classification.2. Differential expression genes analysis between thrombus solution and insolution groups.Based on the data from E and F groups gained from Part 1, to perform group comparison between them, and genes with an opposite expression trend in group E compared with group F (screening criterion: Log2 Ratio≥1 or≤-1) were seleted. Then Gene Cluster 3.0 software was applied in these genes for further analysis.3. An experimetal study of gene targets in LMWH preventing and curing TDVT.(1) Another 150 SD rats were modeled through the modeling method mentioned above. And in which, 50 rats were used for the study of TDVT prophylaxis and the rest 100 were for the drug therapy of TDVT. (2) The 50 rats for TDVT prophylaxis studying were modeled and divided into drug prophylaxis group (n=40) and control group (Y₀ group, n=10). In the drug prophylaxis group, at post-traumatic 6h, LMWH intraperitoneal injection was performed initially (500IU/kg, once a day). According to different time of sampling, drug prophylaxis group was subdivided into group Y₁ (n=10, sampling at 3h after initial LMWH injection) and Y₂ group (sampling at 3h after the last LMWH injection, to observe the states of thrombogenesis and to compare the thrombotic rates between Y₂ and D groups). In the control (group Y₀) rats, the same volume physiological saline was injected, and bilateral femoral veins were cut at post-injection 3h. (3) 100 SD rats used for TDVT treatment were modeled as well as observed until post-traumatic 120h. In which, the rats with thrombogenesis (n≌50) was selected for drug medication studing and subdivided into drug therapy group (n=40) and control group (group Z₀, n= 10). In drug therapy group, LMWH intraperitoneal injection was performed (600IU/kg, once a day). According to different time of sampling, drug prophylaxis group rats were subdivided into group Z₁ (sampling at 3h after initial LMWH injection) and Z₂ group (sampling at 171h after successive medication, to observe the states of thrombogenesis and to compare the thrombogenesis rates between Y₂ and E groups). Group Z₀ rats were injected the same volume physiological saline and the bileteral femoral veins were cut at post-injection 3h. (4) Each 10 rats were selected for Y₀, Y₁, Z₀ and Z₁ groups. Their bilateral femoral veins were cut for RNA extraction and genechip detection. (5) Based on FC analysis results, KEGG Pathway Database was applied to analyze the differential expression genes in Y₁/ Y₀ and Z₁/Z₀ conparisons. (6) Through performing the intersection set among the differential expression genes in E/F, Y₁/Y₀, Z₁/Z₀ conparisons, to further select coexpression genes in them.Results1. Results of TDVT rat model establishing and femoral vein gene expression analysis.(1) In this model, femoral vein thrombogenesis started at post-traumatic 72h; the thrombogenesis crest-time was at the post-traumatic 120h and the rate of thrombogenesis was 50.5%. After that, thrombi began to resolve, and to the post-traumatic 168h, the rate of thrombi solution was 56.7%; the rate of thrombusi insolution was 43.3%. And the state of thrombus insolution would be persistence until more than post-traumatic 240h. (2) The macroscopic and histological observations results of femoral vein thrombosis were conincidence with the corresponding thrombi states. (3)The femoral vein total RNA samples from the 8 groups were no pollution or degradation and their qualities met to the conditions of genechip detection. (4) In the 31042 genes which can be detected by Genechip Rat Genome 230 2.0, group B compared with the control (B/A), 349 genes presented differential expression, in which, 214 were up-regulated and 135 was down-regulated; group C compared with the control (C/A), 2393 genes presented differential expression, in which, 1386 genes were up-regulated and 1007 was down-regulated; group D compared with the control (D/A), 1743 genes presented differential expression, in which, 945 genes were up-regulated and 798 were down-regulated; group H compared with the control (H/A), 2790 genes presented differential expression, in which, 1685 genes were up-regulated and 1105 were down-regulated; group E compared with the control (E/A), 1913 genes presented differeential expression, in which, 1222 genes were up-regulated and 691 were down-regulated; group F compared with the control (F/A), 2564 genes presented differential expression, in which, 1535 genes were up-regulated and 1029 were down-regulated; group G compared with the control (G/A), 1849 genes presented differential expression, in which, 1235 genes were up-regulated and 614 were down-regulated; group D compared with group H (D/A), 805 genes presented differential expression, in which, 51 genes were up-regulated and 755 were down-regulated. The function of these genes involved cell apoptosis, binding, metablism, cell cycle, transcription regulator activity, etc..2. Results of differential expression gene analysis between thrombus solution and insolution groups.(1) In E/F comparison, 229 genes presented differential expression, in which, 111 were up-regulated and 118 were down-regulated. And the differential expression genes with known functions mainly involved cell apoptosis, binding, metablism, etc.. (2) Through Cluster analysis, the genes were divided into 3 clusters: the first cluster included 45 genes such as Mybph, Myf6, Sln, Cox6a2, Alox15, etc., and their expression levels were lower in B, C, D, F groups, but being higher in H, E, G groups; the second cluster included 76 genes such as pcytlb, tfdb2, bpgm, Ca2, etc., and their expression levels were higher only in group E, but being lower in the other phases; the third cluster genes included 108 genes such as mmpl2, tnfaip6, lamp1, pfk1, etc., and the significant expression feature of them was that they presented significantly differential expression in group F and a part of them also presnted up-regualtion in B, H groups.3. Results of the experimetal study of target genes in LMWH preventing and curing TDVT.(1) Post-traumatic 120h, the rate of thrombogenesis was 50.5%; after LMWH prophylaxis, the rate decreased to 16.7%, and there was a significantly statistical difference between them (p=0.008<0.01). (2) At post-traumatic 168h, the rate of thrombi solution was 56.7%; after LMWH therapy, the rate increased to 91.7%, and there was a significantly statistical difference between them (p=0.004<0.01). (3) In Y₁ /Y₀ comparison, 1193 genes presented differential expression, in which, 471 were up-regulated and 722 were down-regulated. (4) In Z₁/D comparison, 1229 genes presented differential expression, in which, 907 were up-regulation and 322 were down-regulation. (5) After LMWH intervention, the diferential expression genes involved several pathways including MAPK, focal adhesion, calcium, cytokine-cytokine receptor interaction signaling pathway, apoptosis, etc.. (6) The intersection set among the differential expression genes in E/F, Y₁/Y₀, Z₁/Z₀ conparisons included 15 genes.Conclusion:1. Applying trauma combined with immobilization can establish a TDVT animal model, which is more close to clinical pathological features of TDVT and can serve as a reliable model for studies of the patho genesis, prophylaxis and cure of TDVT.2. TDVT is a disease related to multiple genes which are mainly involved in cell apoptosis, binding, metablism, cell cycle, signaling transduction, etc... 3. The 45 genes including Mybph, Myf6, Sln, Cox6a2, Alox15, etc are related to nonthrombogenesis and thrombus resolution; the 76 genes including pcyt1b, tfdb2, bpgm, Ca2, etc. are related to thrombogenic prognosis and higher expression levels of them can improve the progress of thrombus resolution; the 108 genes including mmp12, tnfaip6, lamp1, pfk1, etc. play a role in thrombotic persistent insolution states.4. In the mechanism of LWMH preventing and curing TDVT, besides thrombin and Fxa, LWMH also regulates genes related to blood coagulation, anticoagulation and fibrolysis in vascular endothelial cell so as to perforn drug action. In which, Thbd, Plaur, etc. could be the drug action targets.5. The molecular mechanism of LMWH intervention in TDVT mainly involves MAPK, focal adhesion, cytokine-cytokine, calcium receptor interaction, apoptosis, etc. signaling pathways. And thrombosis as well as its prognosis is affected by cell proliferation, differentiation, inflammatory reaction, etc..6. The 15 genes including Actn3, RGD:620059, Mbp, etc., which gained from the intersection set among the differential expression genes in thrombus resolution, thrombus insolution and drug interveion groups, play important roles in the process of thrombus resolution. They will be the drug action targets in LMWH preventing and curing TDVT.