Study On The Gene Expression Changes Of Traumatic Deep Vein Thrombosis And Prevention With Low Molecular Weight Heparin

Objective:Using rats as the model,detect the expression changes of genes during the development of traumatic deep vein thrombosis(TDVT),and identify the differentially expressed genes between thrombosis and non-thrombosis veins. Especially,screen the therapeutic drug target genes which could prevent TDVT.Materials and Methods:PartⅠ:Study on the Gene Expression Changes of Traumatic Deep Vein Thrombosis1.150 SD rats were divided into control(Group A,Oh)and experiment groups randomly.In model rats,beating on bilateral posterior limbs combined with hip spica cast fixation were performed.The experiment group was divided into 7 subgroups according to the different biological phases,i.e.the post-traumatic instant(Group B, 0.5h),the initial period of thrombosis(Group C,72h),the crest-time of thrombosis (Group D,120h),non-thrombosis in post-traumatic 120h(Group H,120h),thrombi solution(Group E,168h),thrombi insolution(Group F,168h)and post-traumatic non-thrombosis sequentially(Group G,168h).Each 10 individuals were selected into corresponding group randomly.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.2.Incise 4 to 5 cm femoral vein of the rats in those different biological phases. About 0.5 cm of each proximate femoral vein was cut for HE staining to confirm the reliability of the previous grouping.The rest of each femoral vein was used to extract total RNA respectively.After RNA quality was assessed,each sample was hybridized to GeneChip(?)Rat Genome 230 2.0 array(Affymetrix)to detect the mRNA expression profiles.3.Based on the Fold Change analysis,the differential expression genes were classified according to GO classification.And the differentially-expressed genes of D vs H group were identified through pathway analysis.PartⅡ:The differential expression genes function assembly analysis between thrombosis and non-thrombosis in traumatic deep vein thrombosisGO enrichment test was further analysed using these differentially-expressed genes olD vs H group,and functional overrepresentation of these genes was identified.PartⅢ:Study on the gene expression of low molecular weight heparin preventing traumatic deep vein thrombosis by time series1.Besides the 150 rats,another 50 rats were selected to modeling by the same method.Then they were divided into drug prophylaxis group(n=40)and control group(Group Y0,n=10).The drug prophylaxis group was injected into abdominal cavity with corresponding dose of LMWH(500 IU/kg)in the sixth hour after modeling,then they were injected with same medicine and by the same method one time a day.The drug prophylaxis group rats were divided into two groups according to the time of sampling.3 hours after the first LMWH injection,the group Y1 (random choose 10 individuals in drug prophylaxis group)were sampled,and the groups Y2(n=30)were sampled in the 120^thhour.HE staining was done in these samples to confirm the reliability,then the RNA was extracted for array preparation.2.Gene expression tendency analytical method was used to detect the gene set which could affect the phenotype significantly.Then the functional significance of these genes was investigated through cluster analysis,finally GO annotation was used to identify the function catalog of the genes with same expression pattern.Results:PartⅠ:Results of the Gene Expression Changes of Traumatic Deep Vein Thrombosis1.In this model,the rate of thrombogenesis was 50.5%at 120 hours after trauma.The rate of non-thrombogensis was 49.5%at 120 hours after trauma.The rate of thrombi solution was 56.7%at 168 hours after trauma,and the other 43.3% remained insoluted.2.The results of the observations of the femoral vein thrombosis through naked eyes and microscope were consistent.3.During TDVT,many genes were differentially-expressed,which were related to apoptosis,binding,metabolism,cell cycle,enzyme regulation,signal transduction and transcription regulation,etc..4.Besides these,the differentially-expressed genes were mainly involved in MAPK, Writ,JAK-STAT,focal adhension,apoptosis signal pathways etc.PartⅡ:Results of the differential expression genes function assembly analysis between thrombosis and non-thrombosis in traumatic deep vein thrombosis1.In D vs H group,806 of them were differentially expressed genes,in which,51 up-regulated and 755 down-regulated.And the differential expression genes with known functions mainly related to binding,catalytic activity,signal transducer activity, enzyme regulator activity,transporter activity,etc..2.The gene function assembly analysis of group D vs H indicated that complement activation,development,growth,morphogenesis,cell motility,localization,protein metabolism etc.and the genes of C4bpα,Bf,Serpinel and Plaur etc.were related to the state of thrombosis and non-thrombosis.PartⅢ:Results of the gene expression of low molecular weight heparin preventing traumatic deep vein thrombosis analysis by time series1.120 hours after the modeling,the rate of thrombogenesis of group D was 50.5%, while the rate of group Y2 treated with LMWH was 16.7%.The comparison of these rate had statistical significance(P＜0.01).2.GO annotation of the genes,which were analyzed by time series in drug prophylaxis groups,identified several GO classification which had significant effect. They were related to voltage-gated ion channel activity,acyltransferase activity, enzyme binding,transferring groups other than amino-acyl groups, hematopoietin/interferon-class(D200-domain)cytokine receptor activity,sugar binding,transmembrane receptor activity etc..And the corresponding genes were Kcna5,Clcn3,Kcnk3,Acat1,Rgd1305719-predicted,Agpat2-predicted,Fez1,Rab3ip, Axin2,Ifngr2-predicted,Loc304091,I16r,Mgl1,Sell,Cspg2,Loc498276 and Fcgr2b. 3.The expression of Sell,Loc498276,Mgl1,Cspg2,Serpinb2,Aebp1_predicted, Ddr1,Pabpc4_predicted,Plaur,Nrp1 etc.were changed after prevention with low molecular weight heparin.Conclusion:1.The differentially-expressed genes during TDVT were related to inflammation, fibrolysis/anti-fibrolysis,blood coagulation/anticoagulation,complement etc..2.The signaling pathways of apoptosis,focal adhension,MAPK,Wnt,JAK-STAT etc.might influence the biological states of TDVT through regulated cell cycle, proliferation,differentiation etc..3.Many genes which were related to cell cycle,binding,metabolism,apoptosis, signal transduction etc.were involved in the process of TDVT.4.The up regulation of Bf,C4bpαand down regulation of Kcna5,Clcn3,Kcnk3, Acatl,RGD1305719_<sub>predicted,Agpat_predicted,Fez1,Rab3ip and Axin2 might decrease the ability of anti-thrombosis,and promote thrombosis.5.Some genes such as Kcna5,Kcnk3 and Clcn3 were not reported as the interrelated genes of TDVT in literatures through PubMed.6.The up regulation of Plaur,Serpinel,I16r,Tg,Loc304091,Fcgr2b, Ifngr2-predicted might be the key step to induce the process of non-thrombosis.7.In the mechanism of LMWH which prevented TDVT,besides thrombin and FXa, Sell,Loc498276,Mgl1,Cspg2,Serpinb2,Aebp1_predicted,Ddr1,Pabpc4_predicted, Plaur,Nrp1 etc.also played an important role in the process.Further studies should be focused whether they were the successful drug target genes to prevent TDVT.8.We first studied the gene expression of LMWH preventing TDVT by time series analytical method.Our work provided the convenient way to screen out genes which were responsible to the thrombosis and non-thrombosis through mathematical computing mode.It also provide a new way to elucidate the molecule mechanism of TDVT.