Expression Of Rac1, Nf-κb And IL-1β In Deep Venous Thrombosis

Expression of Racl, NF-κB and IL-1β in deep venous thrombosis ABSTRACT[Objective]1. Applying gene chip to screen the differential expressed genes in femoral vein wall tissue of rats DVT model, and searching the Ncbi gene and GeneCard database to explore relationship between Racl, NF-κB1, IL-1β mRNA expression change gene and DVT.2. Using color doppler ultrasound to dynamic monitor thrombosis state of rabbit DVT model, accuratly collecting blood samples, and adopting PCR to screen these genes expression in blood of rabbits DVT model, and exploring the relationship between differential expression genes and DVT.3. Through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures, then speculating that these genes may play an important role DVT;4. Applying RCR to detetct these genes expression in DVT patient blood cell, furthermore, make use of real-time RCR to identify these genes expression, so as to explore relationship between these genes mRNA expresseion DVT.[Materials and Methods]Part1:Screening the differential epressed genes in femoral vein wall tissue of rats DVT model by gene chipThe67rats were randomly divided into control group (non-modeling, n=10) and model group (n=57). Rat DVT model was established by combination with clamping both femoral veins and fixing with cast in model group. The incidence and serious degree of thrombus were observed by dissecting rat femoral vein in different time points (2.5hours and25hours after modeling). Model group were further divided into pre-thrombogenesis group (harvested venous tissue at2.5hours after modeling), thrombogenesis group and non-thrombogenesis group (harvested venous tissue at25hours after modeling, and grouped according to whether thrombosis). Acquiring the femoral vein wall tissue and extracting total RNA. Applying the Genechip Rat Genome2302.0to detect genes expression and using FC (Fold Change) to analyse the differential expression genes from experimental data. Searching the Ncbi gene and GeneCard database so as to find out Racl, NF-κB1and IL-1β closely related to ROS (Reactive Oxygen Species) production.Part2:Detecting these genes expression in blood of rabbits DVT model by PCRThe involved18rabbits were randomly divided into sham group (non-modeling, n=5) and model group (n=13). Rabbit DVT model was established by ligaturing bilateral common femoral vein and deep femoral vein. Applying color doppler ultrasound to accurately monitor the thrombosis status of common femoral vein, deep femoral vein and superficial femoral vein, and collecting the blood sample. Adoptting the PCR technology to detecte expression changes of these genes in blood sample, including Racl, IL-1β and so on. Part3:Homology analysis of these gene between rats/rabbits and humanConsulting Ncbi gene and GeneCard database to acertain these gene sequences of rats and rabbits, through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures.Part4:Adoptting PCR and real-time PCR to detecte theses genes expression changes in blood sample of DVT patientsReference "venous thromboembolism prevention NICE guide"(2012) and Diagnosis of DVT:Antithrombotic Therapy and Prevention of Thrombosis,9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (2012,9th version)", formulate the DVT diagnosis standard of this study:DVT comprehensive diagnosis=dangerous factor assessment (foundation risk factors+specialized subject risk factors)+major clinical manifestations+color doppler ultrasound (appendix2). Ruling out other risk factors (pregnancy, diabetes, cancer, immune system disease, and so on), included58research objects were normal control group (20cases, healthy volunteers), non-thrombogenesis group (20cases for lower limb fractures, long bone or spine fracture undergo operation within7day, and without thrombosis after surgery), thrombosis group (18cases DVT patients), during march2010to march2012. Fasting vein blood samples of three groups were collected in early morning. Non-thrombogenesis group was1day after surgery. Thrombosis group was1day after diagnosis. These genes expression changes (including Rac1, NF-κB1, IL-1β, etc) were screened by PCR. The differential expression genes were identified by the real-time. Futhermore, the mutual regulation relationship among Rac1, NF-κB1and IL-1β, and the function influnence on endothelial cell, leucocyte cell and platelet, were analyzed by Ncibi PuMed and GeneCard, so as to explore these genes role in regulating DVT.[Results]1. The test results of gene chip in rats DVT model femoral venous wall found that: The expressions of Rac1, Rac2, NF-κB1and IL-1(3in rat femoral venous wall tissue were significantly up-regulated at2.5hours after modeling (pre-thrombogenesis group was higher than control group), and continued up-regulating at25hours after modeling (thrombogenesis group was higher than the pre-thrombogenesis group, non-thrombogenesis group and control group). But the difference in three groups was not detected.2. The results of adopting color doppler to monitor blood flow signal of common femoral vein, deep femoral vein and superficial femoral vein in rabbit DVT model found that:more than50%of vein have thrombosis at Id after modeling; more than80%of the vein have no blood flow signal at2d after modeling, indicated that venous thrombosis reached peak; Part of the vein began to appear intermittently flow signals at3d after modeling, seggested that venous thrombosis started to organizing; More than50%of the vein appeared intermittently flow signals at8d after modeling, suggest that most of the venous thrombosis emerged organization and recanalization.3. The results of applying PCR to screen differential expression gene in blood samples of rabbit DVT model found that:the mRNA expressions of Rac1, NF-κB2, and IL-1β were significantly up-regulated at1d after modeling and continues to maintain2d,3d,8d after modeling, were higher than Od before modeling of model group and different time points (0d,1d,2d,3d,8d) of sham group (P<0.05); The difference at different time points (1d,2d,3d,8d after modeling) was not deteceted (P>0.05); The Rac2mRNA expressions difference at different time points of two group was not deteceted (P>0.05).4. The BLAST results of Rac1etc gene sequences between rats/rabbits and human found that:Rac1, Rac2, NF-κB1, NF-κB2and IL-1β gene sequences similarity between rats and human respectively were80%,78%,96%,92%and78%; Rac1, Rac2, NF-κB2and IL-1(3gene sequences similarity between rabbits and human respectively were72%,93%,99%,68%.5. The test results of PCR in blood samples of DVT patients found that:The difference of the mRNA expressions of Rac2, Rac3and NF-κB2was not detected in three groups (P>0.05); The results of real-time PCR and PCR showed that the mRNA expressions of Rac1, NF-κB1and IL-1β in thrombogenesis group was significantly higher than the non-thrombogenesis group and control group (P<0.05), and the difference was not detected between nonthrombogenesis group and control group (P>0.05)[Conclusion]1. Upregulated expression of Rac1, NF-κB1and IL-1β in rats DVT modle femoral vein wall tissue closely related to DVT.2. The color doppler ultrasound can accurate dynamic monitoring thrombosis state of the rabbit DVT model;3. Upregulated expression of Rac1,NF-κB2and IL-1β in rabbits DVT modle blood closely crosponded with the progress of thrombosis, and might be played regulated role in DVT.4. Upregulated expression of Rac1,NF-κB1and IL-1β in blood sample of DVT patienet closely related to DVT.