The Expression Changes And Mechanism Of Interleukin-18 And Its Relevant Biomarkers In Deep Vein Thrombosis

In this study we discuss the role and interrelationship of IL-18 and its relevant biomarkers in deep vein thrombosis. Study on the mechanism of deep vein thrombosis based on the research of animal model, Clinical investigation and experiments of HUVECs which use molecular biology techniques.Objective:1. Establish the stasis DVT rat model by straitening the inferior vena cava. Then compare the similarities and differences of IVC thrombosis in experimental groups. Detecting the expression changes of IL-18, NF-κB, vWF and GPIb-a in rat blood. And detecting the same protein’s expression in the blood of DVT patients. Discuss the role and clinical association of those cell factors in deep vein thrombosis.2. Construct the effective IL-18 over-expression lentivector and expression arrest retroviral vector in vitro. Observing the function changes of HUVECs through gene chip assay after HUVECs infected with lentivector and retrovial vector plasmids. Establish the DVT rat models after injected over-expression lentivector and expression arrest retroviral vector plasmids of IL-18. Then compare IVC thrombosis result of different groups. Detecting IL-18 and its relevant genes expression change in venous wall of DVT rat models. Discuss the influence of IL-18 in deep vein thrombosis go a step further.3. Performing HUVECs experiments about the interrelationship between IL-18 and its relevant cell factors in vitro. Investigate the mechanism of those differential expression cell factors with deep vein thrombosis in-depth exploration.Method:1.120 SD-rats were randomized into the DVT-model group (n=40), sham-operated group(n=40) and control group (n=40). Set up 2h,8h,24h and 72h time point after established model according to the classical references and experiences. Animals were sacrificed in different time point, 10 rat-models in each time point of different group. Then observe the difference of IVC thrombosis among three groups. Detecting the protein expression of IL-18, NF-κB, vWF and GPIb-a in the rat blood with ELISA.2. Collected and prepare clinical blood samples of different patients and healthy volunteers. Randomized into the DVT patient group(n=30), control group(n=20) and healthy volunteers group(n=20). Detecting the protein expression of IL-18, NF-κB, vWF and GPIb-a in blood with ELISA.3. Construct the effective IL-18 over-expression lentivectors and expression arrest retroviral vectors of human and rat in vitro. Verify the efficiency of over-expression or inhibition ablity for IL-18. Extract the total RNA of HUVECs after infected with lentivector and retrovial vector plasmids for gene chip assay and observing the function changes of HUVECs.4. Establish the DVT rat models after injected over-expression lentivector and expression arrest retroviral vector plasmids of IL-18. Then compare IVC thrombosis result among different groups. Detecting IL-18 and its relevant genes expression change in venous wall of DVT rat models with real-time PCR and Western blot assay.5. Add exogenous IL-18 into the culture media of HUVECs for preliminary treatment in vitro. Investigate the evidence for activation of NF-κB signaling pathway through the stimulation of IL-18 would influence the normal function of HUVECs and affect the expression of key markers, such as vWF, P-selectin and t-PA. Comprehensive analysis the interrelationship of IL-18 and deep vein thrombosis with all results.Result:1. Establish the stasis DVT rat model by straitening the inferior vena cava is reliable. We could get appropriate rate of thrombosis and survival rate for DVT rat model, the percentage of residual luminal area should be controlled in about 10%. There have formation of completeness thrombus at 8h after rat modeled and stabilized at 24h.2. The protein expression of IL-18, NF-κB, vWF and GPIb-a in ELISA up grade followed by the thrombosis formation especially at 24h after rat modeled(P<0.05). We also find the same performance of those protein expression in DVT patients more than control group and healthy volunteers group(P<0.05).3. We successfully construct the effective IL-18 gene over-expression or inhibition vector by use over-expression lentivector and expression arrest retroviral vector. There are obviously influence on physiological function of HUVECs after the over-expression effect of IL-18 according to the gene chip assay results and a great number of differentially expressed gene were found. Inhibition of IL-18 would lead to different result.4. Comparative analysis the IVC thrombosis weight, length and weight/length ratio-value among different groups. Establish the DVT rat models after injected over-expression lentivector and expression arrest retroviral vector plasmids of IL-18 shows significant influence in thrombus formation(P< 0.05). Gene and protein expression of NF-κB and vWF are appear the same tendency according to the expression of IL-18 in different experiment groups.5. Activation of NF-κB signaling pathway through the stimulation of IL-18 could influence the normal function of HUVECs, increase cell injury and apoptosis in early stage. Affect the expression of key markers, such as vWF, P-selectin and t-PA.Conclusion:1. Deep vein thrombosis occurrence and development is closely related to cytokines of IL-18, NF-κB, vWF, GPIb-a, P-selectin, t-PA;2. Over-expression IL-18 would influence the normal function and apoptosis of HUVECs significantly. NF-κB activation suppression could reduce the HUVECs injury;3. Endothelium injury stimulated by IL-18 could up-regulate the expression of vWF, P-selectin and downregulate the expression of t-PA which lead to easy thrombosis in vein;4. Cell injury and dysfunction caused by activation of NF-κB signaling pathway from IL-18 are closely related to deep vein thrombosis which perhaps is one of the mechanism of DVT.