The Role Of Macrophage Inflammatory Protein-1Alpha In Deep Venous Thrombosis And Resolution

This study was one part of the research of post-traumatic deep venous thrombosis(DVT), based on the research of animal model and gene chip technology, we found that the inflammation and DVT had close relationship. Therefore, through experiments on DVT mouse model and human blood, initial research was made about macrophage inflammatory protein-1alpha (MIP-1α) and matrix metalloproteinase2/9(MMP-2/9) who have close relationship with DVT.Objective:1. Established DVT mouse model by ligating inferior vena cava, set up different time points with reference to the classical literature reports, harvested the blood and inferior vena cava organization, observed the specific time point of DVT formation by HE dyeing, checked MIP-1α expression of blood before and after the formation of DVT.2. Application of MIP-1α neutralizing antibody (rhMIP-1α Ab) with the DVT mouse model by intraperitoneal injection, to further clarify the role of promoting DVT resolution; at the same time, to detected MIP-1α and MMP-2,-9expression in different groups by RT-PCR and ELISA methods, immunohistochemical method to detect surface specific antigen F4/80expressio of macrophages in venous wall and thromboembolus, preliminary cleared the relationship of MIP-1α and DVT.3. Gather blood samples and detect the expression changes of MIP-1, MMP-2,-9and F4/80in DVT group, non-thrombosis group and healthy control, then explor the relationship of gene expression changes and thrombosis states.Method:1.100KunMing mice were randemely divided into control group (n=8) and model group (n=92). Model was built by ligating inferior vena cava. After established model, set up2h,4h,6h,8h,12h,1d,2d,4d,8d,14d and21d etc.11time points with reference to the classical literature. Kill model mice in different time, harvested the blood and about1.0cm inferior vena cava organization. detected MIP-1α expression of the blood samples with RT-PCR and ELISA methods, observed thrombosis by paraffining the inferior vena cava organization.2.200KunMing mice were randemely divided into control group (n=8) and model group (n=192). Model was built by ligating inferior vena cava. Model mice were randomly divided into DVT group (n=96, the mice were without any intervention measures) and DVT+MIP-1α-Ab group (n=96, mice had been intraperitoneal injection MIP-1α neutralizing antibody0.5mL for7days).Set up2d,4d,8d,12d,14d and21d etc. six time points after the model established, and in every time point killed12mice of different groups, harvested about1.0cm long inferior vena cava organization. Evaluated the degree of thrombus resolution by quality/length ratio; detected MIP-1α, MMP-2, and-9expression of thrombi and vein wall by RT-PCR and ELISA method; immunohistochemical method to detect the F4/80expression of vein wall and thrombi organization in corresponding time points.3. Gather human blood samples and divided into DVT group, non-thrombosis group and healthy control group according to diagnosis. Extracte mRNA with Paxgene blood RNA kit, detected MIP-1a, MMP-2,-9and F4/80expression in human blood by RT-PCR, then verify PCR result by sequencing, and quantitatively detected by real-time PCR, then analyze the differences among the groups. Gather clinical data including the results of complete blood count, blood biochemistry, blood coagulation, color Doppler ultrasonography and etc, and then make statistical analysis.Result:1. The method to establish the stasis DVT mouse by ligating inferior vena cava, HE tissue staining displayed that at6h after modeling the inferior vena cava had partial thrombi intravascular filling and at8h it had complete thrombi; Thrombus mainly had uniform distribution of red blood cells, which visible cellulose reticular structure and scattered platelet and leukocyte; At14d the thrombi obviously reduce and organization.2. MIP-1α mRNA and protein expression in the blood of the model mice showed rising trend from2h,4h,6h,8h,12h,1d,2d,4d to8d, and at8d reached peak, then at21d drop to normal levels.3. The quality/length ratio of thrombi showed that was diminishing trend with the extension of time (P<0.05) in both DVT group and DVT+MIP-1α-Ab group; at the same time point, DVT group thromboembolus quality/length ratio is more obvious decrease (P<0.05).4. AT the same time point, F4/80and MMP-9expression in DVT group is obviously higher than that of the DVT+MIP-1α-Ab group (P<0.05), MMP-2expression between the two groups had no difference (P>0.05).5. Gene expression changes of MIP-1a, MMP-2,-9and F4/80were detected by PCR amplificate and electrophoresis, as well as real-time PCR. The gene expressions in DVT group were higher than non-thrombosis group and control group, and there is no difference between non-thrombosis and control group.Conclusion:1. By ligating inferior vena cava method to establish stasis DVT mouse model, at8h there were complete thrombosis in vein, and at14d the thrombi can occur obvious organization.2. MIP-1α expression in model group showed that it rised to the peak after8days, and at21d it droped to the normal levels.3. MIP-1α and deep vein thrombosis, which is closely related to the resolution, and its antibody could weaken the effect of thrombolysis.4. The effect of MIP-1a promoting thrombi resolution were related with monocyte/macrophage gathered and MMP-9expression; the relationship between MMP-2and thrombi resolution may be independent of the MIP-1 a effect.5. MIP-1α, MMP-2,-9and F4/80may play important role in recruit inflammatory cells and thrombosis recanalization.