The Experimental And Clinical Study For Role Of MMP-3,MMP-9 In Traumatic Deep Venous Thrombosis

OBJECTIVE:1. Establish traumatic DVT rat model, then detect gene express changes of femoral vein tissue with gene chip. Key states like prethrombosis, peak of thrombosis and no thrombosis, and etc. were focused to screen MMP-3, MMP-9 that differential expressed significantly, and the expression changes and internal regularities were researched;2. Detect MMP-3, MMP-9 expression changes of traumatic DVT rat blood cell in each group, and made BLAST homology compare with human, to explore its relationship with traumatic DVT process and the homology with human. 3. Detect and analyze MMP-3, MMP-9 expression changes in traumatic DVT human blood by each group,to investigate the possible role in the onset and development of traumatic DVTMETHODS:This research consists of 3 parts:Part 1:Establish traumatic DVT rat model and detect gene expression changes in rat femoral vein tissue with gene chip. Through Fold Change (FC), screen 2458 genes that differential expressed, of which were 1146 up-regulation and 1312 down-regulation. Set Single Log2 ratio=4 and group D and group G=100 as parameters, screening 51 genes like MMP-3, MMP-9, IL-18, and etc. that expressed significantly. Select MMP-3, MMP-9 as research objects after searching for many literatures.Part 2:Reestablish traumatic DVT model, According to different observation phases and biological situations of femoral vein thrombosis,100 SD rats were divided into 4 subgroups:Group A(normal control,n=10), group B(the initial period of thrombosis, n=20), Group C(crest-time of thrombosis, n=20), and Group D (no thrombus at crest-time,n=20). Extract blood total RNA in each group and detect the quality, designing RT-PCR reverse transcription primer by Oligo6.69 software. Reverse transcript total RNA of rat blood to cDNA, and detect MMP-3,MMP-9 expression of blood cells in each rat group through RT-PCR; MMP-3,MMP-9 expression quantities were detected with ELISA. After retrieval in NCBI GenBank and CNKI database, both gene sequences were compared in BLAST, confirming the homology of MMP-3,MMP-9 between rat and human (sequence alignment using the MEGA 4.0 software).Part 3:In clinic, after set up uniform criteria for observation, diagnosis, inclusion and exclusion criteria, collected clinical data according to criteria established by definite person. Experiment groups:normal control group (group A, n=10), thrombosis group (group B,n=10), no thrombosis group (group C, n=10). Collect blood samples and detect expression of MMP-3,MMP-9 with ELISA; taking GAPDH as internal control, detect MMP-3,MMP-9 expression of blood cells in each group through real-time PCR.RESULTS:1. Detection of femoral vein tissue in traumatic DVT rat with gene chip display:2458 differential expressed in total 15864 genes, among which 1146 up regulated, accounting 46.6% of total differential expressed genes; 1312 down regulated, accounting 53.4% of total differential expressed genes. Set parameters and screen MMP-3,MMP-9 as object for future research.2. MMP-3 and MMP-9 expression quantities of blood cells in traumatic DVT rat detected by RT-PCR and ELISA display:in the group of initial thrombosis, MMP-3,MMP-9 began to express high slightly; in the group of crest-thrombosis (group C), the expression were significantly high; in no thrombosis group (group D), the expression were slightly high; group C and D had significant statistical difference. Those verified results had high coordinate with that of gene chip; BLAST homology comparison showed:MMP-3,MMP-9 had comparatively high homologous between rat and human.3. MMP-3,MMP-9 expression results in traumatic DVT human blood detected by real-time PCR and ELISA showed:MMP-3,MMP-9 levels were gradually increased within 24h in group B, at 72h to the peak,120h and 168h maintained at peak level; in no thrombosis group (group C), the content slightly increased within 24h,72h reached the peak, while 120h and 168h gradually decreased. There was significant statistical difference between group B and C. This validated result has high concordance with that of gene chip detect.CONCLUSUON:1. It is an excellent superiority complemented research method that combined gene chip technique which suit for quick, accurate, high-flux and wide gene express screen, and real-time PCR and ELISA which fit important genes and proteins detection.2. Traumatic DVT is associated with an increase in expression of MMP-3,MMP-9 in local venous vascular wall and blood.3. MMP-3,MMP-9 have close correlationship with traumatic DVT, which might be one of the main factors affecting the biological situations of deep vein thrombosis.