Research Of The Changes About P-selectin And PSGL-1in The Rat Model Of Deep Vein Thrombosis

[Purpose]l.To build the rat model of deep vein thrombosis,and observe the formation of blood clots under different time.2. P-selectin mRNA, protein expression, PSGL-1of DVT blood and inferior vena cava wall tissue of rats to detect, analyze P-selectin, PSGL-1expression and correlation of DVT formation.3.Through Bioinformatics P-selectin, PSGL-1regulating leukocyte adhesion and thrombus formation and facilitate contact between that further more targeted research molecular mechanisms of leukocyte adhesion in thrombosis provide experimental basis.[Materials and Methods]1. The88SD rats were randomly divided into control group (n=8), sham group (n=40) and experimental group (n=40), in which the control group without any treatment; experimental group were inferior vena venous stasis Act section (narrow method) Preparation of deep vein thrombosis model; sham operation group, after separating the inferior vena cava, with a ligature to release immediately after intravenous tighten. Respectively,6h,24h,48h,72h and7d of five time points after modeling, modeling after all the rats after8overdose of anesthesia at each time point (renal vein above) inferior vena cava blood, will rats received part of anticoagulated whole blood stored in blood RNA stabilizer RN ALock by PCR assay TF, PSGL1-and gene expression of P-selectin; another part of the anticoagulated whole blood after centrifugation the plasma at-80℃, by ELISA PSGL-1and P-selectin expression in blood; while specimens from the inferior vena cava ligature1.0cm and its contents into two, one placed more than4%paraformaldehyde sent to pathology testing, another part of the wall of the vein immediately separated into liquid nitrogen tank for Western-Blot test.2. Observe the blood clots roughly with naked eyes, judgment criteriarafter laparotomy,check the colour of inferior vena cava, swelling degree, the degree of local tissue reaction,if there is visible intracavitary coagulation.Slice the intravenous tissue of rats,then stain them,observe the thrombosis of different time points.3. After intravenous tissue biopsy tissues of rats HE staining at different time points during venous thrombosis.4. Use Trizol method to extract total RNA of blood,RT-PCR reverse transcribe RNA to be cDNA,design the primers by Primer5.0software,make GAPDH for internal reference, use Real-time PCR method to detect PSGL-1and P-selectin gene expression of quantitative.5. Detect the expression quantity of PSGL-1and P-selectin by ELISA method,and find the expression changes of PSGL-1in venous organization by Western-Blot method.6. Analysis and Discussion thrombotic process PSGL-1and P-selectin expression in leukocytes, endothelial cell adhesion and promote the role of the relationship between thrombosis.[Results]1. Compared with the control group after building rats DVT model,at6h,24h,48h,72h and7d,thrombosis is clearly visible,thrombus formation exists inside the venous wall;HE tissue staining shows that most thrombus formation in inferior vena cava is visible at6h time point,the whole thrombus formation at24h adhered with vascular wall;there are mainly red blood cells in blood clots, fibrous tissue and scattered inflammatory cells are visible;besides the characteristic above,obviously there is inflammatory cells invasion inside the venous wall from48h to7d;no abnormalities in control group and sham operation group.2. It is a rising trend about the expression of PSGL-1and P-selectin mRNA in the blood in experimental group rats at6h,24h,48h,72h and7d (P<0.05),it will come to a peak at48h,no obvious changes at6h and24h (P>0.05),but with a trend of increase;compared with48h it will decline at72h and7d,but still higher than the control group, with statistical significance (P<0.05); Compared control group with sham operation group,there is no significant change (P>0.05).3. The expression of PSGL-1and P-selectin protein appears rising up first then declining in experimental group,increase from6h (P<0.05), come to a peak at48h,and it will decline at72h and7d (P<0.05);Detecting PSGL-1of the rat’s venous wall tissue by Western-Blot,it shows that the DVT model group gets an increasing trend than the control group(P<0.05);no obvious difference between control group and sham operation group.4. According to the analysis of Pathway bioinformatics:the white cell membrane surface expression of PSGL-1(the ligand) and endothelial cell membrane on the expression of P-selectin (receptor) occur in combination with each other, adhesion in the endothelial cells and monocytes, live to activate the ERK pathway in endothelial cells and NF-κB signaling pathways within the nucleus of MCP-1, TF, such as secretion to increase, thus promote thrombosis.[Conclusion]1.6h after building the deep vein thrombosis model by the inferior vena cava partial stasis method (narrow), complete thrombosis appears.2. DVT model rats blood P-selectin, PSGL-1mRNA and protein expression changes related to the process of thrombosis.3. The interaction of the P-selectin, PSGL-1regulate initial adhesion process of mononuclear cells and endothelial cells, and can activate the ERK and NF-κB signaling pathways that TF expression, so as to accelerate the formation of blood clots.