| Background Deep vein thrombosis(DVT)is a very common and dangerous complication in hospital.Among them,orthopedic surgery is a high risk factor for the formation of DVT.Therefore,how to reduce the incidence of perioperative thrombus in orthopaedic surgery is very important and difficult in the area of clinical study and basic research.Venous thrombosis is a multifactorial disease,mainly including vascular endothelial injury,blood hypercoagulability and blood stasis.Vascular endothelial cells play a vital role inside the veins.It prevents VTE by providing a surface that prevents circulating leukocytes and platelets from recruiting and attaching to the blood vessel wall.Thus,it is believed that the activation of endothelial cells is considered to be a key step in the formation of venous thrombosis.In the recent years,studies have shown that exosomes are found to play an important role in maintaining endothelial homeostasis and mediating the development of cardiovascular diseases.However,the relationship between exosomes and deep vein thrombosis has not been determined.Therefore,it is of great significance to explore how exosomes play a role in activating venous endothelial cells and promoting venous thrombosis.It is an important step to carry out research on risk factors for the formation of thrombosis.Abnormal venous structure is also a potentially important factor Among these factors.Since the research on blood vessels mainly relies on imaging examinations,and it is high costive and time confusing,Thus the relationship between these factors are often underestimated.Therefore,there are still lack of studies on the correlation between abnormal venous vessel and postoperative thrombosis.Recently,Some data showed that there was a significant correlation between soleal vein diameter and the formation of postoperative deep vein thrombosis(DVT).However,this phenomenon has not been confirmed in patients with total joint replacement(TJR).Therefore,it may be beneficial to explore this phenomenon for understanding the mechanism of DVT.The prevention and treatment of postoperative thrombosis has been paid enough attention.The guidelines has developed many strategies to reduce thrombosis.for orthopedic patients to reduce thrombosis.However,research on preoperative thrombosis is relatively rare.Therefore,further understanding of preoperative thrombosis is of great help to improve the perioperative thrombosis prevention and treatment system for major orthopedic surgery.Purpose:1.To observe how exosomes in plasma activate endothelial cells and play a role in promoting venous thrombosis,and make a preliminary study on the biological pathways;2.To explore the role of soleal vein dilation in the formation of venous thrombosis after major orthopedic surgery and analyze the population distribution of soleal vein dilation;3.To determine the incidence of thrombosis,risk factors and the relationship on postoperative thrombosis in selected patients before joint replacement.Analysis of preoperative thrombosis and risk factors for typical diseases such as severe knee osteoarthritis;To evaluate the feasibility of anticoagulant therapy with acute proximal deep vein thrombosis without replacement of inferior vena cava filter for femoral neck patients before hip arthroplasty.Methods:1.A rat model of inferior vena cava stenosis thrombus was established,which was divided into Stenosis-Exos and Sham-Exos groups.The blood was collected and exosome was isolated by ultracentrifugation method,the morphology and size of exosome was observed by transmission electron microscopy(TEM);the particle size distribution of exosome was measured by Nanosight particle size analyzer;Western Blot was used to detect the expression of membrane specific proteins on the exosomes and cell source of exosomes was detected by imaging flow cytometry.The exosomes taken up by endothelial cells in vitro were observed under a laser confocal microscope.Western Blot was used to detect the expression of protein in endothelial cells.DAF fluorescence was used to observe the effect of Stenosis-Exos and Sham-Exos on endothelial NO.Fluorescence microscopy was used to determine monocytes bound to endothelial cells.The effect of endothelial cell-monocyte adhesion was also abserved.A rat DVT model was established.Vivo imaging was used to observe whether the Stenosis-Exos can gather more in the thrombus formation site.Moreover,the thrombus site was stained with HE and weight and length of thrombus were compared.Illumina Hi Seq TM 2500 deep sequencing was used to identify the exosome-associated mi RNA target genes between Stenosis-Exos and Sham-Exos.Top10 mi RNAs with statistical differences were selected to predict the target genes of mi RNAs.Additionally,GO function enrichment analysis and the KEGG biological pathway enrichment analysis was performed to analyse target genes and further speculate the biological pathway in which the Stenosis-exogenes act.2.patients who underwent primary knee and hip replacement in our joint center from March 2015 to December 2017 were retrospectively analyzed.Preoperative ultrasound screening was performed 1-3 days before surgery and soleal vein diameter was also assessed preoperatively.Postoperative screening was performed during hospitalization and follow-up.Furthermore,according to the predictive value,a larger sample of patients were enrolled and divided into the dilation group and non-dilation group.Univariate analysis and multivariate logistic regression analysis was used to determine risk factors in DVT formation and soleal vein dilation.3.The study was conducted between January 2013 to March 2017,ultrasound surveillance and venography was used to detect DVT before TJA.Risk factors were assessed via multivariate and logistic regression analysis in patients before TJA.Postoperative screening was conducted and the association between preoperative DVT and postoperative newly developed DVT was assessed.Then,a larger sample study eneolled 521 patients with end-stage knee osteoarthritis showed that the incidence of preoperative DVT was 6.7%.In patients with femoral neck fractures who have comfirmed with acute proximal deep vein thrombosis,all of them received anticoagulant therapy or without placement of inferior vena cava filter before hip arthroplasty.The size of thrombus before and after treatment,changes of coagulation markers,the outcome of thrombosis before surgery,during surgery,postoperatively and during follow-up,the related complications were also recorded.Results:1.A rat model of inferior vena cava stenosis thrombus was established.Stenosis-Exos and Sham-Exos were successfully extracted by ultracentrifugation.Exosome was a typical circular cup-like morphology,expressing exocrine-specific marker proteins.Stenosis-Exos can significantly reduce endothelial NO production and promote the expression of ICAM-1 and VCAM-1.Moreover,Stenosis-Exos can promote endothelial cell-monocyte adhesion.Vivo imaging technique showed that exosome of the experimental group can more easily gather in the thrombosis site.Stenosis-Exo can be found to promote the rapid of development of thrombus in the rat model.Stenosis-Exos and Sham-Exos for sequencing,screen the differentially expressed mi RNAs,predict the target genes of mi RNAs,and then perform GO(Gene Ontology)and KEGG(Kyoto encyclopedia of genes and genomes)on target genes.Stenosis-Exos is involved in the regulation of the NF-kappa B pathway,MAPK pathway,m TOR pathway,PI3K-Akt pathway,HIF-1 pathway.2.A total of 402 patients were enrolled,78 patients(19.4%)were found to have DVT during hospitalization and after discharge.Multiple regression analysis showed that SV diameter were independent predictors of postoperative and symptomatic DVT.And the cut-off value of SV diameter was set at 0.57 cm,a larger sample of 569 cases were enrolled,238 patients(41.8%)were found to have soleal vein dilation.Age and large body mass index were risk factors for preoperative soleal vein dilatation.3.A total of 500 patients were enrolled into this study.Preoperative DVT was detected in 23 cases(4.6%).Multivariable and binary logistic regression analyses indicated that hypertension and abnormal D-dimer level were independent risk factors for preoperative DVT.A larger sample showed that the incidence of preoperative DVT in osteoarthritis patients was 6.7%.And advanced age,preoperative D-dimer≥0.5ug/ml were risk factors for preoperative thrombosis.In patients with hip fracture who have confirmed with proximal DVT,the results showed 5 proximal DVTs have complete disappeared,3 cases significantly improved,1 case had no change but showed stable.After treatment,the length of the proximal thrombus was significantly decreased and D-dimer was significantly lower after treatment.No death,symptomatic pulmonary embolism,bleeding and other adverse events occurred during follow-up.Conclusions1.Stenosis-Exos can promote protein expression in endothelial cell activation,monocyte-endothelial cell adhesion and reduce endothelial NO production.In the rat thrombus model,Stenosis-Exos can promote the formation of venous thrombosis and ICAM-1 expression.This effect may be achieved by Stenosis-Exos involved in the regulation of NF-kappa B,MAPK,m TOR,PI3K-Akt,HIF-1 pathways.2.Soleal vein dilatation is a common phenomenon in patients before joint arthroplasty.The result showed that soleal vein diameter was associated with total and symptomatic DVT after total joint arthroplasty.Age and large body mass index were risk factors for preoperative soleal vein dilatation.3.The incidence of DVT before TJA was not rare.Instrumental screening should be encouraged,especially in subgroups at higher risk for preoperative DVT.It is suggested that the placement with inferior vena cava filter in femoral neck fracture patients with acute proximal venous thrombosis before hip arthroplasty may not be potent.The method of anticoagulant therapy to make the proximal thrombus completely dissolved or stabilized,and then undergo the surgical treatment may be effective. |
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