The Preliminary Study Of The Prevention And Treatment Of Venous Thrombosis With Platelet-derived Growth Factor-C Nano- Sustained Release Microspheres

Objective:(1) To explore the effect of platelet-derived growth factor-C(PDGF-C) on vessel endothelial cells and mesenchymal stem cells cultured in vitro; (2) Constructed a targeted anticoagulant chitosan nanoparticles; (3) To observe the therapy effect on vessel endothelium repair with the targeted anticoagulant chitosan nanoparticles.Methods:(1) The vessel endothelial cells and mesenchymal stem cells were cultured in vitro with different concentration of PDGF-C. Cell counting and MTT assay were used to detect cell proliferation. Flow cytometry was used to detect cell phases. Transwell assay and scarification test were used to detect cell migration. (2) Through the emulsion-chemical cross link and sulfonation reaction compose anticoagulant chitosan nanoparticles. The optical transmission electron microscope was used to characterize its morphous, infrared spectroscopy and coagulation tests were used to characterize its physical and chemical properties. And to evaluate the biological safety by hemolysis, and cells toxicity. (3) Mechanical clip renal vein in mice to establish endothelial damage model, the animal is divided into three groups, both intake the medicine and give heparin anticoagulant therapy to prevent thrombosis in 1, 2,3 days. On 1,3,7 days after transplantation take out the injury renal vein, HE staining and vWF immunofluorescence staining observation vascular injury and repair; Evans blue staining the injury vein, calculate degree of endothelial repair rate by Lucia software.Results:(1) the PDGF-C can significantly stimulate vascular endothelial cells and bone marrow mesenchymal stem cell proliferation, increase the proportion of the two kinds of cells in S phase, into a dose-response relationship on endothelial cell proliferation, the effect on promoting proliferation of mesenchymal stem cells between peak in 20 ng/ml. PDGF-C enhances between endothelial cells and bone marrow mesenchymal stem cells' ability to migrate, mesenchymal stem cell migration effect is stronger than on vascular endothelial cells; (2) scanning electron microscopy (SEM) shows that microcarrier drugs with good morphology, particle size of 50-100nm; Infrared spectrum prompt sulfonated replace; Microcarrier for PDGF-C coating rate was 54.42%. the drug at a rate of 20.33%:Microcarrier 72 hours in vitro slow-release PDGF-C the accumulative release rate 82.25%:Blood coagulation in vitro experiments show that micro drug carrier has significant anticoagulant effect (P<0.05), anticoagulant effect is dose effect relationship, when the concentration of 50 mg/ml its anticoagulant effect compared with heparin, the difference had no statistical significance; Hemolysis test, according to the sample concentration of 10 mg/ml,30 mg/ml,50 mg/ml,1.21%, conform to the national standard about the hemolysis rate of less than 5% of the safety standards; Cell toxicity test show coagulation microcarrier drugs of leaching solution has no obvious cytotoxicity; (3) the microcarrier drug after transplantation, vascular endothelial repair rate increased significantly,7 d repair rate of 93.87%, and significantly reduced micro thrombus and endometrial hyperplasia.Conclusion:(1) the platelet-derived growth factor-C can promote between the cultured vascular endothelial cells, proliferation and migration ability of mesenchymal stem cells; Has targeted anticoagulant function (2) the successful preparation of the carrier, its biological safety meet the national standards; (3) after transplantation microcarrier drug, venous endothelial injury repair accelerated obviously, reduce the related complications.