The Effect Of Inhibiting Autophagy On Organization And Recanalization Of Vein Thrombosis In EPCs

Object: Induced differentiation of endothelial progenitor cells (endothelial progenitorcells, EPCs) in vitro;Transplanted the EPCs which was intervened by the inhibitor ofmacroautophagy(3-MA),then discussed the role of EPCs in recanalization and theneovascularization.So we can provide a new way and experimental basis for the treatmentof deep vein thrombosis.Method:1.BMMNCs were isolated from rat bone marrow through ficoll and culturedwith EGM-2MVmedium cultivation.Observe the features of EPCs.The cells morphologywere observed by microscopy. Also immunocytochemistry performed to analyze theexpression of VEGF-2,vWF and immunofluorescence were performed to analyze theexpression of CD34and progentitor cell marker CD133dynamically. MTT assay was usedto study the activity of proliferation.2.Experimental rat model of chronic deep veinthrombosis were obtained byperforming external banding valvuloplasty between the proximal end of the renal veins andthe proximal part of lumbar veins of inferior vena cava and ferric chloride was applied tothe external vein surfac．The rats were randomly divided into four groups;A(n=10),controlgroup,without any treatment;B(n=10), EPCs group, lml10~6EPCs transplantation；C(n=10)，Injection of1ml3-MA diluent,and the concentration is5mmol/l;D(n=10)transplanted lml EPCs which was disposed by3-MA that the concentration is5mmol/l.0days,7days,14days,28days after transplantation, we used Immunohistochemicalstaining、 HE staining and SME to detect recanalization and the neovascularization．TheSPSS17.0software used for analysis.Results:1.the lately-separated bone marrow mononuclear cells(BMMNCs) showcircular and the size are different.The cells start attaching about3days and formingdifferent shape such as spindle, ovoid, fusiform, triangular or irregular.We can observecell colony and cells arranged in line about one week.The cells get close to coalesce and exhibite the typical cobblestone morphology. Cells growth curve was typical “S”shape.EPCs proliferation activity was rapid from7to13days. Cell growth into theplatform period after14days. Cell growth curve was typical “S” shape. The attachedcells were positively stained for CD34、VEGF、vWF、CD133and change dynamically.2.The EPCs derived from the rat bone marrow were successfully islated in vitro．Theimmunohistochemical of vWF show that capillary density in the intervention group issignificantly higher than the other three groups;Capillary density of the EPCstransplantatio group was higher than group A and group C; there were no differencebetween group A and C; The SME showed that Density of sample tube cavity structure ofgroup D was higher than other three groups.Conclusion: EPCs proliferative capacity enhancement through moderate inhibition ofautophagy,They have improve potentiality to promote angiogenesis and can accelerateorganizationandrecanalization of vein thrombus after transplantation．...