A Study On EPCs From Peripheral Blood Culture In Vitro And The Effect Of Aspirin On Migratory Activity Of EPCs

Background:Endothelial progenitor cells(EPCs),which are considered to include a hematopoietic stem cell population,and were shown to be incorporated into foci of neovascularization.Asahara and his colleague isolated cells from peripheral blood which express CD34,Flk-1,or AC133 antigen-positive in 1997 and identified them EPCs.This finding,that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ,is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization,and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization.Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy.Objective:In this study,we will explore and improve the method of EPCs isolation and culture in vitro from peripheral blood,find out the characteristic of EPCs culture in vitro from peripheral blood,seek the best period for cell intervention, establish the study foundation of EPCs from peripheral blood.At the same time,we will utilize Transwell chamber to imitate EPCs migration in vitro,observe the effect of aspirin on migratory activity of EPCs.We intend to provide information for reasonable application of aspirin in diseases of clinical ischemic blood vessel.Methods:This study can be divided into syntrophic two parts.1.Study on the isolation,culture and identification of EPCs from peripheral blood:MNCs were isolated from the human peripheral venous anticoagulant blood sample(20ml)by Ficoll density gradient centrifugation,we adjust centrifuge rate 2000rpm to 1200rpm.Cell counting,viabilities of MNCs were measured by trypan blue staining.The isolated MNCs were cultured in EndoCult Liquid Medium Kit with 10%excellent FBS on 25μg/ml fibronectin-coated culture dishes.The process of cells growth and morphology change were observed under inverted microscope every day. We evaluated the characteristic and function of EPCs.One,immunofluorescence detection of VEGFR-2,CD133and vWE Two,cells uptake test of ac-LDL and bind test of UEA-1. 2.The effect of aspirin on migratory activity of EPCs:At the basement of the first step of study,cells which culture in 7 days were dissociated,resuspensed, counted and put suspension in upper compartments of Transwell chamber.And then put EndoCult Liquid Medium Kit with FBS contains aspirin of different concentration in lower compartments of Transwell chamber.Put total Transwell chamber into cell incubator.After 24h,take Transwell chamber out and count migratory cells on the low side of membrane.We observe the effect of aspirin on migratory activity of EPCs in different concentration aspirin and the same concentration aspirin in different times.Results:1-2×10~6 MNCs were isolated in one ml peripheral blood by Ficoll density gradient centrifugation when we adjust centrifuge rate to 1200rpm.Trypan blue staining measured the viabilities of MNCs were about 95±3%.In the morning of culture,the most of cells shaped round or oval,mixed with small amounts of fusiform cells.A part of round MNCs attached dishes become fusiform EPCs in the fifth day. About in the seventh day,cell colonies contains several cells emerged.Cells become long-spindle form in the tenth day.After fourteen days,cells begin senesce, disintegration.The detection of endothelial progenitor cell line specific marker VEGFR-2,CD133and vWF are strong positive in seventh day.The function of EPCs for ac-LDL-uptaking and UEA-1 binding were both strong positive in seventh day. We chose seventh day' cells,used Transwell chamber to observe the effect of aspirin on migratory activity of EPCs,count migratory cells under 200×microscope. Statistical analysis confirmed that aspirin obviously decrease migratory cell population of EPCs and these migratory cell populations obviously decrease when aspirin's concentration increase.Conclusions:1.At25～37℃,MNCs can be better isolated from peripheral blood when use 1200rpm by Ficoll density gradient centrifugation.It can satisfy the culture of EPCs.2.The identification of phenotype and mensuration of activity for EPCs exhibit the period of prosperity in the seventh day to tenth day after culture in vitro.3.In the Transwell chamber culture,1mmol/L aspirin can inhibit migratory activity of EPCs,this action obviously depends on aspirin' concentration and action time.