A Method To Establish An Angiogenesis Assay Model By Co-culturing EPCs And Fetal Metatarsal

Objective 1.By the introduction of exogenous endothelial progenitor cells(EPCs)into fetal metatarsal angiogenesis assay,establishing a model of angiogenesis in vitro innovatively.The cellular activities were analyzed by quantificating the endothelial sprouting tube-like structures from fetuses mouse metatarsals,researchers can directly observe the effects of EPCs on angiogenesis of fetal metatarsals,and with the aid of the assay model can better screening or reaction of different experimental targets of EPCs promote or inhibit angiogenesis ability.2.Study the most suitable number of EPCs and the best co-culture time in the EPCs combine with fetal metatarsal angiogenesis assay model tentatively.Methods 1.EPCs were isolated and cultured successfully from six to eight weeks C57BL/6 murine bone marrow by in vitro amplification.2.Dissect the fetal metatarsal from the embryo of 17.5 days pregnant C57BL/6 murine in aseptic condition.3.Fetal metatarsals were treated with various number of EPCs of 5×103,1×104,5×104 and 1×105,co-ciltureed for 12 days,14 days and 16 days,also we set up an blank control group.4.Immunostain the endothelial sprouting tube-like structures with CD31.5.The positive staining endothelial sprouting tube-like structures from fetuses mouse metatarsals were quantified by computerized image analysis software(Image-Pro Plus 6.0).6.The datas were tested and analyzed by SPSS 17.0.Results 1.Using the method of whole bone marrow adherent adherent method can be effective when combined with poor EPCs cell separation and amplification.2.The best co-culture time in the EPCs combine with fetal metatarsal angiogenesis assay model is 14 days.3.The most suitable number of EPCs in the EPCs combine with fetal metatarsal angiogenesis assay model is 5×104.Conclusion We successfully constructed the angiogenesis assay model by co-cultureing EPCs and fetal metatarsal,and EPCs activities were quantificated by analyzing the endothelial sprouting tube-like structures from fetuses mouse metatarsals.