Isolation,Culture and Identification of Endothelial Progenitor Cells from Rat Bone Marrow

Objective To investigate the methods of isolating and culturing endothelial progenitor cells(EPCs) from rat bone marrow,and to explore the differentiation conditions of EPCs into endothelial cells.Methods The mononuclear cells were isolated from rat bone marrow by using density-gradient centrifugation,then the adherent cells were orientation induced for 2 weeks.EPCs were identified with Dil-acLDL combined FITC-UEA-1 double fluorescence,and the expression of CD34,CD133,Flk-1 and vWF were also analyzed by immuocytochemistry and immunofluorescence.Results The adherent cells stretched at 3rd day after inducing culture,endothelial cell colony-forming units(CFU-ECs) and endothelial colony-forming cells(ECFCs) appeared at the 4th～7th day,the proliferation was increased and showed typical cobblestone appearance at 10th～21st d,the cord-like structure were observed and grew as capillary arrangement.CD34,CD133,Flk-1 and vWF of adherent cells were postively expressed in immunofluorescence and Dil-acLDL combined FITC-UEA-1 double fluorescence was also positive.Conclusion The mononuclear cells isolated from rat bone marrow could show partial characteristics of endothelial cells after inducing culture in vitro.