Peripheral blood endothelial progenitor cells of rabbits

Abstract

Objective To investigate the isolation and culture of endothelial progenitor cells(EPCs) from rabbit peripheral blood and the identification of the cells.To explore the feasibility of using enhanced green fluorescent protein (EGFP) to label the cells in order to get ready for subsequent experiments.Methods Male healthy New Zealand white rabbits were selected.Total rabbit peripheral blood mononuclear cells (MNCs) were isolated by Histopaque density-gradient centrifugation and were suspended in endothelial basal medium supplemented with EGM-2 MV BulletKit and then the cells were plated on fibronectin-coated culture dishes.Process of cell growth was observed.EPCs were identified by Dil labeled acetylated low density lipoprotein and FITC labeled UEA-1 lectin.The cells showed red fluorescence were cells phagocytized acetylated low density lipoprotein,while those with green gluorescence were cells bind with BS-1,and the cells double stained showed orange fluorescence.The EPCs were infected by the adenovirus supernatant carrying EGFP,the expression of EGFP were detected by fluorescence microscope.Results Observation of cell morphous showed that new isolated mononuclear cells were round.Attached spindle-like cells were observed at 3 to 4 days' culture ,while cell clusters were observed at 5 to 8 days.Result of EPCs identification by Dil labeled acetylated low density lipoprotein and FITC labeled BS-1 lectin showed that in kytoplasm of EPCs,red fluorescent concentration bind with acetylated low density lipoprotein appeared,with the positive rate of over 95%.Combined rate with UEA-1 lectin nearly reached 100%.Double staining rate reached over 90%.The expression of EGFP began in 24 hours,reached maximum in 4-5 days,maintained stable from week one to week two after infection and then decreased.Transfection efficiency was about 95%.Conclusion Cell colony with the feature of EPCs can be isolated and cultured successfully from peripheral blood of rabbits.The EPCs that transfected by Ad-EGFP can express EGFP efficiently and the transfection efficiency is ralatively high,which proved this method is effective to label EPCs. Key words: Endothelial progenitor cell; Isolation; Transfection; Green fluorescent protein; Identification