Research of the culture condition and biological characteristics endothelial progenitor cells from rabbit marrow and peripheral blood in vitro

Objective:To investigate and establish the methods of in vitro culturing,differentiation and identification of endothelial progenitor cells(EPCs) of rabbit.Methods: The mononuclear cells were gained separately from rabbit bone marrow and peripheral blood by density gradient centrifugation,inoculated in the culture bottle that coated with gelatin and cultured in endothelial cell basel medium-2 then induced differentiation to EPCs.We observe the growth process of both derived EPCs by inverted microscope;measure the survival rate of passaging cells by typan blue exclusion;compare the proliferation status of both derived EPCs by observing the growth curve,MTT method,and testing the cell cycle;observe the express of EPCs-related antigen CD34,CD31,CD133,and von willebrand factor(vWF) by immunofluorescence staining.Results: Both bone marrow-derived EPCs and peripheral blood-derived EPCs are completely adherent after being cultured 3～4 d,both are clone-like growth,form a similar mature vascular endothelial cell morphology after 7～9 d,the number of cells increases gradually,the cell survival rates are 95%.The growth curves of the second generation of Both derived EPCs are S-shaped,MTT method shows that the proliferation of the cells are obvious at 3～5 d,the G0-G1 phase and S + G2 + M phase proportion of peripheral blood-derived EPCs are 96.48% and 3.52%,the G0-G1 phase and S + G2 + M phase proportion of bone marrow-derived EPCs are 97.11% and 1.84%.The result of the identification of the second generation of EPCs by immunofluorescence shows: two sources of EPCs are both positively expressed CD34、CD133 and vWF,but CD31 is weakly expressed.Conclusion: Both derived EPCs could be obtained efficiently stably cultured in vitro.And compared to the traditionally achieved EPCs from bone marrow,achieved EPCs from peripheral blood is reliable and more convenient method,providing the ideal source of cells for tissue engineering.