兔骨髓源性内皮祖细胞的获取研究

Objective: To study in vitro the isolation and cultivation of mononuclear cells(MNCs) from rabbit bone marrow and differentiation with inducible factors, and identify numbers of endothelial progenitor cell(EPCs) among them. Methods : MNCs were extracted from bone marrow by density gradient centrifugation and the method of adherence-sieve. Differentiation into endothelial cell colony unit was controlled by the committed induction with VEGF and bFGF. Analyses with flow cytometry, immunofluresence and immunohisrochemistry assay were carried out after seven-day culture. Results: Adherent cells were round after 24 hours and cell colony were formed by spindle-like cells radiating towards periphery in 72 hours; whirlpool or trabs-like cell colonies were found in 5th day and endothelioid cell colonies link with each other, even showing a tube-like structure in 7th day. CD34 and CD133 were positive in flow cytometry with 2.16±0.09%; the expression rate of KDR of differentiated cells was 72.56±8.76%, while the spindle-like cells were reactive to Dil-ac-LDL and FITC-UEA-1. Conclusion: Bone marrow-derived MNCs, isolated and cultivated with the methods of density gradient centrifugation combined adherence-sieve, can differentiate into endothelioid cells containing small amounts of EPCs with inducible factors in vitro. EPCs cannot be confirmed with the single positive reaction of Dil-ac-LDL and FITC-UEA-1.