In vitro cultivation of rat bone marrow mesenchymal stem cells and establishment of pEGFP/Ang-1 transfection method

ABSTRACTObjectiveTo obtain the bone marrow mesenchymal stem cells (BMSCs), complete phenotypic identification and successfully transfect rat BMSCs by recombinant plasmid pEGFP/Ang-1.MethodsBMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method, and purified. Then the recombinant plasmid pEGFP/Ang-1 was used to transfect BMSCs and the positive clones were obtained by the screen of G418 and observed under light microscopy inversely. Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.ResultsBMSCs cell lines were obtained successfully by adherence screening method and density gradient centrifugation. Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs, which can express Ang-1 for 3 d and decreased after 7 d.ConclusionsAdherence screening method and density gradient centrifugation can be effective methods to obtain BMSCs with high purity and rapid proliferation. Besides, the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.