Experimental Study On The Induced Differentiation Of Bone Marrow Mesenchymal Stem Cells Labelled Through Transfection Of Enhanced Fluorescence Protion Into Cardiomyocyte-like Cell In Vitro

ObjectiveTo study the feasibility of exogenous enhanced green fluorescent protein (EGFP) gene transfecting into marrow mesenchymal stem cells(MSC) and distinguish transgen cells whether they could grow stably and differentiate into cardiomyocyte-like cell in micro–environment in vitro,to find out effect of micro–environment to MSC differentiate into cardiomyocyte-like cell and lay the foundation for further application to label target cells in the transplantation study.MethodsTo extract and identify plasmid of pEGFP gene;to separate and culture rat bone marrow mesenchymal stem cells and neonatal rat cardiac myocytes;to prepare cardiac cell lysate;to transfer EGFP gene in bone marrow mesenchymal; to sieve out postive cells and observe under the microscope;to establish positive cell lines and induce them to differentiate into the myocardial-like cells.To detect expression of Cardiac-specific troponin T (cTnT),α-actin, Connexin 43 and CD31 in the postive cells by Immunohistochemistry and positive rate of transfected cells by Computer Analysis System.ResultsMSC were adherent,elongated,and spindle-shaped in the primary culture after 24 hours of plating,and then became several little clones;During mitosis the cells regained a rounded appearance,and remained loosely attached until division was completed.When they reached 80 % confluence,MSC were subcultured and gained uniform shape and similar growth pattern.MSC transferred with pEGFP by means lipofectamine media methods, the transient expression and transfeetion eficiency were subsequently observed by fluoreseent microscopy,Green fluorescent protein labeling had little effect on proliferation of bone marrow mesenchymal stem cells, The expression of EGFP was initially found in 12h after transfection, reached optimal state in 48～72h, remained strong express within 1 week and then became weak for 4 weeks.The transient efficiency of transferring pEGFP into MSC could achieve to 20%～30%. Transfection efficiency was correlated with the ratio of plasmid to lipofectamine .ConclusionsEGFP transfected by lipofectamine media could be safely expressed in MSC in vitro. Transfection efficiency was correlated with the concentration of plasmid and lipofectamine respectively;transfected mesenchymal stem cells grew well and EGFP gene expressed stably in MSC in vitro ,transfected mesenchymal stem cells can be successfully induced to become cardiac-like cells.