Lentivirus-mediated enhanced green fluorescent protein gene transfection in rabbit bone marrow mesenchymal stem cells in vitro

Objective To establish an effective method of labeling rabbit bone mesenchymal stem cells in vitro.Methods Bone marrow was harvested from the tibias of 2-week old New Zealand white rabbits and cultured by the method of direct adherent culture.Biological characters of bone marrow mesenchymal stem cells (BMSCs) were observed by an inverted microscope.CD44 and CD45 antigens of BMSCs were detected by flow cytometry.The lentiviruses were used to mediate the enhanced green fluorescent protein (EGFP) gene into the BMSCs.Results BMSCs could be observed 5 days after isolation.The primary cells were confluent in single layer after being cultured for 10 days.The 1-5th generation BMSCs were noted to have great potential of proliferation,and the average time for cell doubling was 48 h.The multiplicated ability of the BMSCs began to decrease since the 5th generation.Flow cytometry showed that the markers of BMSCs were CD44 +,CD45-.BMSCs could express green fluorescence stablely after the transfection of EGFP gene by the lentivirus.Conclusion BMSCs cultured by direct adherent culture in vitro showed stable growth and rapid proliferation.Lentivirusmediated enhanced green fluorescent protein gene transfection could integrate EGFP gene into the BMSCs' DNA and express green fluorescence efficiently,and it is an effective labelling method of BMSCs. Key words: Bone marrow mesenchymal stem cells; Cell culture; Enhanced green fluorescent protein; Label