Lentiviral vectors with red fluorescent proteins for transfection of human bone marrow mesenchymal stem cells：the red fluorescent protein can express for 21 consecutive days and exert no effects on cell viability / 中国组织工程研究

ABSTRACT

BACKGROUND:To grasp the optimal multiplicity of infection (MOI) and the time when stronger fluorescence intensities produce can lay the foundation for tracing observation of human bone marrow mesenchymal stem celsin vivo in animal models. OBJECTIVE:To investigate the feasibility of HIV-1 lentivirus carrying enhanced red fluorescent protein to transfect human bone marrow mesenchymal stem cels. METHODS:Human bone marrow mesenchymal stem cels at passage 4 were divided into blank group and MOI 2, 3, 4 groups. After that, the cels were seeded into 12-wel plates at a density of 5.0×105 , and cultured in 1 mL complete medium for adult bone marrow mesenchymal stem cels containing 1% fetal bovine serum. The infectious titer of lentivirus-carried enhanced red fluorescent protein was adjusted to 1.0×10 11 TU/L. Lentivirus solution 10, 15, 20 μL at MOI=2, 3, 4 were respectively added into the MOI 2, 3, 4 groups, and 10μL PBS was added into the blank group. At 24 and 72 hours after transfection, the expression of red fluorescence was observed under an inverted fluorescence microscope and the transfection efficiency was calculated. RESULTS AND CONCLUSION:Enhanced red fluorescent protein expressed stably in bone marrow mesenchymal stem cels. At 24 hours after transfection, red fluorescence could be seen under the inverted fluorescence microscope and achieved the peak at 72 hours after transfection. Within 21 days after transfection, there were no differences in the number of human bone marrow mesenchymal stem cels between the MOI 2, 3, 4 groups and blank group (P > 0.05). These results show that the HIV-1 lentivirus carrying enhanced red fluorescent protein is feasible to transfect human bone marrow mesenchymal stem cels, with the highest transfection efficiency when the MOI=4, which can express at least for 21 days and have no effects on the proliferative activity of labeled cels.