This study was aimed to investigate the transfection efficacy of recombinant adeno-associated virus 2/1 (rAAV2/1) on bone marrow mesenchymal stem cells (BMMSCs) at different multiplicities of infection (MOI) and time, and effect of transfection on growth of rat BMMSCs. The rat BMMSCs cultured in vitro were transfected by using rAAV2/1 with enhanced green fluorescent protein (rAAV2/1-EGFP) at MOI of 1 x 10(4), 1 x 10(5) and 1 x 10(6); the EGFP expression was observed by fluorescent microscopy at 3, 7 and 14 days. The viability, proliferation multiple, differentiation ability of daughter cells were detected for evaluating the effect of rAAV2/1 on survival, proliferation and differentiation of BMMSCs and the fluorescence index (FI) were determined by flow cytometry. The results indicated that after transfection with rAAV2/1 for 24 hours the green fluorescence in BMMSCs were observed, but also the fluorescence gradually was enhanced along with prolonging of time, and reached to steady level after 7 days; the viability, proliferation multiple, differentiation ability of BMMSCs transfected by rAAV2/1-EGFP at different MOI showed no significant changes at 3,7 and 14 days (p > 0.05), meanwhile at same MOI the proliferation multiple obviously increased in comparison between 7 day vs 3 day and 14 days vs 7 days (p < 0.01). The flow cytometric detection showed that the transfection efficacy of rAAV2/1-EGFP on BMMSCs and FI increased significantly as the multiplicity of infection and culture time increased (p < 0.05). It is concluded that rAAV2/1-EGFP is able to transfect into BMMSCs effectively, but the transfection efficiency and fluorescence index increase significantly along with increase of multiplicity of infection and culture time. rAAV2/1-EGFP do not affect viability, proliferation multiple and differentiation ability of BMMSCs. rAAV2/1 is a kind of active vector for gene transfer to reform BMMSCs.
Animals ; Bone Marrow Cells ; cytology ; Dependovirus ; genetics ; Genetic Vectors ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Transfection