Rat ADSCs transfected by lentivirus vector-mediated enhanced green fluorescent protein / 中华显微外科杂志

ABSTRACT

Objective To explore the labeling method of rat adipose-derived stromal cells,and observe the stem cell characteristics and the activities of EGFP-positive adipose-derived stromal cells (EGFP-ADSCs) in vitro and in vivo.Methods ADSCs were transfected for 12 h with enhanced green fluorescent protein gene (EGFP) carried by lentivirus(Lv-EGFP) vector at different value of MOI (0,5,10,25,50,100,respectively).The rate of EGFP expression and fluorescence intensity were evaluated by flow cytometric analysis and fluorescence microscopy,and cell viability was detected by MTT-test after transfection.Secondly,cells were exposed either to adipogenic medium or osteogenic medium,then stained with Oil Red O and Alizarin Red S.Cell growth was investigated on frozen longitudinal sections when EGFP-ADSCs were injected into acellular nerves to build tissue-engineered peripheral nerves repairing sciatic nerve defects in rats for 1 week in vivo.Results EGFP-positive rate and fluorescence intensity peak at 4 days after transfection.The rate of EGFP expression was 0.13％,31.09％,75.33％,92.66％,96.70％,98.38％ for MOI =0,1,5,25,50,100,respectively.The positive rate between the experimental group and control (MOI =0) existed significantly difference (P ＜ 0.05) ; the difference between MOI =1,5 groups and MOI =25,50,100 groups were also observed (P ＜ 0.05).There was no statistical difference in EGFP-positive rate and cell proliferation activity among MOI =25,50,100 groups (P ＞ 0.05).MOI =25 was chosen as best scheme to transfect ADSCs for subsequent experiments.Osteogenic and adipogenic differentiation for 20 days,orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red O staining.At 1 week in vivo,EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections.Conclusion Lv-EGFP transfection does not affect the ADSCs activity and their osteogenic and adipogenic differentiation,so could be as a tracing method for ADSCs-tissue-engineered peripheral nerves repairing nerve defects.