Comparative study of in vitro transfection of R65 ribozyme gene to different human vascular cells mediated by recombinant adeno-associated virus 9 / 中华微生物学和免疫学杂志

ABSTRACT

Objective To evaluate in vitro transfection of anti-nuclear factor-κB ( NF-κB) ribozyme gene to human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) mediated by recombinant adeno-associated virus 9 carrying enhanced green fluorescent protein gene ( rAAV9-EGFP-R65 ) and to study their effects on cell proliferation and NF-κB P65 expression.Methods HUVECs and HASMCs were respectively transfected with rAAV9-EGFP-R65 at different multiplicity of infection ( MOI=1 ×105 , 1 ×106 and 1×107).The expression of EGFP was observed with fluorescence microscopy .Flow cytometry was performed to evaluate the transfection efficiency .Alamar Blue assay was used to measure the proliferation of the transfected cells.Western blot was used to detect NF-κB P65 expression .Results The fluorescence intensity was enhanced along with an increased MOI and an extended time of transfection .HUVECs and HASMCs transfected with rAAV 9-EGFP-R65 began to express EGFP at 24 h after transfection .The expression peak appeared on the sixth day in HUVECs, and the fifth day in HASMCs.The efficiencies of transfection in HUVECs at MOI of 1×105, 1×106 and 1×107 on the sixth day were (1.40±1.20)%, (12.30±1.35)%and (52.80±2.05)%, respectively.The trans-fection efficiencies of HASMCs on the fifth day were (5.30±1.04)%, (18.30±2.24)% and (52.40±3.21)%at MOI of 1×105 , 1×106 and 1×107 .Cell growth and morphology were not affected by transfection .Alamar Blue assay confirmed that there was no significant difference in the absorbance value between the transfected cells and two types of control cells .Western blot assay showed that the expression of NF-κB P65 was decreased by the trans-fection of rAAV9-EGFP-R65 in HUVECs and HASMCs .Conclusion rAAV9-EGFP-R65 can be efficiently trans-fected into two types of human vascular cells .It shows no inhibitory effects on cell proliferation , but can repress NF-κB P65 expression.