Objective To evaluate the transfection efficiency using recombinant adeno-associated virus Serotype 9 mediated Anti-NF-κB ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) to rats H9C2 cells and the effect of Nuclear Factor-κB (NF-κB) activity.
Methods rAAV9-EGFP-R65 was transfected into H9C2 cells at multiplicities of infection (MOI = 1 × 106 v.g./cell). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 cells were treated with TNF-α, rAAV9-EGFP- R65 and PDTC. The DNA binding activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA).
Results The cells with rAAV9-EGFP-R65 transfection at MOI of 1 × 106 v.g./cell began to exhibit EGFP expression 1 day after transfection. The fluorescence intensity increased with the time of transfection. EGFP expression reached the maximum on day 5, at the point of which the transduction efficiency of rAAV9-EGFP-R65 inH9C2 cells was (32.27 ± 3.19)%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-a could active NF-κB, rAAV9- EGFP-R65 and PDCT can efficiently decrease NF-κB activation in rats H9C2 cells.
Conclusions rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition. rAAV9-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro. This study played foundation for further research.