Study On Transfection Of H9C2 Cells With Recombinant Adeno-associated Virus Serotype 9 Mediated Anti-NF-κB Ribozyme In Vitro And Effect Of Nuclear Factor-κB Activity

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 was transfected into H9C2 cells at different multiplicities of infection (MOI=1×105, 1×106, 1×107 v.g./cell). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. rAAV9-EGFP-R65 (MOI=1×106 v.g./cell) was treated by the same methods. 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 transfection at MOI of 1×106and1×107 began to exhibit EGFP expression 1 days after transfection and the cells transfection at MOI of 1×105 began to exhibit EGFP expression 2 days after transfection. the fluorescence intensity increased with the MOI used for transfection. EGFP expression reached the maximum on day 4, at the point of which the transduction efficiency of rAAV9-EGFP in H9C2 cells was (14.1±0.2)%, (35.1±4.8)% and (56.8±0.1)% corresponding to MOIs of 1×105, 1×106and 1×107 respectively. 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 in H9C2 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 after transfection. TNF-αcould active NF-κB, rAAV9-EGFP-R65 and PDCT can efficiently decrease NF-κB activation in rats H9C2 cells. Conclusions: rAAV9-EGFP and rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition. TNF-αcould enhance mobilization of NF-κB in rats H9C2 cells, which suggested that some regulating effect of TNF-αon rats H9C2 cells might be exerted through NF-κB activation pathway. rAAV9-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro. This study played foundation for further research.