RAAV9-EGFP-R65 Transfection Of Rat Cardiac Fibroblasts In Vitro And Effect Of Nuclear Factor-κB Activity

Objective: To assess the transfection efficiency of recombinant adeno-associated virus serotype 9 mediated enhanced green fluorescent protein (rAAV9-EGFP) to rat cardiac fibroblasts and the impact on growth of rat cardiac fibroblasts. And 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 cardiac fibroblasts cells and on the effect of Nuclear Factor-κB (NF-κB) activity. Methods: The rat cardiac fibroblasts were collected and cultured. The rAAV9 carrying enhanced green fluorescence protein (rAAV9-EGFP) gene was transfected into rat cardiac fibroblasts at different multiplicities of infection (MOI=1×10~4, 1×10~5, 1×10~6). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage was determined by flow cytometry. AlamarBlue assay was used to assess the proliferation of the transfected cells. The quantitative analysis of the DNA binding activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA). The cells were divided into controlled group, rAAV9-EGFP-R65 tranfected group, TNF-αtreated group and TNF-αtreated rAAV9-EGFP-R65 tranfected group. Results: The cells with rAAV9-EGFP transfection at MOI of 1×10~6 began to exhibit EGFP expression 24 hours after transfection and the cells transfection at MOI of 1×10~5 and 1×10~4 began to exhibit EGFP expression 48 hours after transfection. The cells growth was normal in all three groups under inverted fluorescence microscope. EGFP expression reached the peak of growth at 120h, at the point of which the transduction efficiency of rAAV9-EGFP in rat cardiac fibroblasts cells was (2.6±0.2)%, (7.3±1.4)% and (45.1±2.7)% corresponding to MOIs of 1×10~4, 1×10~5 and 1×10~6, respectively. The fluorescence intensity increased with the MOI increasing and time varying. AlamarBlue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells after transfection. TNF-αcould active NF-κB activation pathway, rAAV9-EGFP-R65 can efficiently decrease NF-κB activation pathway in cardiac fibroblasts cells. Conclusion: rAAV9 -EGFP can be expressed in cardiac fibroblasts cells stably and efficiently without causing cell growth inhibition. This study makes the further in vitro experiments possible. rAAV9-EGFP-R65 can inhibit NF-κB activation pathway in rats cardiac fibroblasts cells in vitro.