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ASSA13-03-50 Transfection of Rats H9C2 Cells with Recombinant Adeno-Associated Virus Serotype 9 Mediated Anti-NF-kB Ribozyme in Vitro and Effect of Nuclear Factor-kB Activity
  1. Ma Yitong,
  2. Yang Yining,
  3. Liu Fen,
  4. Chen Bangdang,
  5. Xiang Yang
  1. Department of Cardiology, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China

Abstract

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.

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