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[In vitro study on two kind of recombinant adeno-associated virus-mediated transfection efficiency with enhanced green fluorescent protein into rats osteoblasts].

OBJECTIVE
To compare the transfection efficiency of two kind of recombinant adeno-associated virus-mediated transfection to rats osteoblasts with enhanced green fluorescent protein and assess the feasibility of it as a vector for gene therapy of osteoblast lesions.


METHODS
The osteoblasts of rats were isolated, cultured and identified with type I collagen staged digestion method. According to different multiplicity of infection (MOI) (MOI = 1 x 10(3), 1 x 10(4), 1 X 10(4), 5 x 10(5)), rAAV-EGFP was transfected into osteoblasts with rAAV only and rAAV-ADV co-transfection respectively. The expression of EGFP along with the transfection time was observed under inverted fluorescence microscope. The transfection efficiency and fluorescence intensity was evaluated by flow cytometry. The best MOI value was analysed and the cell growth curves were obtained according to the best MOI value to evaluate the toxic effects of rAAV-EGFP.


RESULTS
The cultured cells possessed the biological behaviors of osteoblasts. The transfection efficiency of the rAAV was increased with the increasing of MOI. The EGFP expression reached the maximum on day 5 in ADV(-) group, the transfection efficiency of rAAV2/6-EGFP and rAAV2/9-EGFP was 90.2% and 66.1% respectively when MOI was 1 x 10(5) and no significant increase was observed when MOI was 5 x 10(5). In ADV(+) group, EGFP expression reached its maximum on day 3, the transfection efficiency of rAAV2/6-EGFP and rAAV2/9-EGFP was 47.6% and 30.5% respectively when MOI was 5 x 10(5). And no significant biologic effects on the cyto-activity was observed.


CONCLUSION
The transfection efficiency of two kind of virus vectors was both very high and rAAV2/6's is higher than that of rAAV2/9. This suggested the potential of rAAV-EGFP as a safe and efficient vector for gene therapy.