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| An aldose reductase inhibitor screening model constructed by transfection of pSNAV-AR into HEK293 cells |
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Abstract Abstract：Objective: Aldose reductase gene, involved in the pathogenesis of diabetic complications, was recombinated with an adeno associated virus vector pSNAV2.0, and transfected into human embryonic kidney 293 (HEK 293) cells. The gene engineering produced AR would be used as the target protein to screen aldose reductase inhibitors. Restriction endonuclease digestion and ligation procedures were performed to construct the AR expression plasmid vector pSNAV-hAR. Methods: After confirmation the recombinant plasmid by PCR, restriction endonuclease digestion, and DNA sequencing, pSNAV-hAR was transfected into HEK293 cells. Results: The results of a series of analysis including AR activity assay, Western blot and immunofluorescence analysis shown the expressed protein mediated by the adeno associated virus vector transfecting HEK 293 cells, was functional AR. The traditional aldose reductase inhibitors, Sobinil and Zopolrestat, were used to test and verify the constructed cell model. Conclusion: The established AR expression model can be used in mechanismresearch of activation of polyol pathway on diabetic complications and screening potential aldose reductase inhibitors. Based on previous work, the characteristics of AR expression in the yeast cells, Saccharomyces cerevisiae, and human embryonic kidney 293 cells were introduced. Three kinds of AR activity assay methods and matters need attention were discussed as well.
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Received: 19 January 2009
Published: 10 May 2010
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| Fund: the National Natural Science Foundation of China |
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