Protective effect of adenovirus-mediated vasostatin-1 gene transfection on rat cardiomyocyte hypoxia/reoxygenation injury
10.3724/SP.J.1008.2009.00491
- Author:
Zheng WANG
1
Author Information
1. Department of Cardiothoracic Surgery
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2010;30(5):491-495
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct and identify adenovirus vector containing the chromogranin A 1-76 segment (CGA1-76 ), or vasostatin-1 (VS-1) gene, and to observe its expression in cardiomyocytes of rats, so as to investigate the protective effects of VS-1 transgenic therapy on myocarclial hypoxia/reoxygenation (H/R) injury. Methods: (1) The primers of CGA 1-76 cDNA was designed and used for PCR amplification. The products were then cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme Pme I; the resultant plasmid was co-transfected into E. coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination. Then the recombinant plasmid was identified, linearized and packaged with QBI-293A cells to amplify the recombinant adenovirus Ad-VS-1, which was then used to infect H9c2 cardiomyocytes. Western blotting was used to detect the expression of VS-1 protein expression. (2) Myocardial H/R model was established and the cells were divided into 4 groups: blank group, H/R group, mock infection + H/R group, and Ad-VS-1 infection + H/R group. The viability of cardiomyocytes, the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were measured. Results: (1) The recombinant plasmid was constructed successfully as confirmed by PCR, sequencing and enzyme digestion. Western blotting confirmed the protein expression of VS-1 in the H9c2 cells. (2)The viability of cardiomyocytes and the activity of SOD in H/R group were obviously lower than those of the blank group; the level of MDA was higher than that of the blank group. Transfection with VS-1 increased the cardiomyocytes viability and SOD activity in the H/R model group and decreased the production of M1)A. Conclusion: Ad-VS-1 has been successfully constructed, and transfection with it can protect rat cardiomyocytes from H/R injury, which is related to the anti-oxidation process.
