THE THERAPEUTIC POTENTIAL OF GENE MODIFIED BY ADENO-ASSOCIATED VIRUS COMBINED WITH PDGF-BB IN MI

ObjectivesTo explore the feasibility, safety, and anti-apoptosis effect and to investigate the therapeutic potential of gene modified by adeno-associated virus combined with PDGF-BB in MI.MethodsThe neonatal cardiomyocytes were isolated, cultured, and identified. The AAV9-eGFPs were transfected into the cardiomyocytes to analyse and observe the time of eGFP Expression and Transfection Efficiency. Alamar Blue Assays were used to evaluate the safety of transduction. Likewise, Western Blot Technique was used to analyse PDGF-BB expression and Tunel Assay Method was used to evaluate the function of anti-apoptosis after H2O2-induction. In vivo, 90 Male C57BL/6 mice were used, which were divided into three groups randomly; sham (n = 25), MI (n = 35), and MI + AAV9-PDGF-BB (n = 30). AAV9-PDGF-BB (1.0 × 1011 genome copies each) was injected into the caudal vein two weeks before MI. The hearts of each group were harvested three days and six weeks post MI to assess gene expression, apoptosis, vascular density, infarct area, and cardiac function.ResultsThe Expression of eGFP gradually enhanced and The Transfection Efficiency showed 82.4 ± 2.6% after 5-6 days of transduction. Alamar Blue Assays showed that the reduction ratio of cardiomyocytes were all close to 1.0. After AAV9-PDGF-BB transduction in vitro (three days of MI), overexpression of PDGF-BB in cardiomyocytes resulted in up regulation of Akt phosphorylates and Bcl-2, inactivation of pro-apoptotic proteins; Bad, Bax, and caspase-3, and decrement in the number of Tunel positive cells. Post six weeks of MI, in a group of MI+AAV9-PDGF-BB, PDGF-BB expression not only up regulates the level of ERK phosphorylates, PCNA, and Cyclin D1 but also increases vascular density and survival of cardiomyocytes in the infarct area. Decrease in size of MI and significant improvement in cardiac function were also seen in a group of AAV9-PDGF-BB transduction.ConclusionsAdeno-associated virus serotype-9 can be effectively transfected into the cardiomyocytes without significant toxicity. Overexpression of PDGF-BB with improvement in function of mice MI heart may be through the induction of angiogenesis and cardiomyocyte proliferation, activation of prosuvival pathways, and reduction of cell apoptosis.