ASSA13-03-40 The Study of Cardiomyocytes Gene Modified by Recombinant Adeno-Associated Virus-9 Combined with PDGF-B in Vitro

Objective To explore the feasibility, safety and anti-apoptosis of using recombinant adeno-associated virus-9 which contained platelet-derived growth factor-B (rAAV9-PDGF-B) transduction to rat cardiomyocytes in vitro. Methods The primary neonatal rat cardiomyocytes were isolated and cultured, and cardiomyocytes were transduced with rAAV9-PDGF-B at MOI (multiplicities of infection) = 5 × 105, and collected cardiomyocytes from different times after transduction, respectively at1, 3, 6, 14, 21, 28 days. Proteins were extracted from the cells for Western Blot analyse and observed the expression of PDGF-B, the transfection efficiency were measured by cell immunofluorescence. Alamar Blue assays were used to evaluate the safety of rAAV9-PDGF-B transduction to cardiomyocytes. And explore the cardiomyocytes which over expressed PDGF-B whether own the capacity of protecting cells against H2O2 –induced apoptosis by the test of apoptosis-related proteins and the ratio of Tunel-positive cell. Results Cardiomyocytes were over expressed PDGF-B at the third day after transduction, and the expression of PDGF-B was increased gradually. After days 5, the expression would be stable and sustaining, such expression can last for 28 days. Cell immunofluorescence showed that approximately 78.6% cardiomyocytes overexpressed PDGF-B at days 6 after transduction. Alamar Blue assays, which evaluated the toxicity of rAAV9-PDGF-B transduction to cardiomyocytes, showed that the reduction ratios of cardiomyocytes at different times were all close to 1.0. Moreover, in Cardiomyocytes which over expressed PDGF-B, H2O2 –induced apoptosis was accompanied by the down-regulation of Bax and Tunel-positive cells, by the up-regulation of Bcl-2 and P-Akt, there was no difference between group PDGF-B+ H2O2 (P > 0.05). Conclusions rAAV9-PDGF-B could effectively transduce cardiomyocytes cultured in vitro and persistently express PDGF-B at least 28 days. rAAV9-PDGF-B was no significant toxic effects on cardiomyocytes. Furthermore, the cardiomyocytes which over expressed PDGF-B also own the capacity of protecting cells against H2O2 –induced apoptosis, these effects might be mediated by phosphorylation of Akt signalling in cardiomyocytes.