The Research Of The Therapeutic Potential Of Gene Modified By Adeno-associated Virus Combined With PDGF-BB In Myocardial Infarction

Objective: To explore the feasibility, safety and anti-apoptosis of using recombinantadeno-associated virus-9which contained platelet-derived growth factor-BB(AAV9-PDGF-BB) transduction to cardiomyocytes in vitro, and investigated thetherapeutic effect of PDGF-BB gene via adeno-associated virus delivered to mouse beforemyocardial infarction. Methods: The cultured neonatal cardiomyocytes were isolated,cultured and identified. With AAV9-eGFP transfected cardiomyocytes to analysed andobserved the times of eGFP expression and the transfection efficiency, Alamar Blueassays were used to evaluate the safety of transduction. After AAV9-PDGF-BB transferedto cardiomyocytes, used Western Blot and Tunel to analysed PDGF-BB expression ofcardiomyocytes, and H2O2-induced apoptosis in order to evaluate the function ofanti-apoptosis. In vivo,90Male C57BL/6mice were used, and divided three groupsrandomly, sham(n=25); MI (n=35); MI+AAV-PDGF-BB(n=30), AAV9-PDGF-BB (1.0×1011genome copies per injection) into caudal vein2weeks before myocardialinfarction(MI), hearts were harvested after3days and6weeks post-MI, to assess geneexpression levels, apoptosis, vascular density, infarct area, and cardiac function, by meansof Western Blot, RT-PCR, Tunel, tissue histology, and echocardiography. Results:(1)The expression of eGFP was gradually enhanced, and reached a peak after5-6days, andthe transfection efficiency showed that approximately82.4±2.6%; Alamar Blue assays,which evaluated the toxicity of AAV9transduction to cardiomyocytes, showed that thereduction ratios of cardiomyocytes at different times were all close to1.0.(2) Overexpression of PDGF-BB after transduction, and up-regulation of Akt phosphorylates andBcl-2, inactivates the pro-apoptotic proteins Bad, Bax and caspase-3, meanwhile decreases the number of Tunel-positive cell both in vitro and in3days post-MI.(3)In6weekspost-MI, PDGF-BB upregulates the level of ERK phosphorylates, PCNA and cyclin D1,PDGF-BB also increases vascular density and the survival of cardiomyocytes in infarctarea.(4) Decreasing in MI size and a significant improvement in cardiac function in groupof AAV9-PDGF-BB transduction. Conclusion: These findings indicate thatadeno-associated virus serotype9can be effectively transfected into cardiomyocyteswithout significant toxicity. Overexpression of PDGF-BB significantly improves theefficiency of anti-apoptosis in vitro and in the post-MI heart. PDGF-BB not only increasesvascular density and the survival of cardiomyocytes in infarct area and the improvementof cardiac function.