Platelet-derived growth factor-B gene transfection reduces ischemia and hypoxia-induced myocardial apoptosis / 中国组织工程研究

ABSTRACT

BACKGROUND:Platelet-derived growth factor-B (PDGF-B) is an effective pro-angiogenic growth factor, and adeno-associated virus type 9 (rAAV9) has a strong cardiomyocyte targeting affinity, which is an ideal vehicle for ischemic heart disease gene therapy. OBJECTIVE:To explore the PDGF-B gene transfection of in vitro neonatal rat myocardial cells mediated by rAAV9 against ischemia and hypoxia-induced cardiomyocytes apoptosis. METHODRat neonatal myocardial cells were isolated and cultured, and then transfected by rAAV9-PDGF-B and empty virus, rAAV9 with enhance green fluorescent protein (eGFP), under multiplicity of infection (MOI) of 105, 106 and 107, respectively. We observed the expression of eGFP under fluorescence microscopy every day, and used flow cytometry to measure transfection efficiency of vector rAAV9. Western blot and immunofluorescence were used to evaluate protein expression of PDGF-B. Myocardial ischemia and hypoxia injury model was established in vitro on the 5th day of transfection of rAAV9-eGFP and rAAV9-PDGF-B with 107 MOI. The number of myocardial apoptosis was measured by TUNEL assay. Western blot was employed to detect the protein expression of Bax and Caspase-3 which were related apoptosis, and the effect and its possible mechanism of PDGF-B gene overexpression against myocardial apoptosis were explored. RESULTS AND CONCLUSION:rAAV9 vector can efficiently transfect neonatal rat myocardial cells. eGFP and PDGF-B protein expressed in myocardial cells correctly and efficiently, and the expression intensity increased gradual y with the increasing of time course and MOI. The expression became stable on the 5th day, and the transfection efficiency showed significant difference among these groups (P<0.01). Myocardial apoptosis rate was significantly reduced in the rAAV9-PDGF-B group than the rAAV9-eGFP group (P<0.05), and protein levels of Bax and Caspase-3 in the rAAV9-PDGF-B group were significantly lower than those of the rAAV9-eGFP group (P<0.05). These data indicate that overexpression of PDGF-B gene can effectively reduce ischemia and hypoxia-induced myocardial apoptosis, and the possible mechanism might be by inhibiting Bax and Caspase-3 protein expression, which can provide evidence of rAAV9-PDGF-B vector in the gene therapy of ischemic heart diseases.