Objective:To study the protective effect of adenovirus-mediated heat shock protein 70 (HSP70) gene transfection against hypoxia/reoxygenation injury of cultured neonatal rat myocardiocytes.Methods: The neonatal rat cardiomyocytes were isolated,cultured,and transfected with Ad.EGFP.The transfection efficiency and safety were examined at various titrations by fluorescence microscope and flow cytometer.The cultured cardiomyocytes were then infected with adenovirus vector harboring HSP70 cDNA.Protein expression of HSP70 was evaluated by ELISA,Western blotting analysis and immunohistochemical methods.The cytoprotective effect of adenovirus-mediated HSP70 gene transfection was studied using in vitro myocardiocyte hypoxia/reoxygenation model.Results: High purity neonatal rat cardiomyocytes were obtained by primary culture.The infection efficiency and the EGFP expression increased with the increase of MOI.Over 90% of myocardiocytes were infected at MOI 50.The growth of myocardiocytes was not inhibited at MOI 200.ELISA and Western blotting analysis showed that the transfected myocardiocytes overexpressed HSP70 under normal physiological condition.Immunohistochemical analysis showed prominent overexpression of HSP70 in the cytoplasm and nuclei in HSP70 transfection group.Results of hypoxia/reoxygenation injury model showed that the cell vitality,MTT metabolism,cell apoptosis rate,and changes of myocardial enzyme spectrum were significantly improved in the gene transfection group compared with the control group (P<0.05).Conclusion: Recombinant Ad.HSP70 can efficiently and safely infect myocardiocytes in vitro,leading to expression of HSP70 protein.Overexpression of HSP70 induced by adenovirus-mediated transfection has prominent protective effect against hypoxia/reoxygentaion injury of neonatal rat myocardiocytes.