RESUMEN
Objective To investigate the feasibility of recombinant adeno-associated virus encoding secrected forms of human acidic fibroblast growth factor transfecting endothelial progenitor cells.Methods sp-haFGF was obtained through combining signal peptide sequence of FGF-4 with native aFGF gene by PCR.sp-haFGF was cloned into AAV vector plasmid pAAV-IRES-hrGFP.Recombinant AAV encoding sp-haFGF was packaged through co-transfecting HEK293 cells with plasmid sp-haFGF-pAAV-IRES-hrGFP,pAAV-RC and pHelper.Ex vivo cultured EPCs were infected with concentrated rAAV.Expression of green fluorescent protein(GFP) in infected EPC was observed by fluorescence microscope and expression of sp-haFGF in EPCs was verified by RT-PCR and western blot analysis.Results Recombinant AAV encoding sp-haFGF was obtained.After EPCs being infected with rAAV,green fluorescence was found in about 20～30% EPCs,gene of(560 bp) was generated from EPCs by RT-PCR method,and(sp-haFGF) protein was detected in infected cells.Conclusion EPC was efficiently infected by rAAV encoding(sp-haFGF) and(haFGF) was expressed by EPCs,which establishes a basis for therapeutic angiogenesis by transgenic EPCs transplantation.