Construction of recombinant adenovirus vectors encoding angiopoietin-1 gene and its transfection on bone marrow mesenchymal stem cells

Objective To construct recombinant adenovirus vectors encoding rat angiopoietin-1(ANG-1)gene and to evaluate the effects of ANG-1 gene transfection on proliferation of bone marrow mesenchymal stem cells(BMSCs)in vitro.Methods The ANG-1 gene sequence was amplified by RT-PCR and then subcloned into shuttle plasmid pAdTrack-CMV,and transformed into BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination.The recombinant vector was identified by digestion with Pac I,and was packaged and amplified in 293 cells.The successfully packaged virus were transfected to BMSCs.The expression of ANG-1 protein in BMSCs were monitored by Western Blot assay,and the effects of ANG-1 on the proliferation of BMSCs under hypoxia and normoxia conditions in vitro were evaluated by MTT assay.Results The recombinant plasmid pAdEasy-1-ANG-1 was identified to be confirmed by double digestion and DNA sequencing.The expression of ANG-1 protein in BMSCs was detected by Western Blot after the transfection of pAdEasy-1-ANG-1.MTT assay suggested that the expression of ANG-1 protein in BMSCs had no significant effect on the proliferation of BMSCs before and after transfection(hypoxia+ANG-1 vs hypoxia:P 0.05;normoxia+ANG-1 vs normoxia:P0.05).Conclusion The recombinant adenovirus vector named pAdEasy-1-ANG-1 carrying ANG-1 gene have been constructed successfully.The transfection of ANG-1 has no significant effect on the proliferation of BMSCs in vitro.