ABSTRACT. The aim of this study was to investigate the expression of bone morphogenetic protein-2 (BMP-2) in bone marrow stromal stem cells (BMSCs) and the in vivo and in vitro osteogenic activity of BMSCs transfected with the adenovirus plasmid, Ad-GFP-hBMP-2. The Ad-GFP-hBMP-2 plasmid was packaged and transfected into rabbit BMSCs to determine the transfection rate. The alkaline phosphatase (ALP) activities of Ad-GFP-hBMP-2-transfected BMSCs (experimental group) and untransfected BMSCs (control group) were detected. In situ hybridization of type I collagen and Western blot were used to determine the BMP-2 gene and protein expressions. The transfected and untransfected BMSCs were respectively inoculated into nude mice to observe in vivo osteogenesis. The decalcified bovine cancellous bone scaffold was respectively combined with transfected and untransfected BMSCs and implanted into ulnar defects in rabbits to repair the bone. The adenovirus titer was 1.2 x 10 10 pfu/mL. Green fluorescent protein expression appeared 48 h after transfection with the adenovirus plasmid, and the transfection rate was 71.1%. The ALP activity was higher in 4457 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (2): 4456-4465 (2014) Osteogenic activity the experimental group than the control group at each time point after transfection. The gene and protein expressions of BMP-2 were higher in the experimental group than the control group. The positive rates of in vivo osteogenesis in the experimental and control groups were 90% and 40%, respectively. The bone defect repair effects differed markedly between the two groups. The BMP-2 gene can be highly expressed in BMSCs to successfully induce osteogenic differentiation. BMSCs can be used as seed cells for bone tissue engineering.

ABSTRACT. The aim of this study was to construct an adenoviral expression vector for vascular endothelium growth factor 121 (VEGF 121 )-FLAG and humanized Renilla reniformis green fluorescent protein (hrGFP-1) genes, and to observe their expressions in bone marrow mesenchymal stem cells. Using pTG19T-VEGF 121 as a template, polymerase chain reaction technology was adopted to mutate the VEGF 121 gene by removing the stop codon and inserting NotI and XhoI restriction sites both before and after the gene sequences. The resultant gene was then subcloned into a pMD19-T plasmid, the pMD19-T-VEGF 121 and pShuttle-CMV-IRES-hrGFP-1 plasmids were doubledigested, and small and large fragments were linked after gel recovery to complete the construction of recombinant adenovirus vectors. After titer determination, the recombinant adenovirus vectors were used to affect Bone marrow mesenchymal stem cells rabbit bone marrow mesenchymal stem cells, and fluorescence intensity was observed under fluorescence microscopy. Enzyme digestion identification and sequencing confirmed that the recombinant plasmids were successfully constructed, and observations under fluorescence microscopy showed significant expression of green fluorescent protein in recombinant adenovirus-infected bone marrow mesenchymal stem cells. The constructed adenoviral gene expression vectors carrying VEGF 121 -FLAG and hrGFP-1 can be expressed in eukaryotic cells, which may be used for gene therapy of ischemic disorders.