Construction of exogenous recombinant eukaryotic expression vector pcDNA3.1-hBMP-7 and transfection into rabbit bone marrow stromal cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Under the in vitro conditions of cell harvesting, culture, and transplantation, whether bone marrow stromal cells (BMSCs) can be effectively applied in local gene therapy remains unclear.OBJECTIVE: To construct a recombinant eukaryotic expression plasmid carrying human bone morphogenetic protein-7 (hBMP-7) gene, and to expect to enhance osteoinductive properties of rabbit BMSCs transfected.DESIGN, TIME AND SETTING: A cell-genomics in vitro observation was performed at the Laboratory of Scientific Research, Second Affiliated Hospital of Harbin Medical University between July 2006 and July 2007.MATERIALS: Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University.METHODS: hBMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying hBMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. BMSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-hBMP-7-transfected, pcDNA3.1 -transfected, and untransfected. 5×106 BMSCs were inoculated into a 60 mm3 flask containing antibiotic-free medium 1 day prior to transfection.MAIN OUTCOME MEASURES: RT-PCR and immunohistochemistry were employed to detect hBMP-7 expression in BMSCs, alkaline phosphatase activity, hydroxypreline content, and osteocalcin production in each group. RESULTS: After 72-hour transfection, a 1.3 kb fragment was seen in the pcDNA3.1-hBMP-7-transfected group, showing brown granules in the endochylema, but not seen in the pcDNA3.14ransfected and untransfected groups. ALP activity in the pcDNA3.1-hBMP-7-transfected group significantly increased at 2 days after transfection, peeked at 8 days, and still increased at 10 days. At each time point, alkaline phosphatase activity, hydroxyproline content, and osteocalcin production were significantly higher in the pcDNA3.1-hBMP-7-transfected group than in the pcDNA3.1 -transfected and untransfected groups (P<0.05 or P<0.01).CONCLUSION: Recombinant eukaryotic expression vector pcDNA3.1- BMP-7 was constructed successfully. Results indicated that hBMP-7 was expressed in BMSCs sufficiently and was involved in inducing differentiation of BMSCs into osteoblasts. The method would provide substantial basement for hBMP-7 gene therapy.