Osteoblastic differentiation of rabbit adipose-derived stem cells transfected by polyethylenimine-mediated bone morphogenetic protein-2 gene in vitro

Abstract

Objective To investigate the expression of human bone morphogenetic protein-2 (BMP-2) gene transfected to rabbit adipose-derived stem cells (ADSCs) mediated by polyethylenimine and assess the effect of osteoblastic differentiation of the cells.Methods A total of 10 g adipose tissue was collected from the neck of adult Japanese white rabbits,and after isolation and culture in vitro ADSCs were obtained,and identified phenotypically.BMP-2 was transfected into rabbit ADSCs mediated by polyethylenimine (13 × 103).Positive cell clones were selected with G-418.The expression of BMP-2 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) respectively.The expression of osteocalcin and collagen Ⅰ was detected by Western blotting.Alkaline phosphatase was detected with ALP kit,and stained with alizarin red staining.The untransfected groups served as the control groups.Results ADSCs were successfully isolated from rab bit adipose tissue.ADSCs at passage 3 and 6 highly expressed CD29 and CD44 (the positive rate being more than 90％) and low expressed CD34 and CD45 (the positive rate being less than 5％).The expression of BMP-2 mRNA and protein was detectable and that of alkaline phosphatase,osteocalcin and collagen I increased in transfected ADSCs,and Alizarin red staining was postive.Conclusion It is feasible that polyethylenimine-mediated BMP-2 gene can be transfected into rabbit ADSCs.BMP-2 gene was effectively expressed in ADSCs and induce directional osteogenic differentiation of ADSCs. Key words: Polyethylenimine; Bone morphogenetic protein-2; Adipose-derived stem cells; Gene transfection