Study On Mechanism Of Expression And Function Of Human Bone Morphogenetic Protein-2 Gene Transferred In Rabbit Bone Marrow-Derived Mesenchymal Stem Cells In Vitro

Objectives: (1) To isolate and culture mesenchymal stem cells(MSCs) obtained from rabbit bone marrow and to study their biological characteristics and potential of osteogenic differentiation under controlled condition. (2) To extract human bone morphogenetic protein 2 gene(hBMP-2) cDNA from human osteosarcoma tissue and construct an Eukaryotic Expression Vector recombinated with this gene (pIRES2-EGFP-hBMP2). (3) To investigate the expression of hBMP-2 via transfection of pIRES2-EGFP-hBMP2 into rabbit MSCs and explore its mechanism of inducing osteogenesis in vitro.Methods: (1) Rabbit MSCs isolated from bone marrow of ilia and tibia by density gradient centrifugation were purified and expanded in vitro, their biological characteristics was observed under inverted phase contrast microscope. Alkaline phosphatase (ALP) activity and calcifed nodules were observed in the second passage MSCs cultured in osteogenesis-inducing media. (2) The hBMP-2 cDNA was extracted from human osteosarcoma cells by RT-PCR method and then inserted into cloning vectors pMD18-T plasmids. The positive clones were screened out and amplified after transforming the recombinant plasmids into E. coli. strain DH5α , and the recombinant plasmids were confirmed by nucleotide sequence analysis. The hBMP-2 cDNA digested from pMD18T-BMP2 was directionally inserted into the pIRES2-EGFP eukaryotic expression vectors, and the...