[Construction of human bone morphogenetic protein-7 gene fluorescent eukaryotic cell expression vector and test of bioactivity in vitro].
OBJECTIVE
To construct fluorescent eukaryotic cell expression vector with human bone morphogenetic protein-7 (hBMP-7) gene and to transfect mouse stromal cell line W-20-17 to detect the bioactivity of pEGFP-hBMP-7 in vitro.


METHODS
pEGFP-hBMP-7 plasmid was constructed by subcloning technique and identified by enzyme cutting and electrophoresis. W-20-17 cells were transfected with pEGFP-hBMP-7 by means of lipofectamine-2000 media methods. Transfection efficiency and gene expression were evaluated by fluorescent microscopy. ALP, von Kossa and osteocalcin (OC) were tested to determined the phenotypes of osteoblast.


RESULTS
After 48 hours, the gene transfection efficiency was 40%. Based on GFP and immunofluorescence of pEGFP-hBMP-7, there was the expression of aim gene. After gene transfection, there were not significant different of cell morphology feature and cell proliferation. ALP activity, the number of calcium nodules and the expression of OC increased.


CONCLUSIONS
pEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteoblasts.
Yang
Xue-chao
XC
Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School of Stomatology, Wuhan University, Wuhan 430079, China.
Fan
Ming-wen
MW
D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010012:Osteogenesis; D017154:Stromal Cells / Q000166:cytology*; D023822:Tissue Engineering; D014162:Transfection
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7