Construction of human bone morphogenetic protein-7 gene fluorescent eukaryotic cell expression vector and test of bioactivity in vitro / 中华口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>pEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteoblasts.</p>