人骨形成蛋白7荧光真核表达载体的构建及体外生物学活性检测

Construction of human bone morphogenetic protein-7 gene fluorescent eukaryotic cell expression vector and test of bioactivity in vitro

目的构建携带人骨形成蛋白7(hBMP-7)基因的绿色荧光蛋白真核表达载体pEGFP-hBMP-7,体外转染小鼠基质细胞系W-20-17,检测相关生物学活性的变化。方法应用亚克隆法构建真核表达载体pEGFP-hBMP-7,酶切电泳鉴定。脂质体法转染W-20-17细胞,检测瞬时转染效率及目的基因的表达,观察细胞形态及生长情况,检测碱性磷酸酶(ALP)、钙结节及骨钙素等成骨细胞表型。结果48h后,基因转染效率达到40%,报告基因及免疫荧光证明目的基因的表达。目的基因转染后细胞形态未见明显变化,增殖能力无明显改变,ALP活性明显增高,钙结节增多,骨钙素表达增强。结论成功构建了具有生物学活性的pEGFP-hBMP-7,基因转染后能诱导W-20-17细胞向成骨细胞表型转化。

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 , yon Kossa and osteocalcin （OC） were tested to determined the phenotypes of osteoblasL 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. Condusions pEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteobIasts.