【摘要】 目的通过克隆、构建pEGFP-BMP-2表达质粒载体,观察骨形态发生蛋白-2(BMP-2)基因在血管内皮祖细胞(EPCs)中的表达与分布。方法采用密度梯度法分离兔骨髓的单个核细胞,应用细胞荧光化学法及DAPI染细胞核法鉴定培养的细胞;BMP-2重组于pEGFP-C3中,经酶切、测序分析鉴定后,将其转染入EPCs内并筛选稳定转染细胞。结果鉴定培养的细胞为EPCs;pEGFP-C3-BMP-2表达质粒载体经双酶切鉴定、测序分析证实其构建成功;成功转染的EPCs中绿色荧光弥散分布于细胞胞质内,表明pEGFP-C3-BMP-2蛋白高表达,表达率为46%。结论分离与培养出EPCs;构建的pEGFP-C3-BMP-2表达质粒载体转染后,BMP-2蛋白高效表达于EPCs胞质中。更多还原

【Abstract】 Objective To consctructed the bone morphogenetic protein-2(BMP-2) expression vector,thus to investigate the original and post-transfection characteristics of endothelial progenitor cells(EPCs) for the expression and distribution of BMP-2 in EPCs.Methods Rabbit bone marrow mononuclear cells were isolated through density gradient separation,while immunohistochemistry and immunofluorescence cytochemistry assays were used to identify the surface markers and biological functions of EPCs.BMP-2 was engineered into the pEGFP-C3 vector as pEGFP-C3-BMP-2,which was confirmed by enzyme cutting and sequencing analysis.Results The cultrued EPCs were confirmed characteristicly and the pEGFP-C3-BMP-2 vector was also successfully constructed.Abundant expression of BMP-2 was detected in EPCs transfected with pEGFP-C3-BMP-2.Under fluorescence microscope,dispersed green fluorescence was observed in the cytoplasm of EPCs,indicating the high expression of pEGFP-C3-BMP-2 protein with the expressing rate of 46%.Conclusion The EPCs are successfully cultured.Efficient expression of BMP-2 is proved in pEGFP-C3-BMP-2 expression vector transfected EPCs.更多还原