转染骨形态发生蛋白-2基因的血管内皮祖细胞的构建

Construction of bone morphogenetic protein-2 expressing endothelial progenitor cells

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。

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 immunohistoehemistry 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 characteristiely and the pEGFP - C3 - BMP - 2 vector was also successfully constructed. Abundant expression of BMP - 2 was detected in EPCs transfeeted 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.