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背景:采用GatewayTM技术构建腺病毒载体,显著弥补了外源性细胞因子局部应用的缺陷。目的:观察人骨形态发生蛋白2重组腺病毒表达载体(Ad-BMP-2/GFP)转染兔骨髓间充质干细胞后骨形态发生蛋白2的表达情况。方法:密度梯度离心联合贴壁培养法,分离、培养、纯化兔骨髓间充质干细胞;GatewayTM技术构建的Ad-BMP-2/GFP腺病毒载体转染兔骨髓间充质干细胞。结果与结论:RT-PCR检测转染诱导后的细胞表达成骨细胞特异性产物骨钙素;转染后兔骨髓间充质干细胞在mRNA水平和蛋白水平均有人骨形态发生蛋白2的表达。结果表明构建的Ad-BMP-2/GFP可高效转染兔骨髓间充质干细胞,骨形态发生蛋白2在细胞中能高效表达。
Background: The construction of adenovirus vectors using GatewayTM technology significantly overcomes the drawbacks of topical application of exogenous cytokines. OBJECTIVE: To observe the expression of bone morphogenetic protein-2 after transfection of human bone morphogenetic protein-2 recombinant adenovirus vector (Ad-BMP-2 / GFP) into rabbit bone marrow mesenchymal stem cells. Methods: Rabbit mesenchymal stem cells were isolated, cultured and purified by density gradient centrifugation and adherent culture. Rabbit mesenchymal stem cells were transfected with Ad-BMP-2 / GFP adenovirus vector constructed by GatewayTM. RESULTS AND CONCLUSION: Osteocalcin was expressed by RT-PCR in osteoblast-specific products after transfection. The expression of human bone morphogenetic protein-2 was detected at both mRNA and protein levels in transfected rabbit bone marrow mesenchymal stem cells. The results showed that the constructed Ad-BMP-2 / GFP transfected rabbit bone marrow mesenchymal stem cells, bone morphogenetic protein 2 in the cells can be highly expressed.