人骨形态发生蛋白2基因重组腺病毒表达载体转染兔骨髓间充质干细胞*

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。

Human bone morphogenetic protein-2 recombinant adenovirus expression vector transfection of rabbit bone marrow mesenchymal stem cells

BACKGROUND: The adenovirus vector structured by GatewayTM has made up the deficiencies of exogenous cytokine application.  OBJECTIVE: To observe the target gene expression and osteoblastic differentiation of human bone morphogenetic protein-2 (hBMP-2) recombinant adenovirus expression vector (Ad-BMP-2/GFP) after transfected with bone marrow-derived mesenchymal stem cells (rBMSCs). METHODS: To isolate and culture, purify rBMSCs through the density gradient centrifugation combined with adherent cultivation; the Ad-BMP-2/GFP adenovirus vector constructed by GatewayTM technology was transfected with rBMSCs.RESULTS AND CONCLUSION: Osteocalcin was the osteoblast-specific product which could be expressed on the transfected rBMSCs detected by RT-PCR method; hBMP-2 gene was positive at mRNA and protein levels. Rabbit bone marrow stromal cells were successfully separated and purified. Ad-BMP-2 can efficiently transfect rBMSCs and the BMP-2 can be expressed in BMSCs efficiently.