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  

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

作　　者：何春耒[1] 姬广林[1] 刘午阳[1] 吴东宝[1] 何澄[1] 王茂源[1] 叶勇军[1] 莫建文[1] 高辉[1] 

机构地区：[1]赣南医学院第一附属医院骨科,江西省赣州市341000

出　　处：《中国组织工程研究》2012年第10期1817-1821,共5页Chinese Journal of Tissue Engineering Research

基　　金：江西省教育厅科学技术项目(GJJ09327)~~

摘　　要：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 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.

关 键 词：腺病毒载体 骨形态发生蛋白类 骨髓间充质干细胞 GatewayTM技术 转染 

分 类 号：R394.2[医药卫生—医学遗传学]