中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (10): 1817-1821.doi: 10.3969/j.issn.1673-8225.2012.10.024

• 干细胞转基因表达 transgenic expression in stem cells • 上一篇    下一篇

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*★

何春耒，姬广林，刘午阳，吴东宝，何  澄，王茂源，叶勇军，莫建文，高  辉   

  1. 赣南医学院第一附属医院骨科，江西省赣州市 341000
  • 收稿日期:2011-08-08 修回日期:2011-09-22 出版日期:2012-03-04 发布日期:2012-03-04
  • 通讯作者: 高辉，教授，硕士生导师，赣南医学院第一附属医院骨科，江西省赣州市341000 gaohui3721@163.com
  • 作者简介:何春耒★，男，1983年生，江西省赣州市人，汉族， 2010年广州医学院毕业，硕士，医师，主要从事创伤骨科及运动医学的研究。chunleihe1983@126.com
  • 基金资助:

    江西省教育厅科学技术项目(GJJ09327)。

     

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

He Chun-lei, Ji Guang-lin, Liu Wu-yang, Wu Dong-bao, He Cheng, Wang Mao-yuan, Ye Yong-jun, Mo Jian-wen, Gao Hui   

  1. Department of Orthopedics, First Affiliated Hospital of Gannan Medical College, Ganzhou  341000, Jiangxi Province, China
  • Received:2011-08-08 Revised:2011-09-22 Online:2012-03-04 Published:2012-03-04
  • Contact: author: Gao Hui, Professor, Mater’ s supervisor, Department of Orthopedics, First Affiliated Hospital of Gannan Medical College, Ganzhou 341000, Jiangxi Province, China gaohui3721@163.com
  • About author:He Chun-lei★, Master, Physician, Department of Orthopedics, First Affiliated Hospital of Gannan Medical College, Ganzhou 341000, Jiangxi Province, China chunleihe1983@126.com
  • Supported by:

    Science and Technology Project of Jiangxi Education Bureau, No. GJJ09327*

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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。

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

Abstract:

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.

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