中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (23): 4273-4278.doi: 10.3969/j.issn.1673-8225.2012.23.019

• 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和人成纤维细胞生长因子2双基因共表达腺病毒载体转染人骨髓基质干细胞后的细胞增殖

郭伟韬1，王 辉1，刘思景2，曾 荣1，肖启贤1，陈子秋1，黄 云1，王 斌1，胡资兵1   

  1. 1广东医学院附属医院骨科, 广东省湛江市 52400；
    2广州中医药大学附属中山医院，广东省中山市 528400
  • 收稿日期:2011-11-03 修回日期:2012-01-10 出版日期:2012-06-03 发布日期:2013-11-06
  • 作者简介:郭伟韬☆，男，1969年生，河南省禹州市人，汉族，2005年华中科技大学同济医学院毕业，博士，主任医师，主要从事骨科基础与临床研究。 Guoweitao2000@sina.com
  • 基金资助:

    广东省自然科学基金资助项目(S2011010004096)；广东省医学科研基金资助项目(A2009477，A2010431)

In vitro proliferation of human bone marrow stromal cells after transfection by denovirus vector co-expressing human bone morphogenetic protein-2 and human fibroblast growth factor 2 genes

Guo Wei-tao1, Wang Hui1, Liu Si-jing2, Zeng Rong1, Xiao Qi-xian1, Chen Zi-qiu1, Huang Yun1, Wang Bin1, Hu Zi-bing1   

  1. 1Department of Orthopedics, Affiliated Hospital of Guandong Medical College, Zhanjiang 524001, Guangdong Province, China;
    2Affiliated Zhongshan Hospital of Guangzhou University of Traditional Chinese Medicine, Zhongshan 528400, Guangdong Province, China
  • Received:2011-11-03 Revised:2012-01-10 Online:2012-06-03 Published:2013-11-06
  • About author:Guo Wei-tao☆, M.D., Chief physician, Department of Orthopedics, Affiliated Hospital of Guandong Medical College, Zhanjiang 524001, Guangdong Province, China Guoweitao2000@sina.com

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316

被引次数

3

摘要：

背景：利用重组腺病毒载体转染外源性基因到组织工程骨的种子细胞是骨缺损基因治疗研究的热点。
目的：用人骨形态发生蛋白2和人成纤维细胞生长因子2双基因共表达腺病毒载体转染人骨髓基质干细胞，以探讨基因转染对人骨髓基质干细胞增殖的影响。
方法：将Ad-hBMP2-IRES-hFGF2转染至人骨髓基质干细胞中，荧光显微镜观察转染效果，RT-PCR方法观察人骨形态发生蛋白2 cNDA和人成纤维细胞生长因子2 cNDA在人骨髓基质干细胞中的转录情况，Wester blot 方法检测人骨形态发生蛋白2和人成纤维细胞生长因子2蛋白表达情况，锥虫蓝测定细胞活力，流式细胞仪分析其对细胞增殖的影响。
结果与结论：转染后人骨形态发生蛋白2和人成纤维细胞生长因子2基因在mRNA水平和蛋白水平均有表达，细胞活力无明显变化，流式细胞仪分析细胞周期中增殖细胞比例明显增加。说明该双基因可高效转染人骨髓基质干细胞，且促进细胞增殖。

关键词: 腺病毒, 人骨形态发生蛋白2, 人成纤维细胞生长因子2, 骨髓基质干细胞, 基因转染, 干细胞

Abstract:

BACKGROUND: Transfection of exogenous gene into tissue-engineered seed cells via recombinant adenovirus vector is the key to gene therapy of bone defects.
OBJECTIVE: To investigate the effect of gene transfection on the proliferation of human bone marrow stromal stem cells that tranfected with adenovirus vectors co-expressing human bone morphogenetic protein-2 (hBMP-2) and human fibroblast growth factor 2 (hFGF-2).
METHODS: The Ad-hBMP2-IRES-hFGF2 plasmids were transfected into human bone marrow stromal stem cells. The efficiency of transfection was evaluated by fluorescence microscope. Reverse transcriptase polymerase chain reaction was used to observe the successful transcription of hBMP-2 and hFGF-2 cNDA in bone marrow mesenchymal stem cells. Western blot assay was used to identify the protein expression of hBMP-2 and hFGF-2 genes. The cellular viability was determined by trypan blue staining and the changes of the cell proliferation were observed by flow cytometry assay.
RESULTS AND CONCLUSION: The expression of hBMP-2 and hFGF-2 genes was confirmed at mRNA and protein levels. The cell viability was not changed after transfection. The flow cytometry assay showed that the percentage of proliferating cells was increased significantly in cell cycle. The Ad-hBMP2-IRES-hFGF2 plasmids could transfect human bone marrow stromal stem cells with high transfection efficiency and promote cell proliferation.

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