中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1847-1852.doi: 10.3969/j.issn.1673-8225.2010.10.029

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

非病毒载体介导脑源性神经营养因子基因转染骨髓间充质干细胞：脂质体及电穿孔转染法的比较

陈观贵1，刘谦虚2，谢鼎华3   

  1. 1广州医学院第二附属医院耳鼻咽喉科，广东省广州市   510260；
    2珠海市人民医院耳鼻咽喉科，广东省珠海市  519000； 
    3中南大学湘雅二医院耳鼻咽喉科，湖南省长沙市   410011
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 作者简介:陈观贵，男，1975年生，广东省吴川市人，汉族，2009年中南大学毕业，博士，主治医师，主要从事耳科学基础与临床的研究。 entcgg@yahoo. com.cn

Non-virus vector methods in hBDNF gene transfected bone marrow mesenchymal stem cells: Lipofectamine versus electroporation

Chen Guan-gui1, Liu Qian-xu2, Xie Ding-hua3   

  1. 1Department of Otorhinolarygology, Second Affiliated Hospital of Guangzhou Medical College, Guangzhou   510260, Guangdong Province, China;
    2Department of Otorhinolaryngology, Zhuhai People’s Hospital, Zhuhai   519000, Guangdong Province, China;
    3Department of Otorhinolaryngology, Second Xiangya Hospital of Central South University, Changsha   410011, Hunan Province, China
  • Online:2010-03-05 Published:2010-03-05
  • About author:Chen Guan-gui, Doctor, Attending physician, Department of Otorhinolarygology, Second Affiliated Hospital of Guangzhou Medical College, Guangzhou 510260, Guangdong Province, China entcgg@yahoo.com.cn

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摘要：

背景：基因转染细胞有病毒及非病毒载体法，因病毒载体面临安全性、免疫排斥等问题，实验探讨脂质体及电穿孔转染法。

目的：比较脂质体及电穿孔法介导人脑源性神经营养因子基因转染骨髓间充质干细胞的细胞转染特性和体外表达情况，建立基因工程细胞。

方法： ①脂质体法：取体外分离、培养的第3代豚鼠骨髓间充质干细胞，将质粒-脂质体混合物加入含细胞的培养基中培养6 h，再加入胎牛血清的培养基，孵育48 h 后行免疫组织化学检测，即为瞬时表达。48 h后加入含G418培养基筛选。②电穿孔法：取骨髓间充质干细胞，胰酶消化，用无血清培养基重悬细胞，将细胞悬液加入电转化池中，加入质粒，将电转化池移至电极间放电转导。转染48 h后，检测目的基因瞬时表达。48 h后加入含G418培养基筛选。用免疫组织化学及RT-PCR检测两种方法脑源性神经营养因子基因表达情况。

结果与结论：免疫组织化学显示脂质体介导转染的脑源性神经营养因子瞬时表达率约为5.80%，电穿孔法约为24.29%。脂质体法转染筛选14 d后细胞几乎全部死亡；电穿孔法转染后筛选并扩大培养建立工程细胞，免疫组织化学显示工程细胞脑源性神经营养因子阳性表达率达90%以上，RT-PCR扩增产物电泳证实目的基因阳性条带。结果表明，用电穿孔法可成功建立脑源性神经营养因子基因修饰骨髓间充质干细胞的工程细胞，并用免疫组织化学和RT-PCR方法证实细胞体外表达目的基因。

关键词: 脂质体法, 电穿孔法, 骨髓间充质干细胞, 基因转染, 非病毒载体

Abstract:

BACKGROUND: Gene transfection of cells includes virus and non-virus vector. As virus vector has some issues, such as safety and immunological rejection, the present study explored lipofectamine and electroporation transfection methods.   

OBJECTIVE: To establish genetic engineering cells using human brain-derived neurotrophic factor (hBDNF) gene transfected bone marrow mesenchymal stem cells (BMMSCs) by lipofectamine or electroporation, and explore its characteristics and expression in vitro.

METHODS: Lipofectamine method: The BMMSCs were obtained from the tibias and femurs of the guinea pigs. The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours, followed by fetal bovine medium for 48 hours. Immunohistochemistry was performed for transient expression. G418 was added after 48 hours. Electroporation method: BMMSCs were trypsinized and resuspended with serum-free medium. Cell suspension was added into electrotransformation pool, and plasmid was added. The electrotransformation pool was moved between electrodes. After transfection for 48 hours, gene transient expression was detected. G418 was added after 48 hours. Brain-derived neurotrophic factor expression was detected by immunohistochemistry and RT-PCR.

RESULTS AND CONCLUSION: Immunohistochemistry showed that BDNF transient expression was 5.80% by lipofectamine and 24.29% by electroporation. Cells almost died at 14 days following lipofectamine transfection. Stable expression cell lines of BDNF engineered BMMSC were successfully established by electroporation, with 90% expressive rate by immunohistochemistry and expression of BDNF mRNA by RT-PCR. Genetic engineering cells using BDNF transected BMMSC were established by electroporation whereas failed by lipofectamine, and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.

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