Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (10): 1847-1852.doi: 10.3969/j.issn.1673-8225.2010.10.029

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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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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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