Non-virus vector methods in hBDNF gene transfected bone marrow mesenchymal stem cells :Lipofectamine versus electroporation / 中国组织工程研究
Chinese Journal of Tissue Engineering Research
;
(53): 1847-1852, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-402700
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:
Lipofectaminemethod:
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.Electroporationmethod:
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 ANDCONCLUSION:
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Tissue Engineering Research
Year:
2010
Type:
Article
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