树突状细胞电转染的优化条件及影响因素

唐岩;唐军民; 战军;刘影;常昕莹;王国涛;单铁英;苏安英

解剖学报 ›› 2007, Vol. 38 ›› Issue (2) : 209-212.

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解剖学报 ›› 2007, Vol. 38 ›› Issue (2) : 209-212.
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树突状细胞电转染的优化条件及影响因素

  • 唐岩1; 唐军民1*; 战军1 ;刘影1 ;常昕莹1 ;王国涛1 ;单铁英2 ;苏安英2
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P>THE INFLUENTIAL FACTORS AND OPTIMUM CONDITIONS OF ELECTRIC TRANSFECTION ON DENDRITIC CELLS/P>

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Abstract

Objective To investigate the method and optimum conditions of electric transfection, and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line (Bel 7402). Purified monocytes as precursor DCs (pDCs) were separated from human peripheral blood cells (PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method, pDCs were incubated in RPMI-1640 medium containing rhGM-CSF (8×10SUP>5/SUP>IU/L) and rIL-4 (5×10SUP>5/SUP>IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs). The total RNA human hepatocarcinoma cell and green fluorescent protein (GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages, times of impulse, cell concentrations, temperatures and electroporation buffers. Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope, and visible light microscope. One day after the electric transfection, the cells were stained with 0.4% trypan blue, and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value, up to about 49.7% when imDCs with the concentration of 5×10SUP>6 /SUP>cells/ml were mixed with 40μg-total RNA of human hepatocarcinoma cell, the electric voltage of electroporation apparatus was set at 300V, and the time of impulse was 500Us. Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs. The major influential factors of the electransfection efficiency were electric voltage and impulsing time. As receptor cells, the imDCs growing condition was also an

关键词

电转染 / 人肝癌细胞RNA / 树突状细胞 / 电转染率 /

Key words

Electric transfect / Human hepatocarcinoma cell RNA / Dendritic cell / Electransfection efficiency / Human

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唐岩;唐军民; 战军;刘影;常昕莹;王国涛;单铁英;苏安英. 树突状细胞电转染的优化条件及影响因素[J]. 解剖学报. 2007, 38(2): 209-212
P>THE INFLUENTIAL FACTORS AND OPTIMUM CONDITIONS OF ELECTRIC TRANSFECTION ON DENDRITIC CELLS/P>[J]. Acta Anatomica Sinica. 2007, 38(2): 209-212
中图分类号: R329.3   

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