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| 电穿孔法转染人树突状细胞条件的优化 | |
| 引用本文： | 单铁英,苏安英,唐军民. 电穿孔法转染人树突状细胞条件的优化[J]. 长治医学院学报, 2008, 22(4) |
| 作者姓名： | 单铁英 苏安英 唐军民 |
| 作者单位： | 1. 河北工程大学医学院人体解剖学与组织胚胎学系,056002 2. 北京大学基础医学院人体解剖学与组织胚胎学系 |
| 摘 要： | 目的:通过电穿孔法将带有增强型绿色荧光蛋白基因(enhanced green fluorscent proteinEGFP)的真核表达载体pIRES2-EGFP(IRES2 internal ribosome entrysite 2 内部核糖体进入位点2)质粒导入未成熟树突状细胞(Immature Dendritic Cell,imDC),探讨不同电穿孔条件对imDC电转染率和存活率的影响.方法:通过密度梯度离心法分离人外周血单核细胞,采用细胞因子体外培养诱导其成为imDC.在大肠杆菌中扩增pIRES2-EGFP质粒,选择不同的电压、脉冲时间、细胞浓度、DNA剂量等.应用电穿孔仪将pIRES2-EGFP质粒导入未成熟树突状细胞,荧光显微镜下和光镜下分别计算绿色荧光阳性细胞数和总细胞数.0.4%锥虫蓝(trypan blue)染色,计数电转染后细胞存活率.结果:当imDC浓度为5×10<'6>/mL与6/μg DNA质粒混合,设定电转染仪电压为300 V、脉冲时间为500 μs时,其电转染率到最高,细胞存活率最高,转染后24 h明显表达,48 h后表达最强.结论:在适当的电压、脉冲时间下,选择适当的细胞浓度、DNA剂量,在转染后的一定时间范围内,电穿孔法转染imDC可使目的基因获得较高的转染率,且细胞死亡最少.电转染提供了外源基因转染imDC的可行技术. |
| 关 键 词： | 电穿孔 转染 绿色荧光蛋白 树突状细胞 人 |
Optimum Conditions of Tansfection on Dendritic Cells by Electroporation |
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| Shan Tieying,Su Anying,Tang Junmin. Optimum Conditions of Tansfection on Dendritic Cells by Electroporation[J]. Journal of Changzhi Medical College, 2008, 22(4) | |
| Authors: | Shan Tieying Su Anying Tang Junmin |
| Abstract: | Objective:pIRES2-EGFP was successfully transfacted into immature dendritic cells by means of electroporation in order to investigate the effect of electransfection efficiency and survival rate of immature dendritic cells in different conditions.Methods:Monocytes obtained form human peripheral blood by density guadient centrifugation are induced into imDCs in the presence of cytokines in vitro.pIRES2-EGFP was amplified in E.coli.imDCs were transferred with pIRES2-EGFP by means of electroporation with differe... |
| Keywords: | Electroporation Transfect Green fluorescent protein Dendritic cell Human |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ | |