电穿孔法转染人树突状细胞条件的优化

电穿孔法转染人树突状细胞条件的优化

(作者：___________ 单位： __________ 邮编：___________ )

作者：单铁英苏安英唐军民

【摘要】目的：通过电穿孔法将带有增强型绿色荧光蛋白基因

(enhan ced gree n fluoresce nt protein EGFP )的真核表达载体plRES2-EGFP( IRES2 in ternal ribosome en trysite 2 内部核糖体

进入位点2)质粒导入未成熟树突状细胞( Immature Den dritic

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plRES2-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5X 106/mL 与6卩g DNA质粒混合，设定电转染仪电压为300 V、脉冲时间为500 卩s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

Abstract Objective:plRES2-EGFP was successfully tran sfacted

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 con diti on

s.Methods:M ono cytes obta ined form huma n peripheral blood by density guadient centrifugation are induced into imDCs in the presenee of cytokines in vitro.plRES2-EGFP was amplified in

E.coli.imDCs were tran sferred with pIRES2-EGFP by means of electroporati on with differe nt electric voltages,times of impulse,cell concen trati onsand the doses of DNA.Numbers of green fluorescence positive cells and the total cell number were coun ted respectively un der fluoresce nt microscope and visible light microscope.the cells were stained with 0.4% trypan blue,electransfection efficiency and the cell survival

rate were counted.Results:Electransfection efficiency was

in creased to the highest value whe n imDCs with the

concentration of 5X 106 cells/mL were mixed with 60 卩g-total DNA,the electric voltage of electroporation apparatus was set at 300

V,and the time of impulse was 500 卩s.There was

sig ni fica nt and the highest expressi on of plRES2-EGFP respectively at 24 h and at 48 h after tran sferri ng.C on clusio n:A higher tra nsfecti on efficie ncy of target genes can be obta ined by means of electroporati on with suitable electric conditions.Electric transfection provides a

tech ni cal possibility to make DNA into imDCs.

Key words Electroporati on; Tra nsfect;Gree n fluoresce nt protein;Dendritic cell;Human

树突状细胞（dendritic cell,DCs ）作为体内惟一能活化初始T细胞的抗原递呈细胞（antigen presentingcell,APC），是激发机体特

异性免疫反应的关键，其在抗肿瘤免疫治疗领域中的作用受到广泛关注，以DCs为基础构建的各种类型的疫苗在多种肿瘤免疫治疗的基础和临床研究中显示了旺盛的生命力和良好的前景[1,2] 。本研究

应用电穿孔仪将plRES2-EGFP质粒导入未成熟树突状细胞，探索外源基因转染imDC的优化条件。

1材料与方法

1.1材料与器材

PIRES2-EGFP质粒（Clontech 公司）、Wizard 质粒抽提试剂盒