树突状细胞电转染的优化条件及影响因素  

THE INFLUENTIAL FACTORS AND OPTIMUM CONDITIONS OF ELECTRIC TRANSFECTION ON DENDRITIC CELLS

作　　者：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] 

机构地区：[1]北京大学基础医学院组织学与胚胎学教研室,北京100083 [2]河北工程大学临床医学院,邯郸056029

出　　处：《解剖学报》2007年第2期209-212,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(39970380);河北省科技厅基金资助项目(05276101D-28)

摘　　要：目的探讨树突状细胞(DC)电转染的方法、优化条件以及影响DC电转染率和存活率的主要因素。方法提取人肝癌细胞(Bel 7402)RNA,利用淋巴细胞分离液密度梯度法分离人外周血单个核细胞(PBMC),并通过贴壁法获取单核细胞作为树突状细胞前体(pDC)。将pDC置含rhGM-CSF和rIL-4的RPMI-1640培养液内联合培养诱导7d,使之充分分化为未成熟的DC(imDC)。应用电穿孔仪(electroporation apparatus)分别将人肝癌细胞总RNA和绿色荧光蛋白(GFP)电转染至imDC,并设定不同的电压、脉冲时间、细胞浓度、温度和电转染缓冲液等条件。荧光镜下和光镜下分别计算绿色荧光阳性细胞数和总细胞数。电转染1d后,用0.4%锥虫蓝染色分别计算其电转染率和存活率。结果当imDC浓度为5×106/ml与40μg的人肝癌细胞总RNA混合,设定电转染仪电压为300V、脉冲时间为500μs时,其电转染率达到最高值,imDC存活率约49.07%。结论电转染提供了一种使人肝癌细胞RNA电转染至imDC的可行技术。影响imDC电转染率的最主要因素是受体细胞imDC的生长状况、电转染的电压及脉冲时间。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 DC, （pDC,） 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× 10^5 IU/L） and rIL-4 （5 × 10^5 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 × 10^6 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 important influential factor.

关 键 词：电转染 人肝癌细胞RNA 树突状细胞 电转染率 人 

分 类 号：R329.3[医药卫生—人体解剖和组织胚胎学]