High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer
High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer
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DOI：
10.1189/jlb.0906561
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发表时间：
2007-10-01
影响因子：
5.5
通讯作者：
Littel-van den Hurk, Sylvia van Drunen
中科院分区：
文献类型：
--
作者：
Landi, Abdolamir;Babiuk, Lorne A.;Littel-van den Hurk, Sylvia van Drunen
树突状细胞（Dendritic cells，DCs）是骨髓来源的专职抗原捕获细胞和抗原提呈细胞，可作为疫苗的载体。虽然已经用病毒载体实现了人DC的有效转染，但病毒基因产物可能影响细胞功能。相反，非病毒方法通常导致低效的基因转移、低水平的基因表达和/或低细胞活力。单核细胞来源的DC是体外研究和体内应用中最常见的DC来源。我们假设减少产生未成熟DC（iDC）的时间可能导致转染后更高的存活率。因此，我们建立了在3天内从CD 14（+）单核细胞产生人iDC的方案。这些“快速”iDC在表型和功能上与常规iDC无区别，显示出高内吞能力和低抗原呈递能力。此外，快速iDC正常成熟，并具有与常规成熟DC相似的抗原提呈能力。为了优化iDC的转染，我们比较了质粒DNA和体外转录（IVT）RNA与转染试剂、电穿孔和核转染的非病毒转染。用Amaxa Co. Nucleofector的X1程序核转染IVT RNA导致最有效的转染，平均93%转染的iDC、优异的长期存活力和强蛋白表达。此外，IVT RNA转染的iDC保留了iDC的所有表型和功能特征。该方法适用于大多数目的，包括体外功能测定、体内DC免疫治疗和基于DC的疫苗。
Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.