Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells
Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells
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DOI：
10.1038/sj.gt.3301347
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发表时间：
2000-12-01
期刊：
影响因子：
5.1
通讯作者：
Ott, G
中科院分区：
文献类型：
--
作者：
Denis-Mize, KS;Dupuis, M;Ott, G
树突状细胞（DC）在抗原呈递和特异性免疫的激活中起着关键作用。目前的许多研究集中于利用DC的效力用于疫苗、基因治疗和癌症免疫治疗应用。然而，DC不容易通过传统的非病毒技术进行体外转染。一种新的DNA疫苗制剂用于确定DC是否在体外转染。该配方由质粒DNA吸附在由可生物降解聚合物聚乳酸-羟基乙酸酯（PLG）和阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）组成的阳离子微粒上组成。利用在PLG-CTAB微颗粒上配制的荧光标记质粒DNA制剂，研究巨噬细胞和树突状细胞在体外和体内的内化，我们发现大部分（但不是全部）荧光集中在内体腔室中。此外，通过RT-PCR检测，小鼠骨髓来源的树突状细胞摄取编码HIV p55 gag的质粒DNA，吸附在PLG-CTAB微粒上，导致靶基因表达。抗原随后被处理并呈递，导致H-2k(d)限制性、gag特异性T细胞杂交瘤的刺激。通过IL-2的产生检测，该杂交瘤的激活在0.1-20马克杯DNA（10-2000马克杯PLG）范围内呈剂量依赖性，并在转染后持续5天。因此，质粒DNA在PLG-CTAB微粒上的吸附为树突状细胞的体外转染提供了一种潜在有用的非病毒方法。
Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2k(d)-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1-20 mug DNA (10-2000 mug PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells.