Denis-Mize K S, Dupuis M, MacKichan M L, Singh M, Doe B, O'Hagan D, Ulmer J B, Donnelly J J, McDonald D M, Ott G
Department of Anatomy, and Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA.
Gene Ther. 2000 Dec;7(24):2105-12. doi: 10.1038/sj.gt.3301347.
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-2kd-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 microg DNA (10-2000 microg 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. Gene Therapy (2000) 7, 2105-2112.
树突状细胞（DC）在抗原呈递和特异性免疫激活中起关键作用。当前许多研究聚焦于利用DC的效力用于疫苗、基因治疗和癌症免疫治疗应用。然而，传统的非病毒技术在体外不易转染DC。一种新型DNA疫苗制剂被用于确定DC在体外是否能被转染。该制剂由吸附在阳离子微粒上的质粒DNA组成，阳离子微粒由可生物降解的聚合物聚乳酸-羟基乙酸共聚物（PLG）和阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）构成。使用在PLG-CTAB微粒上配制的荧光标记质粒DNA制剂，来研究巨噬细胞和树突状细胞在体外和体内的内化情况，我们发现大部分（但不是全部）荧光集中在内体区室。此外，通过逆转录聚合酶链反应（RT-PCR）检测发现，小鼠骨髓来源的树突状细胞摄取吸附在PLG-CTAB微粒上的编码HIV p55 gag的质粒DNA后导致了靶基因表达。随后该抗原被加工并呈递，从而刺激了一种H-2kd限制性的、gag特异性的T细胞杂交瘤。通过白细胞介素-2产生检测到的杂交瘤激活在0.1 - 20微克DNA（10 - 2000微克PLG）范围内呈剂量依赖性，并且在转染后可持续长达5天。因此，质粒DNA吸附在PLG-CTAB微粒上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。《基因治疗》（2000年）7卷，2105 - 2112页 。
