Yoshinaga Takaharu, Yasuda Kei, Ogawa Yoshiyuki, Takakura Yoshinobu
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto, Japan.
Biochem Biophys Res Commun. 2002 Dec 6;299(3):389-94. doi: 10.1016/s0006-291x(02)02648-7.
In spite of the important roles of dendritic cells in DNA-based therapies, the cellular uptake mechanism of plasmid DNA (pDNA) in dendritic cells is poorly understood. The present study was undertaken to investigate the binding and uptake of pDNA in vitro using a murine dendritic cell line, DC2.4 cells. A significant and time-dependent cellular association of [32P]pDNA with DC2.4 cells was observed at 37 degrees C and this fell markedly at 4 degrees C. The binding and uptake of [32P]pDNA were significantly inhibited by cold pDNA, polyinosinic acid (poly[I]), dextran sulfate, or heparin, but not by polycytidylic acid (poly[C]), dextran, or EDTA, suggesting that a specific mechanism mediated by a receptor like the macrophage scavenger receptor may be involved. The TCA precipitation experiments showed that DC2.4 cells rapidly endocytosed and degraded a significant amount of [32P]pDNA at 37 degrees C and released the degradation products into the medium. The pDNA degradation was also significantly inhibited by poly[I], but not poly[C]. The rate of pDNA degradation by DC2.4 cells was significantly higher than that by macrophages. A confocal microscopic study using fluorescein-labeled pDNA confirmed the rapid internalization and degradation of pDNA by the dendritic cells. Taken together, these results indicate that pDNA is efficiently taken up and rapidly digested by the dendritic cells via a specific mechanism. These findings may suggest the important role of the dendritic cells in the innate immune system for host defense.
尽管树突状细胞在基于DNA的治疗中发挥着重要作用，但人们对质粒DNA（pDNA）在树突状细胞中的细胞摄取机制了解甚少。本研究旨在利用小鼠树突状细胞系DC2.4细胞体外研究pDNA的结合和摄取情况。在37℃观察到[32P]pDNA与DC2.4细胞有显著的时间依赖性细胞结合，而在4℃时这种结合明显下降。[32P]pDNA的结合和摄取受到冷pDNA、聚肌苷酸（聚[I]）、硫酸葡聚糖或肝素的显著抑制，但不受聚胞苷酸（聚[C]）、葡聚糖或EDTA的抑制，这表明可能涉及一种由类似巨噬细胞清道夫受体的受体介导的特定机制。三氯乙酸沉淀实验表明，DC2.4细胞在37℃迅速内吞并降解大量[32P]pDNA，并将降解产物释放到培养基中。聚[I]也显著抑制了pDNA的降解，但聚[C]没有。DC2.4细胞对pDNA的降解速率明显高于巨噬细胞。使用荧光素标记的pDNA进行的共聚焦显微镜研究证实了树突状细胞对pDNA的快速内化和降解。综上所述，这些结果表明pDNA通过一种特定机制被树突状细胞有效摄取并迅速消化。这些发现可能提示树突状细胞在宿主防御的固有免疫系统中发挥重要作用。
