Takagi T, Hashiguchi M, Mahato R I, Tokuda H, Takakura Y, Hashida M
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Biochem Biophys Res Commun. 1998 Apr 28;245(3):729-33. doi: 10.1006/bbrc.1998.8521.
The binding and uptake of plasmid DNA encoding luciferase reporter gene (pCMV-Luc) were studied in vitro using cultured mouse peritoneal macrophages. A significant and time-dependent cellular association of [32P]pCMV-Luc with resident macrophages was observed at 37 degrees C and this decreased at 4 degrees C. The binding at 4 degrees C was saturable and a Scatchard plot gave a maximum binding capacity of 0.81 microgram/mg-protein and a dissociation constant of 0.30 microgram/ml. The binding of [32P]-pCMV-Luc was inhibited by polyinosinic acid, dextran sulfate and salmon sperm DNA, but not by polycytidylic acid, dextran and EDTA. A confocal microscopic study demonstrated that fluorescein-labeled pCMV-Luc was internalized at 37 degrees C while only cell surface binding occurred at 4 degrees C. No significant luciferase gene expression was obtained after incubation with a high concentration (100 micrograms/ml) of pCMV-Luc. These data suggest that plasmid DNA is taken up by macrophages via a mechanism mediated by a receptor like the macrophage scavenger receptor.
利用培养的小鼠腹腔巨噬细胞在体外研究了编码荧光素酶报告基因的质粒DNA（pCMV-Luc）的结合与摄取。在37℃观察到[32P]pCMV-Luc与驻留巨噬细胞有显著的时间依赖性细胞结合，而在4℃时这种结合减少。4℃时的结合是可饱和的，Scatchard图给出的最大结合容量为0.81微克/毫克蛋白，解离常数为0.30微克/毫升。[32P]-pCMV-Luc的结合受到多聚肌苷酸、硫酸葡聚糖和鲑鱼精DNA的抑制，但不受聚胞苷酸、葡聚糖和EDTA的抑制。共聚焦显微镜研究表明，荧光素标记的pCMV-Luc在37℃时被内化，而在4℃时仅发生细胞表面结合。用高浓度（100微克/毫升）的pCMV-Luc孵育后未获得显著的荧光素酶基因表达。这些数据表明，质粒DNA通过一种类似于巨噬细胞清道夫受体的受体介导的机制被巨噬细胞摄取。
