尾静脉注射质粒巨噬细胞-粒细胞集落刺激因子诱导扩增小鼠肝脏CD11c＋树突状细胞

Injection of gene macrophage and granulocyte colony stimulating factor plasmid via tail vein for ex- pansion of large number of hepatic CD11c dendritc cells

目的探讨载粒巨噬细胞-粒细胞集落刺激因子（GM—CSF）基因质粒在小鼠体内扩增肝脏CDllc’树突状细胞（DC）的方法。方法采用快速尾静脉推注法将含20μgGM．CSF基因质粒的生理盐水2．0ml导入小鼠肝脏内，对照组注射增强型绿色荧光蛋白（EGFP）基因。第7天取肝，胶原酶消化、密度梯度离心、磁珠标记等分选出DC。Giemsa染色作形态学观测，采用流式细胞术分析其免疫表型。结果实验组小鼠肝脏非实质细胞（NPC）大量增殖，主要分布于门静脉及其周围区域，质粒处理组中CD11c’DC比例较对照组显著增多（29．31％比6．77％，P〈0．05）。Giemsa染色显示两组DC细胞核形状不规则，胞质无颗粒，然而实验组DC较对照组有明显凸起。流式细胞仪检测结果显示实验组DC较对照组明显成熟，CD40、CD80、CD86表达显著增高（P〈0．05）。结论经快速尾静脉推注质粒GM-CSF的方法可显著可靠地扩增肝脏CD11c’DC的数量。

Objective To induce liver-derived CDllc＋ dendritic cells （DCs） proliferation by transfecting plasmid macrophage and granulocyte colony stimulating factor （GM-CSF） genes into mouse liv- er, and compare the biological characteristics of liver DCs between plasmids-treated group and control group. Methods We adopted an established approach to transfeet a gene into mouse livers by tail-vein rapid injection of naked plasmids carrying the gene. Plasmids carrying genes encoding GM-CSF （20 μg） in 2.0 ml saline were injected at 0. 3 ml/s into mouse tail veins 7 days prior to isolation, then DCs were isola- ted from NPC by digestion in 20 ml collagenase IV and Pereoll density gradient centrifugation as described previously, and ultimately purified using magnetic beads. At 3rd, 5th, and 7th day after injection, the he- patic sections were stained by hematoxylin ＆ eosin staining and examined under a microscope. At the 7th day, the number of NPC got the peak. Morphology of DCs was studied microscopically with Giemsa stai- ning, and immunological characteristics of DCs were studied by flow eytometry. Results Liver nonparen- chymal cells were dramatically increased in the liver portal and periportal areas in the treated group, but not seen in the control group. At 7th day after treatment, the proportion of DCs in liver NPC was 35% , while that was 5% in control group. Morphologically, the CDs in both the control and treated groups had a- bundant cytoplasm with few granules, and irregular and eccentric nuclei, however the later had more typi- cal dendritic processes. Flow cytometry showed the expression levels CIMO, CDdO, and CD86 were higher in the treated group than in the control group. Conclusion CD11 c ＋ DCs were expanded in vivo in the mu- rine livers by rapid tail-vein injection of plasmids carrying genes encoding GM-CSF in a large amount.