中文摘要：

目的观察改良大鼠肝脏Kupffer细胞（KCs）分离方法获取KCs的效果。方法参照Akira提供的方法进行以下改进：①前灌注液在体灌注，Ⅳ型胶原酶离体灌注消化；②Percoll分离液不连续密度梯度离心；③台盼蓝染色检测分离细胞的活度；④选择性贴壁法纯化获取的细胞；⑤吞墨实验、DAB染色及CD163细胞免疫荧光法鉴定所分选细胞。结果肝脏KCs的获得量为（3±1．5）×10^5／g鼠肝，细胞活度〉92％；光镜下细胞呈圆形，培养24h后呈梭形或多角形；具有较强的吞噬能力，DAB染色呈“煎蛋”样，荧光显微镜下〉99％为KCs。结论改良的大鼠肝脏KCs分离方法较Akira法能够获取更高纯度的KCs，简捷经济，值得椎广。

英文摘要：

Objective To observe the isolation effect of kupffer cells （KCs） by an improved isolation method of KCs from a single rat liver. Methods The isolation method was based on Akira＇ s method with some improvements as followed： ① After primarily perfused in vivo, the liver was perfused with collagens type IV and digested in vitro; ② Discontinuous density gradient centrifugation in Percoll was used; ③ The viability of isolated ceils were determined by trypan blue staining; ④ The purification of isolated cells by selective cell adherence ; ⑤ Phagocytosis test , DAB dyeing and CD163 immunofluorescence staining were used to identified isolated KCs. Results The number of acquired cells was （3 ± 1.5） × 10^5 per gram rat liver, and the viability of cells was more than 92%. The morphology of cells was roundness, and became spindle or polygonal after 24 h cultured. The cell had strong phagotrophic ability, and appeared ＂fried eggs＂ by DAB dyeing. More than 99% ceils were identified as KCs by immunofluorescence staining. Conclusion The improved isolation method of KCs is more productive of high purity of KCs, it is simple and economic