一种改良大鼠肝脏Kupffer细胞分离方法

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;② Percoll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)×105/g鼠肝,细胞活度＞92%;光镜下细胞呈圆形,培养24 h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,值得推广.%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 cells 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) x 105 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% cells were identified as KCs by immunofluorescence staining. ConclusionrnThe improved isolation method of KCs is more productive of high purity of KCs, it is simple and economic.