一种简便实用的大鼠Kupffer细胞的分离与鉴定方法

目的：研究一种简便实用的大鼠Kupffer细胞（KCs）的分离与鉴定方法.方法：原位两步灌流法对大鼠肝脏进行冲洗消化;利用Percoll液进行不连续密度梯度离心分离KCs;;选择性贴壁纯化KCs;台盼蓝染色法鉴定细胞存活率;吞噬实验鉴定细胞功能;ED1单克隆抗体免疫荧光细胞化学鉴定KCs;显微镜下观察KCs形态变化.结果：获取的KCs数量为（2.41±0.32）×107/只,其中活细胞数量占（92.3±2.12）％;吞噬实验显示（95.2±2.58）％的细胞内存在碳素颗粒;免疫荧光化学检测证明KCs纯度为（96.3±1.46）％;在显微镜下观察KCs形态,36h细胞形态变得不规则,3d后呈星形或多角形,体外培养可以存活7～10d.结论：此种KCs分离方法操作相对简便,获取的细胞数量、活性功能、纯度等方面均能达到进一步的实验要求,值得推广.

A simple and practical method for isolating and identifying rat liver Kupffer cells

Objective： To introduce a simple and practical method for isolating and identifying rat liver Kupffer cells （KCs） . Methods： The liver was perfused and digested in situ by two ways of liver perfusion. Discontinuous density gradeint centrifugation in Percoll was used to isolating KCs. KCs was purifide by selective adherence. Using trypan blue dye method to measure activity of KCs, phagocytosis experiments to function, ED1 immunofluorescence cytochemistry to purity. Morphological changes of KCs were observed under microscope. Results ： KCs activity was measured ： the number of alive ceils was （92. 3 ± 2. 12）%. Total amount of KCs isolated in each rat was （2. 41 ±0. 32） × 107 in average. KCs phagocytic experiment showed that the number of cells （95.2 ±2. 58 ）% had function. ED1 immunofluorescence cytochemistry staining showed cell purity was （96. 3 ±1.46） %. The morphology of KCs became irregular after 36 hours , spindle or polygonal after 3 days. KCs could keep alive 7 - 10 days in vitro culture. Conclusion： The isolation methods of KCs was relatively simple. The acquire cells were quite up to the standare for further experiments in quantity, activity and purity.