大鼠肝库普弗细胞的分离、鉴定及纯度、活性分析

按Higgins等方法制作大鼠2／3肝切除（partial hepatectomy，PH）模型，用两步灌流法分散肝脏细胞，用60％Percoll梯度离心结合免疫磁珠方法分离库普弗细胞（Kupffer cell，KC），用外胚层发育不良抗原2（ectodermal dysplasia antigen2，ED2）和溶菌酶（lysozyme，LYZ）的免疫组织化学方法定性、定位再生肝（regenerating liver，RL）、分散的肝脏细胞及分离的库普弗细胞，用RT—PCR定量库普弗细胞的ED2和LYZmRNA,用蛋白免疫印迹方法定量库普弗细胞的ED2和LYZ蛋白．初步证实，分离的肝库普弗细胞中ED2和LYZ阳性细胞占96％以上，从PH后各时间点分离的库普弗细胞ED2和LYZmRNA量稳定，相应的蛋白量亦稳定。表明改进的分离库普弗细胞方法具有收率和纯度高、活性好等优点，值得采用．

Isolation, Purification and Identification of Hepatic Kupffer Cells in Rat Liver

Rat 2/3 hepatectomy model was made following Higgins et al. Hepatic cells were scattered by two-step perfusion,and Kupffer cells(KCs)were isolated and purified by density gradient centrifugation with 60 % percoll and immuno-magnetic beads. Immunocytochemistry method was used to qualitify and localize ectodermal dysplasia antigen 2(ED 2)and lysozyme(LYZ)in liver tissue,the isolated hepatic cells,and the purified Kupffer cells. The exrpressions of ED 2 and LYZ were quantified using RT-PCR. The results showed that ED 2 and LYZ positive Kupffer cells account up more than 96 % of the total hepatic Kupffer cells;mRNA levels of ED 2 and LYZ in the purified Kupffer cells were stable in the purified hepatocytes of rat regenerating liver,and also was the content of the corresponding proteins,indicating the modified method for Kupffer cells purification in this study has the adventage of high hepatic Kupffer cells harvest,high purification and survival rate.