大鼠肝卵圆细胞的分离、鉴定及纯度、活性分析

按Higgins等方法制作大鼠2/3肝切除（parital hepatectomy,PH）模型,用两步灌流法分散肝脏细胞,用60%Percoll梯度离心给合免疫磁珠分离方法卵圆细胞（oval cell,OC）.用卵圆细胞标志蛋白OC2和OV6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卵圆细胞,用RT-PCR定量卵圆细胞的OC2和OV6 mRNA,用蛋白免疫印迹方法定量卵圆细胞的OC2和OV6蛋白.初步证实分离的肝卵圆细胞中,OC2和OV6阳性细胞占96%以上,从PH后各时间点分离的肝卵圆细胞的OC2和OV6 mRNA量稳定,相应的蛋白量亦稳定.表明改进的分离肝卵圆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 Oval Cells in Rat Liver

Rat 2/3 hepatectomy model was made following Higgins et al.Hepatic cells were scattered by two-step perfusion,and hepatic oval cells were isolated and pruified by density gradient centrifugation with 60 % percol1 and immune-magnetic beads.Immunocytochemistry method was used to qualitify and localize OC2 and OV6 in liver tissue,the isolated hepatic cells,and the purified hepatic oval cells.The expressions of OC2 and OV6 were quantified using RT-PCR.The results showed that OC2 and OV6 postive hepatic oval cells account up more than 96 % of the total hepatic oval cells,mRNA levels of OC2 and OV6 in the purified hepatic oval cells were stable in the purified hepatic cells of rat regenerating liver,and also was the content of the corresponding proteins,indicating the modified method for hepatic oval cells purification in this study has the adventage of high hepatic oval cell harvest,high purification and survival rate.