大鼠肝窦内皮细胞的分离、鉴定及纯度、活性分析  

Isolation,Purification and Identification of Sinusoidal Endothelial Cells in Rat Liver

作　　者：王泽[1,2] 王磊[1,2] 谢来峰[1,2] 樊晋宇[2] 徐存拴 

机构地区：[1]河南师范大学生命科学学院,河南新乡453007 [2]河南省-科技部共建细胞分化调控重点实验室,河南新乡453007

出　　处：《河南科学》2009年第8期930-934,共5页Henan Science

基　　金：河南省重大公益性科研计划项目(081100910700)

摘　　要：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梯度离心和免疫磁珠分离肝窦内皮细胞(hepatic sinusoidal endothelial cell,SEC),用分化抗原簇14(cluster of differentiation 14,CD14)和内皮素-1(endothelin-1,ET-1)的免疫组织化学定性、定位再生肝(regenerating liver,RL)、分散的肝脏细胞及分离的窦内皮细胞,用RT-PCR定量窦内皮细胞的CD14和ET-1的mRNA,用蛋白免疫印迹方法定量窦内皮细胞的CD14和ET-1蛋白.初步证实,分离的窦内皮细胞中CD14和ET-1阳性细胞占95%以上,从PH后各时间点分离的窦内皮细胞的CD14和ET-1mRNA量稳定,相应的蛋白量亦稳定.表明改进的分离窦内皮细胞方法具有收率和纯度高、活性好等特点,值得采用.Rat 2/3 hepatectomy （PH） model was made following Higgins et al., hepatic cells were scattered by two-step perfusion, and sinusoidal endothelial cells were isolated by density gradient centrifugation with 60 % percoll and immuno-magnetic beads. Immunocytochemistry method was used to qualitify and localize cluster of differentiation 14 （CD14） and endothelin-1 （ET-1） in liver tissue, the isolated hepatic cells,and the purified sinusoidal endothelial cells. The expressions of CD14 and ET-1 were quantified using RT-PCR. The results showed that CDI4 and ET-1 postive sinusoidal endothelial cells account up more than 95 % of the total sinusoidal endothelial cells： mRNA levels of CDI4 and ET-1 in the isolated sinusoidal endothelial cells were stable in rat regenerating liver （RL）, and also was the content of the corresponding proteins, indicating the modified method for sinusoidal endothelial cell isolation in this study has the advantage of high sinusoidal endothelial cell harvest, high purification and survival rate.

关 键 词：大鼠 细胞分离和鉴定 窦内皮细胞 免疫磁珠 标记蛋白 

分 类 号：Q2-33[生物学—细胞生物学]