大鼠正常肝和再生肝的肝细胞分离、鉴定及纯度、活性分析  

Isolation,Purity and Identification of Hepatocytes in Rat Normal Liver and Regenerating Liver

作　　者：王文博[1,2] 谢来峰[3,2] 王望[3,2] 王磊[3,2] 徐存拴 

机构地区：[1]中国海洋大学水产学院,青岛266100 [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 [3]河南师范大学生命科学学院,河南新乡453007

出　　处：《河南科学》2008年第12期1492-1498,共7页Henan Science

基　　金：国家973计划前期研究专项(2006CB708506)

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密度梯度离心分离肝细胞,用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肝细胞中白蛋白(albumin,ALB)和葡萄糖-6-磷酸酶(glucose-6-phosphatase,G-6-P),用蛋白免疫印迹方法定量肝细胞的ALB和G-6-P,用RT-PCR定量肝细胞的ALB和G-6-P mRNA.初步证实纯化的细胞中,ALB和G-6-P阳性细胞占96%以上;PH后各时间点纯化的肝细胞的ALB和G-6-P mRNA量稳定,相应的蛋白量亦稳定.表明改进后的分离肝细胞方法具有收率高、纯度高、活性好等特点.Rat 2/3 hepatectomy model was made by the method of Higgins et al., hepatic cells were scattered by two-step perfusion, and hepatocytes were isolated and pruified by density gradient centrifugation with 60 % percoll. Immunocytochemistry method was used to qualitify and localize albumin （ALB）and glucose-6-phosphatase （G-6-P） in liver tissue,the dispersed hepatic cells, and the purified hepatoctyes. The exrpressions of alb and G-6-P were quantified using RT-PCR. The results-primarily showed that ALB/G-6-P postive hepatocytes account up more than 96 % of the total hepatocytes; mRNA levels of ALB and G-6-P in the purified hepatocytes were stable in the Purificl hepatocytes of rat regenerating liver, and also was the content of the corresponding proteins, indicating the modified method for hepatocytes purification in this study has the adventage of high hepatocyte harvest,high purity and survival rate.

关 键 词：大鼠 细胞分离和鉴定 肝细胞 基因表达 标记蛋白 

分 类 号：Q2-33[生物学—细胞生物学]