大鼠肝星形细胞的分离、鉴定及纯度、活性分析  

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

作　　者：张永晶[1,2] 谢来峰[1,2] 王磊[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6期670-674,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相结合分离肝星形细胞,用结蛋白(desmin,DES)和波形蛋白(vimentin,VIM)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肝星形细胞,用RT-PCR定量肝星形细胞的DES和VIM mRNA,用蛋白免疫印迹定量肝星形细胞DES和VIM.初步证实,分离的肝星形细胞中DES和VIM阳性细胞占95%以上,从PH后各时间点分离的肝星形细胞的DES和VIM mRNA量稳定,相应的蛋白量亦稳定.表明改进的分离肝星形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 hepatic stellate cells were isolated and purified by density gradient centrifugation with 60 % percoll and immunomagnetic beads. Immunocytochemistry method was used to qualitify and localize desmin （DES） and vimentin （VIM） in liver tissue, isolated hepatic cells, and purified hepatic stellate cells. The expressions of DES and VIM were quantified using RT-PCR. The results showed that DES and VIM positive hepatic stellate cells account up more than 95 % of the total hepatic stellate cells; mRNA levels of DES and VIM were stable in the purified hepatic stellate cells in rat regenerating liver （RL）, and also was the content of the corresponding proteins, indicating the modified method for hepatic stellate cells purification in this study has the adventage of high hepatic stellate cells harvest, high purity and survival rate.

关 键 词：大鼠 细胞分离和鉴定 肝星形细胞 免疫磁珠 标记蛋白 

分 类 号：Q2-33[生物学—细胞生物学]