大鼠胆管上皮细胞的分离、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,在32%～90%Perco11密度梯度离心法的基础上进一步用免疫磁珠分离胆管上皮细胞(biliary epithelial cell,BEC),用γ-谷氨酰转肽酶(γ-glutamyl transferase,GGT)和角蛋白19(cyto-keratin19,CK19)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胆管上皮细胞,用RT-PCR定量胆管上皮细胞的GGT和CK18mRNA,用蛋白免疫印迹定量胆管上皮细胞的GGT、角蛋白18(cytokeratin 18,CK18).初步证实分离的细胞中GGT和CK19阳性细胞占96%以上,从PH后各时间点分离的胆管上皮细胞的GGT和CK18mRNA量稳定,相应的蛋白量亦稳定.表明改进的分离胆管上皮细胞方法具有收率和纯度高、活性好等特点,方便适用.

Isolation,Purification and Identification of Biliary Epithelial Cell in Rat Liver

Rat 2/3 hepatectomy model was made following Higgins et a1.,hepatic cells were scattered by two step perfusion,and biliary epithelial cell(BEC)were isolated and purified by density gradient centrifugation with 32 %～ 90 % percoll and immune-magnetic beads. Immunocytochemistry method was used to qualitify and localize γ-glutamyl transferase(GGT)and cytokeratin 19(CK19)in liver tissue,the isolated hepatic cells,and the purified BEC. The expressions of GGT and cytokeratin 18(CK18)were quantified using RT-PCR. The results showed that GGT/CK19 positive BEC account up more than 96 % of the total BEC;mRNA levels of GGT and CK18 in the purified BEC were stable in the purified hepatocytes of rat regenerating liver,and also was the content of the corresponding proteins,indicating the modified method for BEC purification in this study has the advantage of high BEC harvest, high purity and survival rate.