小鼠肝内胆管上皮细胞的分离与鉴定

Isolation,Identification and Cultivation of Intrahepatic Bile Duct Epithelial Cells from Mouse

为建立一种简单可靠的小鼠肝内胆管上皮细胞(MIBECs)分离培养方法,用Ⅳ型胶原酶进行门静脉灌注,取出肝内胆管,用DNaseⅠ、pronase E和Ⅳ型胶原酶分类消化,小鼠肝内胆管组织细小、均匀的细胞团培养于含100mL/L胎牛血清(FBS)和10ng/mL表皮生长因子(EGF)、肝细胞生长因子(HGF)和胰岛素转铁蛋白硒(ITS)的DMEM/F12培养基中。细胞贴壁后,利用细胞角蛋白19免疫荧光法鉴定目的细胞。在培养的过程中,用CCK8测定细胞的生长曲线。结果显示,成功分离出MIBECs,细胞纯度约95%。细胞培养3d^7d内呈对数生长状态。以上结果表明本分离培养方法是一种简单可靠的小鼠肝内胆管上皮细胞分离纯化培养技术。

To establish a simple and reliable method for isolation and cultivation of intrahepatic bile duct epithelial cells（MIBECs）from mouse, the portal vein was perfused with type 1V collagenase, subsequently intrahepatic bile duct was taken out and then digested with DNase I , pronase E and IV type collagenase into small and uniform cells which were culured in DMEM/F12 medium containing 100 mL/L FBS and 10 ng/ mL EGF, HGF and ITS. After cells attached to the sidewall, cells were identified by immunofluorescence assay of cytokeratin 19. During cell growth process, cell growth curve was determined by CCKS. The re-suits showed that we have successfully isolated MIBECs by this method, and cell purity is about 95%. And CCK8 tests showed MIBECs were in logarithmic growth state within cultured 3 d-7 d. These results suggest that this method is simple and reliable for isolation and purification and culture assay of MIBECs.