原代小鼠肝脏细胞的高效分离纯化与培养  

Isolation,Purification and Primary Culture of Mouse Hepatocytes with High Viability

作　　者：张钰[1] 付亮[1] 鲁超[1] 锁涛[1] 宋陆军[1] 

机构地区：[1]复旦大学附属中山医院普外科,上海200032

出　　处：《现代生物医学进展》2014年第6期1005-1008,共4页Progress in Modern Biomedicine

基　　金：上海市科学技术委员会基金项目(09ZR1405900)

摘　　要：目的:建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法:应用改良的Seglen二步法原位灌注和机械离心分离肝脏细胞,并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率,倒置显微镜动态观察肝脏细胞形态变化,应用免疫荧光技术对肝脏细胞进行Albumin染色。结果:每只小鼠可获取肝脏细胞的总产量平均为1.35×106/g体重,肝脏细胞存活率>90%。倒置显微镜下观察贴壁前肝细胞直径为35.14μm±4.35μm,肝脏细胞在接种后3 h基本完成贴壁;肝脏细胞接种后24h,所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin,肝细胞纯度>95%。结论:改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。Objective： A stable method was established for isolation and primary culture of mouse hepatocytes with high purity and viability. Methods： The C57BL/6 mouse hepatoeytes were isolated by a modified two-step collagenase perfusion and low-speed mechanical centrifugation. The hepatocytes were cultured in modified DMEM with high level of glucose. The viabilities of cells were evaluated by Trypan blue exclusion before cells were seeded. Cellular morphological changes were observed using a phase contrast microscopy. The Albumin and CK19 staining were performed on hepatocytes by immunofluorescence. Results： The total cell yield of hepatocytes was 1.35~ l06 cells/g wet weight. Total cell viability was more than 90%. The diameter ofhepatocytes was 35.14/xm：t： 4.35 Ixm before adherence. The hepatocytes with enlarged and flattened round shape were attached to the surface of collagen-coated dishes 3 hours after seeding. Albumin was positively expressed in all hepatocytes at 24 h after attachment, and the purity of hepatocytes was more than 95%. Conclusion： Mouse hepatocytes with high quantity, purity and survival rate were obtained by the improved method for isolation, purification and culture.

关 键 词：肝脏细胞 原代细胞培养 分离 纯化 小鼠 

分 类 号：Q953[生物学—动物学] Q813.11