一种改良的简易灌注分离小鼠肝细胞的方法  

A MODIFIED SIMPLE PERFUSION METHOD FOR THE ISOLATION OF MOUSE HEPATOCYTES

作　　者：宋荣景 司霞[1] 陈月[1] 冯婉玉[1] SONG Rongjing;SI Xia;CHEN Yue;FENG Wanyu(Department of Pharmacy, Peking University People’s Hospital, Beijing 100044, China)

机构地区：[1]北京大学人民医院药剂科,北京100044

出　　处：《精准医学杂志》2018年第6期539-541,545,共4页Journal of Precision Medicine

基　　金：国家卫生计生委药政司委托研究课题(药政[2016]41号)

摘　　要：目的探讨一种改良的简易灌注分离小鼠肝细胞的方法。方法采用改良的简易两步灌注法分离小鼠肝细胞,即使用连有头皮针的注射器进行肝门静脉插管并灌流冲洗,再用Ⅳ型胶原酶灌流消化;分离肝细胞后采用室温静止法进行纯化;利用锥虫蓝染色法鉴定细胞存活率;培养板预先用鼠尾胶原铺备,利用倒置显微镜观察细胞形态;细胞贴壁后1～10d检测培养液上清液中的白蛋白的分泌情况,判断肝细胞的生物学活性和功能。结果成功建立了一种简单易行的分离与培养小鼠原代肝细胞的方法。培养的小鼠肝细胞在接种后4h基本完成贴壁,细胞可稳定贴壁至少1周,成活率可达85%以上且功能良好,可用于后续实验。结论改良的简易两步灌注法是一种简单易行、经济高效的分离纯化小鼠肝细胞的方法,适合小鼠肝细胞的分离。Objective To investigate a modified simple perfusion method for the isolation of mouse hepatocytes.Methods Mouse hepatocytes were isolated by the modified two-step simple perfusion method.A syringe with scalp needle was used for hepatic portal vein intubation and perfusion,and then type Ⅳ collagenase was used for digestion.The hepatocytes were purified by room-temperature immobilization after isolation.Trypan blue staining was used to measure cell viability; the culture plate was preliminarily prepared with rat tail collagen,and then an inverted microscope was used to observe the morphology of the cells; the secretion of albumin in the supernatant of culture medium was measured at 1-10 days after cell attachment to determine the biological activity and function of hepatocytes.ResultsA simple and easy method for the isolation and culture of primary mouse hepatocytes was successfully established.The cultured mouse hepatocytes basically adhered to the wall at 4 h after inoculation,with stable attachment for at least 1 week.Cell viability reached more than 85% and the cells had good function and could be used for subsequent experiments.ConclusionThe modified two-step simple perfusion method is a simple and cost-effective method for the isolation and purification of mouse hepatocytes and is suitable for the isolation of mouse hepatocytes.

关 键 词：肝细胞 细胞分离 原代细胞培养 小鼠 

分 类 号：R446.113[医药卫生—诊断学] R329-33[医药卫生—临床医学]