小鼠原代肝细胞的分离培养及体外脂肪变性模型构建方法的优化  

Optimization of methods for isolation and culture of primary mouse hepatocytes and establishment of a steatosis model

作　　者：隋鲜鲜 邱燕燕 SUI Xianxian;QIU Yanyan(Center for Experimental Medical Science Education,Shanghai Jiaotong University School of Medicine,Shanghai 200025;National Demonstration Center for Experimental Teaching,School of Basic Medicine,Fudan University,Shanghai 200032,China)

机构地区：[1]上海交通大学医学院基础医学实验教学中心,上海200025 [2]复旦大学基础医学院国家级实验教学示范中心,上海200032

出　　处：《基础医学与临床》2024年第10期1428-1435,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(82000405);上海交通大学教育发展基金(CTLD23J0031)。

摘　　要：目的优化改进原代肝细胞分离培养方法并建立肝脂肪变性(hepatic steatosis)的细胞模型,以提升实验效率与准确性。方法在原代肝细胞培养及已有的脂肪变性模型上进行优化,对逆向灌注插管及固定方式进行细化,精确小鼠肝脏灌注消化的速度与时间,并在撕去肝脏包膜及过滤过程中应用含2%胎牛血清(FBS)的培养基;探索诱导脂肪变性细胞模型选用的游离脂肪酸种类、浓度与比例,精确诱导时间。结果所得小鼠原代肝细胞存活率达90%以上,量足、形态好、状态佳;所构建的脂肪变性细胞模型胞质内脂滴明显、糖脂代谢能力下降,并表现出轻度炎性反应及胰岛素抵抗状态。结论优化完善的技术手段,帮助得到稳定且模拟活体生理状态的脂肪变性细胞模型。Objective To improve the isolation and culture methods for primary hepatocytes and to establish a hepatic steatosis cell model for enhancing experimental efficiency and model precision.Methods Improvements were made upon existing techniques for primary hepatocyte cultivation and steatosis model establishment.The technology oriented to optimize the retrograde cannulation and fixation procedures,meticulously calibrating the perfusion speed and duration for mouse liver digestion.The culture medium was supplemented with 2%fetal bovine serum(FBS)during the removal of liver capsules and filtration steps.Additionally,the induction parameters for the steatosis cell model were refined,including the selection of free fatty acid(FFA)types and optimize their concentrations,ratios,and precise induction durations.Results The optimized protocol yielded mouse primary hepatocytes with a viability exceeding 90%,demonstrating ample quantity,favorable morphology,and excellent overall condition.The steatosis cell model exhibited prominent cytoplasmic lipid droplets,impaired glucose and lipid metabolism,as well as mild inflammation and insulin resistance,closely mimicking key aspects of the disease in vivo.Conclusions The refined techniques facilitated the establishment of a stable and physiologically representative in vitro steatosis cell model,which may support further research of pathogenesis of the disease,identification of potential therapeutic targets.

关 键 词：肝脂肪变性 原代肝细胞 分离培养技术 脂肪变性细胞模型 

分 类 号：R31[医药卫生—基础医学]