Aim To establish and evaluate a cell model of non-alcoholic fatty liver disease (NAFLD) in vitro for clarifying the molecular mechanisms of NAFLD occurrence and development, and high throughout screening of drugs. Methods The method of two-step collegenase perfusion technique was used for solation and culture of primary mice hepatocytes. Different concentrations of free fatty acids (FFAs) were added to the medium. After 24 hours co-culture with FFAs, hepatocytes were dyed with oil red O and concentration of triglyeride in cytoplasm was examined. The MTS assay and flow cytometry were used to analyze the influence of FFAs to the proliferation and apoptosis of hepatocytes. The MAPK related signal pathway was analyzed by Western-blot assay. Real-time PCR was used to detect the expression of bim and fas. Results The extent of lipid accumulation in the liver parenchymal cells was significantly increased in accompany with the rising concentration of FFAs added(P<0. 05). Cell proliferation of hepatocytes was decreased by FFAs via promoting the cellular apoptosis. The strength of JNK signal pathway wasincreased, whereas the strength of ERK signal pathway was decreased as stimulation time went on. The expressions of bim and Fas were also increased. Conclusion These results reveal that we successfully established a NAFLD cell model, which could simulate lipid accumulation in the liver parenchymal cells and hepatocellular apoptosis in vitro.%目的 建立小鼠非酒精性脂肪肝病(Non-alcoholic fatty liver disease,NAFLD)体外细胞模型,为进一步体外药物高通量的筛选以及深入探讨NAFLD发生发展的分子机制奠定基础.方法 胶原酶灌注分离并培养小鼠原代肝实质细胞,随后给予不同浓度的游离脂肪酸(free fatty acids,FFA)刺激,进行油红染色以及三酰甘油测定;通过MTS法以及流式细胞术分析游离脂肪酸对肝实质细胞增殖以及细胞凋亡的影响,使用 Western印迹法检测肝实质细胞内的MAPK相关信号通路的变化,实时定量PCR检测细胞中bim与fas的表达.结果 肝实质细胞内的脂质累积程度随加入的FFA浓度上升而呈明显增加(P<0.05);肝实质细胞的细胞增殖受到明显抑制,细胞凋亡明显增加,肝实质细胞内JNK信号通路随刺激时间延长强度增加,ERK信号通路随刺激时间延长强度减弱,细胞内bim和fas的表达量上升.结论 成功建立了非酒精性脂肪肝病的体外细胞模型,能够很好地模拟体内肝实质细胞内脂肪累积以及细胞凋亡现象.
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