游离脂肪酸联合LDL-C诱导脂肪性肝炎细胞模型的建立

Establishment of a cellular model of steatohepatitis induced by low-density lipoprotein cholesterol combined with free fatty acid

目的:探讨建立人肝细胞系L-02脂肪性肝炎细胞模型的方法。方法:体外培养人正常肝细胞系L-02,分为对照组、脂肪变性组和模型组。对照组使用DMEM高糖培养液,脂肪变性组施加200μmol/L油酸(OA),而模型组则施加200μmol/L OA和50 mg/L低密度脂蛋白胆固醇(LDL-C)孵育36 h,观察不同处理对细胞活力、脂质沉积和炎症反应的影响。结果:0~100 mg/L LDL-C干预对细胞活力无显著影响。以50 mg/L LDL-C干预细胞0~48h,36 h时细胞内总甘油三酯(TG)含量趋于稳定,总胆固醇(TC)含量及丙氨酸氨基转移酶(ALT)释放量达到最大值,提示36 h为最佳造模时间。油红O和菲律宾菌素染色结果显示,模型组胞内TG和游离胆固醇蓄积情况更为严重。此外,模型组NLRP3炎症小体关键组件caspase-1、白细胞介素1β(IL-1β)和IL-18的mRNA和蛋白表达水平显著高于脂肪变性组(P<0.05)。结论:应用游离脂肪酸联合LDL-C可以诱导建立类似脂肪性肝炎病理特征的细胞模型。该模型具有较高细胞活力和可重复性,可以用于病理机制及药物筛选研究。

AIM:To establish a cellular model of steatohepatitis using human hepatocyte cell line L-02.METHODS:The L-02 cells were divided into 3 groups:control group,steatosis group and model group.The cells in control group were cultured in high-glucose DMEM,those in steatosis group were treated with 200μmol/L oleic acid(OA),and those in model group were treated with 200μmol/L OA and 50 mg/L low-density lipoprotein cholesterol(LDL-C)for36 h.The effect of LDL-C on cell viability,intracellular triglyceride(TG)and total cholesterol(TC)content,and the inflammatory response were investigated.RESULTS:Treatment with 0 to 100 mg/L LDL-C had no significant effect on the viability of L-02 cells.During the time course of LDL-C treatment,the cellular TG and TC content,and alanine aminotransferase(ALT)released from L-02 cells reached the maximum values at 36 h,indicating that 36 h was the optimal time for modeling.Consistently,intracellular lipid droplets and free cholesterol accumulation in model group were significantly increased,as indicated by oil red O staining and filipin fluorescence staining,respectively.Furthermore,the mRNA and protein of the key components of NLRP3 inflammasome,caspase-1,interleukin-1β(IL-1β)and IL-18,were highly expressed in the model group(P<0.05).CONCLUSION:The application of free fatty acid combined with LDL-C induces a cellular model with pathological characteristics similar to steatohepatitis.This model has high cell viability and reproducibility,and can be used for the study of pathological mechanism and drug screening.