一种用于脂肪肝脂毒性药理研究的体外模型

目的 建立和复制一种适合大鼠药物血清进行脂肪肝脂毒性药理研究的体外模型.方法 以大鼠血清替代胎牛血清培养HepG2细胞,添加长链游离脂肪酸(油酸1 mmol/L、棕榈酸0.5 mmol/L)刺激,观察上清液中肿瘤坏死因子(TNF)α含量、细胞内甘油三酯含量,细胞脂肪油红染色及电镜下观察超微结构变化;同时观察细胞TNF α蛋白及其基因表达,细胞组织蛋白酶B(ctsb)表达和分布的变化.结果 游离脂肪酸刺激24 h后,HepG2细胞内甘油三酯显著沉积,高达627.24 mg/g(t=23.6,P＜0.01);上清液中TNF α含量显著升高,增至52.04 pg/mg(t=2.6,P＜0.05);细胞ctsb、TNF α的蛋白表达及其mRNA表达均显著增强.结论 在大鼠血清培养环境下,游离脂肪酸可通过对ctsb作用显著诱导HepG2细胞脂肪变性和TNF α分泌,方法简易经济,可作为一种较理想的抗脂肪肝脂毒性药理研究模型.

An in vitro model applicable for fatty liver lipotoxicity pharmacological research

OBJECTIVE: To establish an in vitro model applicable for fatty liver lipotoxicity pharmacological research. METHODS: HepG2 cells were cultured with rat serum instead of fetal bovine serum and with long-chain free fatty acid (FFA) added. The tested indices were: the content of serum TNFa, cellular triglycerides (TG) content, Oil Red staining and ultrastructural changes; protein expression and gene expression of cellular TNFa, and the expression and distribution of cathepsin B (Ctsb). RESULTS: After incubation with FFA for 24 hours, the TG deposition of HepG2 in the model group increased markedly and TG content was 627.24 mg/g protein (t = 23.6, P less than 0.01), TNFa content in the cell supernatant also increased to 52.04 pg/mg protein (t = 2.6, P less than 0.05). Compared with those of the normal group, the protein expression and mRNA expression of cellular TNFa and Ctsb also increased significantly. CONCLUSION: FFA could induce a model of HepG2 steatosis with TNFa secretion through the Ctsb signal pathway using rat serum in the culture media. The method is simple and economical, which is an ideal model applicable for fatty liver lipotoxicity pharmacological research.