一种用于脂肪肝脂毒性药理研究的体外模型

An in vitro model applicable for fatty liver lipotoxicity pharmacological research

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1mmol／L、棕榈酸0．5mmol／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TNFα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24h后，HepG2细胞内甘油三酯显著沉积，高达527．24mg／g（t=23．6，P〈0．01）；上清液中TNFα含量显著升高，增至52．04pg／mg（t=2．6，P〈0．05）；细胞ctsb、TNFα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TNFα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。

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-chum free fatty acid （FFA） added. The tested radices were： the content of serum TNF α, cellular triglycerides （TG） content, Oil Red staining and ultrastructural changes; protein expression and gene expression of cellular TNF α, 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 〈 0.01）, TNF α content in the cell supematant also increased to 52.04 pg/mg protein （t = 2.6, P 〈 0.05）. Compared with those of the normal group, the protein expression and mRNA expression of cellular TNF α and Ctsb also increased significantly. Conclusion FFA could induce a model of HepG2 steatosis with TNF α 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 pharmaeologieal research.