目的 构建适宜于评价中药以“肝脂调控”为核心的调血脂活性和评价的“多细胞来源”“稳定性好”的非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）体外细胞模型，以利于精准发现中药脂代谢调控活性物质并评价其调血脂药理学作用特征。方法 选择人肝癌HepG2细胞系及小鼠肝实质AML12细胞系，对造模试剂（油酸及棕榈酸）、造模方法、作用浓度以及作用时间和溶媒选择进行文献和实验研究，并以CCK-8及油红O实验确定最佳造模浓度，进而循形人体肝脂代谢轮廓，采用Western blotting法检测细胞中脂代谢相关蛋白的表达情况，作为模型评价标准。结果 经文献和实验研究发现，10%无脂肪酸牛血清白蛋白（fatty acid free bovine serum albumin，BSA）为油酸溶媒，20% BSA为棕榈酸溶媒稳定性好；500 μmol/L油酸及500 μmol/L油酸＋棕榈酸（体积比2∶1）造模，作用24 h，可显著增加细胞内脂质积累（P＜0.001），且不影响细胞活性，证明造模成功；单独使用棕榈酸造模，对2种细胞损伤都较大，模型不适宜于调血脂物质发现与评价；500 μmol/L油酸及500 μmol/L油酸＋棕榈酸造模后，脂肪从头合成相关蛋白固醇调节元件结合蛋白-1（sterol regulatory element-binding protein-1，SREBP-1）、乙酰辅酶A羧化酶（acetyl-CoA carboxylase，ACC）及脂肪酸合酶（fatty acid synthase，FASN）的表达无显著变化，脂肪分解相关蛋白三酰甘油脂肪酶（adipose triglyceride lipase，ATGL）的表达显著升高（P＜0.05、0.001），激素敏感脂肪酶（hormone sensitive lipase，HSL）的磷酸化显著降低（P＜0.05、0.01），脂肪酸氧化相关蛋白过氧化物酶体增殖物激活受体α（peroxisome proliferator-activated receptor alpha，PPARα）的表达无显著差异，肉碱棕榈酰转移酶1A（carnitine palmitoyl transferase 1A，CPT1A）的表达显著增加（P＜0.05）。结论 500 μmol/L油酸及500 μmol/L油酸＋棕榈酸（体积比2∶1）造模24 h，均可显著增加HepG2、AML12细胞中的脂质积累，成功构建NAFLD体外细胞模型，且具有稳定、经济、高效的特点。

Objective To construct an in vitro cell model of non-alcoholic fatty liver disease (NAFLD) with “multi-cell origin” and “good stability”, which is suitable for lipid-regulating activity and evaluation with “liver lipid regulation” as the core of traditional Chinese medicine, so as to facilitate the accurate discovery of lipid-regulating active substances of traditional Chinese medicine and evaluate their pharmacological effects of lipid regulation. Methods HepG2 cell line of human hepatocellular carcinoma and AML12 cell line of mouse liver parenchyma were selected, modeling reagents (oleic acid and palmitic acid), modeling methods, action concentration, action time and solvent selection were studied by literature and experiments. CCK-8 and oil red O experiments were used to determine the optimal modeling concentration, and then the contour of human liver lipid metabolism was followed. The expressions of lipid metabolism-related proteins in cells was detected by Western blotting as a model evaluation standard. Results It was found that 10% fatty acid free bovine serum albumin (BSA) was oleic acid solvent and 20% BSA was palmitic acid solvent with good stability. 500 μmol/L oleic acid and 500 μmol/L oleic acid + palmitic acid (volume ratio 2∶1) treated for 24 h could significantly increase the intracellular lipid accumulation (P < 0.001) without affecting the cell activity, which proves that the modeling is successful. Palmitic acid modeling alone caused great damage to both kinds of cells, so the model was not suitable for the discovery and evaluation of lipid-lowering substances. After modeling with 500 μmol/L oleic acid and 500 μmol/L oleic acid + palmitic acid, expressions of de novo lipogenesis synthesis related proteins sterol regulatory element-binding protein-1 (SREBP-1), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) were not significantly increased, but the expression of lipolysis related protein adipose triglyceride lipase (ATGL) was significantly increased (P < 0.05, 0.001), and phosphorylation of hormone sensitive lipase (HSL) was significantly decreased (P < 0.05, 0.01), and the expression of peroxisome proliferator-activated receptor α (PPARα), a protein related to fatty acid oxidation, had no significant difference, while the expression of carnitine palmitoyl transferase 1A (CPT1A) was significantly increased (P < 0.05). Conclusion 500 μmol/L oleic acid and 500 μmol/L oleic acid + palmitic acid (volume ratio of 2∶1) can significantly increase the lipid accumulation in HepG2 and AML12 cells, and successfully construct the cell model of NAFLD in vitro, which is stable, economical and efficient.

R285.5

国家重点研发计划项目（2022YFC3502100）；国家自然科学基金面上项目（82274197）；国家自然科学基金面上项目（81773997）；济南市科研带头人工作室项目（202228099）