【摘要】 为探究棕榈酸（palmitic acid,PA）诱导HepG2细胞建立代谢相关脂肪性肝病（metabolic-associated fatty liver disease,MAFLD）的最适浓度及其对自噬的影响，该试验采用细胞计数试剂盒-8(cell counting kit-8,CCK8)检测、油红O染色、甘油三酯（triglyceride,TG）含量检测、免疫荧光标记自噬标志物LC3及免疫印迹等进行综合评估。结果表明：0.4～1.0 mmol/L的PA均明显降低细胞活力，并且0.2～1.0 mmol/L的PA显著增加细胞中的脂滴数量和TG含量，并减少了LC3荧光点的数目，0.6～1.0 mmol/L的PA显著降低磷酸化腺苷酸活化蛋白激酶/腺苷酸活化蛋白激酶（phosphorylated AMP-activated protein kinase/AMP-activated protein kinase,P-AMPK/AMPK）的比值，1.0 mmol/L的PA增加了磷酸化雷帕霉素靶蛋白/雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin/mammalian target of rapamycin,P-mTOR/mTOR）的比值。基于脂质积累、脂毒性及自噬结果，最终选用0.6 mmol/L棕榈酸进行MAFLD细胞模型的构建。50、200、800μg/mL的灵芝孢子粉水提物可降低脂滴数量和TG含量，增加LC3荧光点数目。因此，0.6 mmol/L PA诱导的MAFLD模型可用于靶向自噬MAFLD药物的筛选，灵芝孢子粉水提物能够通过恢复PA抑制的自噬来改善细胞死亡和脂质积累。更多还原

【Abstract】 To investigate the optimal concentration of palmitic acid(PA)for inducing HepG2 cells to establish metabolic-associated fatty liver disease(MAFLD)and its effect on autophagy,this experiment adopted methods such as cell counting kit-8(CCK8),oil red O staining,triglyceride(TG)content detection,immunofluorescence labeling of autophagy marker LC3,and immunoblotting for comprehensive evaluation. The results showed that PA at concentrations of 0.4-1.0 mmol/L significantly reduced cell viability. Moreover,PA at concentrations of 0.2-1.0 mmol/L significantly increased the number of lipid droplets and TG content in cells and reduced the number of LC3 fluorescent dots. PA at concentrations of 0.6-1.0 mmol/L significantly reduced the ratio of P-AMPK/AMPK,and PA at a concentration of 1.0 mmol/L increased the ratio of P-mTOR/mTOR. After comprehensive consideration of lipid accumulation,lipotoxicity,and autophagy,PA at a concentration of0.6 mmol/L was finally selected for the construction of the MAFLD cell model. The water extracts of Ganoderma lucidum spore powder at concentrations of 50,200,and 800 μg/mL reduced the number of lipid droplets and TG content and increased the number of LC3 fluorescent dots. Therefore,the MAFLD model induced by PA at a concentration of 0.6 mmol/L could be used for the screening of drugs targeting autophagy in MAFLD.The water extracts of Ganoderma lucidum spore powder could improve cell death and lipid accumulation by restoring autophagy inhibited by PA.更多还原