[Establishment and optimization of hepatocyte steatosis model].

Objective: To establish a new model of hepatic steatosis cells by optimizing the original ethanol or high fat, the present study proposed an in vitro hepatocyte steatosis model for the study of fatty liver. Methods: Oil red O staining was used to observe the effects of fetal bovine serum, oleic acid and ethanol on lipid accumulation in human liver cell line L02 in a concentration- and time-dependent manner. RT-PCR was used to detect the mRNA expression levels of PPAR-γ and AP-2, and the suitable conditions for the establishment of hepatocyte steatosis model were screened out. A t-test was used for comparison between the two groups, and one-way Analysis of Variance (ANOVA) was used in more than three groups. Results: Oil red O staining showed the number of reddish-orange lipid droplets in L02 cells gradually increased with the increase of fetal bovine serum, oleic acid and ethanol in a concentration - and time-dependent manner. Compared with 0.00% oleic acid and 2% ethanol, the count value of red particle was 100.00% ± 17.63% at the beginning and after 24 h, 0.003% oleic acid and 2% ethanol jointly acted in L02 cells. After incubation for 48 hours with 2% ethanol and serum-free DMEM medium, the accumulation of lipid droplets was the highest with a count value of 802.38%+71.06%(t = 42.36, P < 0.001). RT-PCR analysis showed the lipid accumulation induced by this method was positively correlated with the mRNA expression of PPAR-γ and AP-2. Conclusion: L02 cells were successfully exposed to high fat and ethanol, and the hepatocyte steatosis model was established and optimized, suggesting that the occurrence of hepatic cell steatosis was related to the up-regulation of PPAR-γ and AP-2.目的： 在原有的乙醇或高脂诱导的细胞模型上进行优化，拟建立新的肝脂肪变性细胞模型，为研究脂肪肝提供更为高效、便捷的体外模型方法。方法： 采用油红O染色法观察血清、油酸和乙醇在不同作用时间和作用浓度下对人源肝细胞株L02中脂滴蓄积的影响。同时采用RT-PCR法检测过氧化物酶体增殖物激活受体（PPAR）-γ、脂肪酸转运蛋白-2（AP）-2的mRNA表达，筛选出肝脂肪变性细胞模型建立的合适条件。两组之间比较采用t检验，三组以上采用单因素方差分析。结果： 油红O染色显示随着血清、油酸和乙醇作用时间和作用浓度的增加，L02细胞中的桔红色颗粒（脂滴）逐渐增多；并确定，与0%油酸和2%乙醇刚开始作用诱导的红色颗粒计数值100.00%±17.63%相比，0.003%油酸和2%乙醇共同作用于L02细胞24 h后，再用2%乙醇和无血清的DMEM培养基继续培养48 h的条件下，细胞中脂滴蓄积水平最高，计数值为802.38%±71.06%（t = 42.36，P < 0.001）。RT-PCR检测显示，诱导的脂滴蓄积与脂肪化相关因子PPARγ、AP-2的mRNA表达呈正相关。结论： 成功将高脂、乙醇等复合因素共暴露L02细胞，建立并优化了肝脂肪变性的细胞模型，提示肝细胞脂肪化的发生与PPARγ、AP-2的上调有关。.