非酒精性脂肪性肝炎AML-12体外模型建立

Establishment of AML-12 cell model of non-alcoholic steatohepatitis

目的分析并评价不同比例浓度梯度OA/PA诱导的非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)炎症体外模型。方法通过采用不同比例浓度梯度OA/PA(2∶3或2∶1)诱导AML-12细胞建立NASH炎症体外模型;收集细胞OA/PA(500μmol/L∶250μmol/L)诱导AML-12株CCK-8检测细胞毒性或存活率;油红O染色观察其胞内脂肪变性;qRT-PCR或Western blotting检测细胞内TNF-α、IL-6和TGF-β1 mRNA含量。结果得出不同比例浓度的OA/PA混合药物刺激24 h或48 h后,OA/PA以2∶1比例(500μmol/L+250μmol/L)等摩尔比混合后,细胞毒性明显低于2∶3的OA/PA(200μmol/L+300μmol/L)浓度(P<0.05);刺激48 h后,模型组细胞内脂滴及IL-6和TNF-αmRNA和蛋白表达水平均显著升高(P<0.05)。结论以2∶1比例的OA(500μmol/L)/PA(250μmol/L)混合药物诱导下成功建立NASH炎症AML-12体外模型。

Objective To analysis and evaluate the in vitro models of non-alcoholic steatohepatitis(NASH)inflammation induced by different proportional concentration gradients of OA/PA.Methods AML-12 cells were induced by different concentrations of OA/PA(2∶3 or 2∶1)to establish NASH model in vitro,and CCK-8 cells were induced by OA/PA(500μmol/L∶250μmol/L)to detect cytotoxicity or survival rate.The mRNA contents of TNF-α,IL-6 and TGF-β1 in the cells were measured by qRT-PCR or Western blotting.Results It was concluded that after 24 hours or 48 hours of stimulation with a mixture of different concentrations of OA/PA,OA/PA were mixed in a 2∶1 ratio(500μmol/L+250μmol/L)in equal molar ratio,the cytotoxicity was significantly lower than that of 2∶3 of OA/PA(200μmol/L+300μmol/L)(P<0.05).After 48 hours of stimulation,the expression levels of IL-6 and TNF-αmRNA and protein in the model group were significantly increased(P<0.05).Conclusion In vitro model of NASH inflammation AML-12 was successfully established with a 2∶1 ratio of OA(500μmol/L)/PA(250μmol/L).