对乙酰氨基酚诱导HepG2细胞氧化应激损伤模型的构建

Establishment of Oxidative Stress Injury Model Induced by Acetaminophen in HepG2 Cells

目的建立以对乙酰氨基酚(Acetaminophen,APAP)诱导的HepG2细胞氧化应激损伤模型。方法噻唑蓝〔MTT〕法检测HepG2细胞活力;微板法检测细胞培养液中天门冬氨酸氨基转氨酶(AST)、丙氨酸氨基转氨酶(ALT)和乳酸脱氢酶(LDH)的含量,细胞中超氧化物歧化酶(SOD)及过氧化氢酶(CAT)活性;利用荧光探针(DCFH-DA)法对HepG2细胞内活性氧的水平(ROS)进行检测;JC-1染色法检测不同浓度APAP对HepG2细胞线粒体膜电位的影响;Hoechst染色观察细胞凋亡情况。结果随着APAP处理时间和浓度增加,HepG2细胞相对活力明显降低,细胞数量减少,并伴随着皱缩、变圆甚至漂浮等形态学变化;细胞培养液中的ALT、AST和LDH含量显著升高,而细胞中的CAT和SOD活性明显下降;另外,胞内ROS水平和线粒体膜电位发生显著变化,细胞凋亡显著增加。诱导Hep G2细胞产生氧化应激损伤模型最佳条件为APAP浓度30mmol·L-1,细胞作用时间12h。结论本研究构建了APAP体外氧化应激细胞损伤模型,为保肝活性药物筛选及其作用机制研究提供细胞学模型。

OBJECTIVE To establish a model of oxidative stress injury induced by Acetaminophen(APAP) in HepG2 cells.METHODS The cell viability of HepG2 cells was detected by MTT assay.The activity of AST,ALT and LDH in cell culture medium was detected by microplate method,as well as SOD,CAT activity in cells.The level of ROS in cells was detected by fluorescent probe(DCFH-DA) method;Mitochondrial membrane potential was detected by JC-1 fluorescence probe;apoptosis induced by APAP was observed by Hoechst staining.RESULTS After treated by APAP with increased concentration and time,the relative cell viability of HepG2 cells decreased significantly,along with the obviously decreased number of cells and morphological changes such as shrinkage,rounding and even floating.The notably increased contents of ALT,AST and LDH in cell culture medium were observed,as well as prominently decreased CAT,SOD and increased levels of ROS and apoptosis in cells.Therefore,the oxidative stress injury in HepG2 cells can be induced by exposure with 30 mmol·L-1 of APAP for 12 h.CONCLUSION APAP was used to induce oxidative stress injury in cells successfully,providing the cell model for investigating hepatoprotective activity and action mechanism of hepatoprotectant.