肝星状细胞氧化应激模型的构建、检测与分析

Construction,detection and analysis of the oxidative stress models of hepatic stellate cells

目的:建立更为全面的肝星状细胞体外氧化应激模型,为检验药物在肝纤维化氧化应激中发挥的具体作用提供有效工具。方法:分别用Fe-NTA/AA及过氧化氢作用于大鼠原代肝星状细胞及人肝星状细胞系LX-2细胞以造成不同类型的氧化应激反应,以超氧化物阴离子荧光探针检测细胞内产生的超氧化物水平,并分别用结晶紫染色或MTT检验来检测不同氧化刺激物对两种肝星状细胞体外增殖性的影响。结果:铁过载及过氧化氢都会引起两种细胞产生氧化应激反应,铁过载可加速原代肝星状细胞激活,对已完全活化的LX-2细胞产生明显毒性作用;过氧化氢则对两种细胞都会造成明显的细胞毒性作用。结论:处于不同激活状态的肝星状细胞对不同氧化刺激物的反应存在差异,因而在检验肝纤维化药物有效性时,应根据不同的病理、生理需求选择恰当的细胞及氧化应激模型。

AIM：To develop correct in vitro oxidative stress models of hepatic stellate cells for the analysis of anti-fibrosis drugs. METHODS：Two representative oxidative models in the liver,the iron-overload model and the H2O2 model were used to generate different oxidative-stress reactions on the primary rat hepatic stellate cells and the immortal cell line of LX-2 cells.The level of intracellular superoxide was detected using dihydroethidium probe and the proliferation status was tested by crystall violet staining and the MTT assay,respectively.RESULTS：Both the iron-overload model and the H2O2 model could generate oxidative reactions in the two kinds of cells.Iron-overload treatment could accelerate the activation of primary hepatic stellate cells,but exerted significant cytotoxicity to LX-2 cells.H2O2 led to completely cytotoxicity to both kinds of cells.CONCLUSION：Hepatic stellate cells at different activation phase would have discrepant responses to the stimulation of different oxidation,thus suitable type of cells and appropriate oxidative-stress related models should be chosen in order to analyse the anti-fibrosis drugs meeting with different physiological and pathological requirement.