第三丁基过氧化氢诱导MIN6细胞损伤

Tert-butyl Hydroperoxide Induces Oxidative Damage Model in Murine Pancreatic β-cell MIN6

目的通过第三丁基过氧化氢(t-BHP)诱导小鼠胰腺β细胞,模拟体外氧化应激的细胞模型,观察氧化损伤对β细胞生长的影响。方法体外培养小鼠β细胞株MIN6,MTT法测定细胞存活率,AO-EB荧光显微镜观测,Annexin-Ⅴ-PI染色流式细胞技术定量定性分析细胞凋亡率及坏死率。结果MTT显示,t-BHP(12.5～200μmol/L)作用不同时间后可明显抑制MIN6细胞的生长,并呈现一定的时效和量效关系;AO-EB荧光显微镜及Annexin-Ⅴ-PI流式细胞检测证实,25μmol/L浓度的t-BHP诱导β细胞的损伤作用以凋亡为主,而更高浓度的t-BHP使细胞由凋亡转向坏死。结论t-BHP可用于体外模拟β细胞氧化应激损伤的模型。

Objective To investigate effects of oxidative stress on pancreatic β-cells by exposure of tert-butyl hydroperoxide（t-BHP）. Methods Murine pancreatic β-cell MIN6 was cultured in vitro and cell viability was measured by MTT assay. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of apoptosis or necrosis cell was determined by flow cytometric assay after Annexin-Y-PI staining. Results Exposured of 12.5-200 μmol/L t-BHP to MIN6 cells for 15-60 min, the viability of cells were gradually reduced in dose-dependant and time-dependant mannar. The percentage of apoptotic and necrotic cells were increased gradually significantly by AO-EB stain assay. Flow cytometric assay further confirmed the most component of demaged cells were apoptosis when t-BHP at the dose of 25 mol/L. The higher concentration of t-BHP（over 50 mol/L） made MIN6 cell membrane integrity lost as the death process shifted from apoptosis to necrosis. Conclusion t-BHP can use as a good reagent for oxidative stress mimic model in inducing apoptosis or necrosis of MIN6 cell in vitro.