HepG2细胞胰岛素抵抗模型的建立及其应用

为建立方便、可靠、重复性好的胰岛素抵抗细胞模型,研究胰岛素受体底物-2(IRS-2)蛋白磷酸化与胰岛素抵抗的关系以及不同铬制剂对细胞葡萄糖代谢的作用。试验采用高胰岛素、高糖联合诱导Hep G2细胞建立胰岛素抵抗模型,通过葡萄糖氧化酶法及噻唑蓝比色法(MTT)分析受试物对细胞葡萄糖代谢及活性的影响,优化最佳造模条件;同时,利用蛋白免疫印迹法(WB)分析IRS-2的蛋白表达及其蛋白磷酸化,验证模型的胰岛素抵抗性。在此基础上,将模型应用于分析葡萄糖耐量因子(GTF)及其它铬制剂的降糖效果。结果表明,高胰岛素(10-6mol·L~(-1))和高糖培养基(25 mmol·L~(-1))处理细胞48h,细胞存活率达96%,与对照组相比,葡萄糖消耗量降低了5.7%,IRS-2含量减少31.02%,胰岛素抵抗效果显著,且该胰岛素抵抗模型可稳定维持48 h。相比于吡啶酸铬和三氯化铬,GTF对细胞葡萄糖代谢有显著的改善作用,其最佳作用浓度为1.0μg·m L-1。利用HepG2细胞建立体外胰岛素抵抗模型,方法简单可靠、重复性好,可广泛应用于天然活性物质的降糖功能研究。

Establishment and Application of Insulin-resistant HepG2 Cell Model

To establish a convenient,reliable and reproducible insulin resistance cell model,this study attempted to establish the HepG2 cells model by the method of high glucose and high insulin induced in vitro,and analyzed the results with the method of glucose-oxidase and MTT.At the same time,in order to verify the insulin resistance of the model,the IRS-2 and its phosphorylation were analyzed by Western Blot (WB).On the basis,the model was applied to analyze the hypoglycemic effect of glucose tolerance factor (GTF) and other chromium complexes.The results showed that the cell survival rate arrived at 96％ after incubated with the high glucose medium (25 mmol· L-1) and high concentration of insulin (10-6 mol· L-1) for 48 h.Compared with the control group,the glucose consumption was decreased by 5.7％ and the protein content of IRS-2 was decreased by 31.02％.The insulin-resistant model could maintain 48 h.Besides,when the concentration arrived at 1.0 μg·mL-1,the GTF had a significant effect on the glucose metabolism in the cells.In summary,the method is simple,reliable and reproducible,and it could be widely used in the study of hypoglycemic activity of natural active substances.