高糖诱导的人近端肾小管上皮细胞损伤及机制的研究

目的:观察高糖诱导人近端肾小管上皮细胞(HK-2)损伤及机制。方法:细胞生长融合至约80%时,用DMEM低糖培养基无血清同步化48 h,然后分为以下7组。①正常糖对照组(5.5 mM低糖DMEM培养基,NG);②高糖1组(15 mM高糖DMEM培养基,HG1);③高糖2组(30 mM高糖DMEM培养基,HG2);④高糖3组(45 mM高糖DMEM培养基,HG3);⑤高糖4组(60 mM高糖DMEM培养基,HG4);⑥等渗1组(30 mM甘露醇DMEM培养基,HM1);⑦等渗2组(45 mM甘露醇DMEM培养基,HM2)。观察不同糖浓度(5.5~45 mM)刺激HK-2细胞24 h,观察IκBα和p65蛋白表达规律。MTT比色法检测细胞活力。Western blot法检测IκBα和p65蛋白表达。结果:高糖2组、高糖3组、高糖4组浓度呈依赖性对HK-2活力存在显著的抑制作用(P0.05,P0.01);高糖2组、高糖3组浓度的甘露醇对HK-2活力没有明显的抑制作用(P0.05)。IκBα蛋白表达随着葡萄糖浓度的升高而依次降低,而p65蛋白表达呈浓度依赖性升高,高糖2组、高糖3组的葡萄糖与正常糖对照组比较,差异有显著性或非常显著性意义(P0.05,P0.01)。结论:高糖诱导的HK-2毒性损伤呈一定的浓度依赖关系,其机制与NF-κB通路的激活有关,与渗透压作用无明显关联。

Study on High Glucose-induced Human Proximal Renal Tubular Epithelial Cell Injury and Its Mechanism

Objective： To investigate the toxic effects of high glucose on HK-2 cells and to explore its mechanism. Methods- When the cell fusion of HK-2 cells reached 80%, HK-2 cells were incubated with low sugar serum-free DMEM for 48 hours, and then were divided into 7 groups, normal glucose control group（5.5 mM low glucose DMEM）, high glucose groups 1-4（DMEM with HG at the concentrations of 15mM, 30mM, 45mM, 60mM, respectively）, and isotonic mannitol groups 1-2（DMEM with mannitol at the concentrations of 30 mM, 45mM, respectively）. After stimulation with glucose at the concentrations of 5. 5-45mM for 24h, the cell viability of HK-2 cells was detected with M-IT assay, and the protein expression levels of IκBα and p65 were measured by Western blotting. Results： High glucose groups 2-4 had an significant inhibitory effect on cell viability of HK-2 cells in a dose-dependent manner（P〈 0.05, P〈 0.01）. Isotonic mannitol groups 1-2 had no significant inhibitory effect on cell viability of HK-2 cells（P 〉 0.05）. IκBα protein expression was decreased with the increase of glucose concentration, p65 protein expression was increased with the increase of glucose concentration, and the differences were significant between high glucose groups 2-3 and normal glucose control group（P〈 0.05, P 〈 0.01）. Conclusion= The toxic effect of high glucose on HK-2 ceils is dose-dependant, which has no significant correlation with the osmotic pressure. NF-KB pathway may be activated in high glucose-induced HK-2 cell injury.