人肾小球血管内皮细胞胰岛素抵抗模型的建立

Establishment of Insulin Resistance Model in Human Glomerular Endothelial Cells

目的:采用不同浓度的棕榈酸与葡萄糖在体外诱导建立人肾小球内皮细胞(Human glomerular endothelial cells,HRGEC)胰岛素抵抗模型。方法:以人肾小球内皮细胞为研究对象,不同浓度棕榈酸(100,200,300,400,500μmol/L)与不同浓度的葡萄糖(20,30,40,50,60 mmol/L)分别作用细胞24小时和48小时,应用MTT法和葡萄糖氧化酶法检测棕榈酸和葡萄糖对HRGEC存活率与葡萄糖消耗量的影响,蛋白免疫印迹法检测P-IRS、IRS、AKT和p-AKT(Ser473)的影响。结果:1、当棕榈酸500μmol/L干预细胞24小时,与正常组比较,细胞活性显著下降(P<0.01),棕榈酸浓度大于或等于300μmol/L干预细胞48小时,细胞存活率显著降低(P<0.01)。与空白组比较,300μmol/L、400μmol/L、500μmol/L棕榈酸干预细胞24小时能够明显的降低细胞的葡萄糖消耗(P<0.05);200μmol/L、300μmol/L、400μmol/L、500μmol/L干预细胞48小时能够明显的降低细胞的葡萄糖消耗(P<0.01)。2、不同浓度葡萄糖刺激人肾小球内皮细胞(HGREC)24小时和48小时,与空白组比较,各组细胞的存活率与对照组比较均无显著变化(P>0.05)。与空白组比较,40mmol/L、50 mmol/L、60 mmol/L葡萄糖干预细胞24小时能够降低人肾小球内皮细胞的葡萄糖消耗(P<0.05)。与空白组比较,30mmol/L、40mmol/L、50 mmol/L、60 mmol/L葡萄糖干预细胞48小时能够明显降低人肾小球内皮细胞的葡萄糖消耗量(P<0.01)。3、不同浓度的葡萄糖刺激人肾小球内皮细胞(HGREC)24小时后,结果显示,50 mmol/L、60 mmol/L葡萄糖刺激细胞24小时能降低P-IRS/IRS和p-AKT/AKT(Ser473)的水平(P<0.01),而其他组无明显显著变化(P>0.05)。结论:高糖诱导方法能够建立HRGEC细胞胰岛素抵抗模型,具有建模周期短、容易重复、可控性强的优点,可用于糖尿病胰岛素抵抗机制的研究和中药成分的筛选研究。

Objective:To establish Human glomerular endothelial cells(Human glomerular endothelial cells,HRGEC)model of insulin resistance by the different concentrations of palmitic acid and glucose in vitro induced.Methods:Human glomerular endothelial cells as the research object,the different concentration of palmitic acid(100,200,300,400,500μmol/L)and different concentrations of glucose(20,30,40,50,60 mmol/L)for 24 hours and 48 hours respectively,to detect palmitic acid and glucose effects on glucose consumption and HRGEC survival determined by MTT method and glucose oxidase method,to detect P-IRS,the IRS,AKT and P-AKT(Ser473)by protein immunoblot method.Results:1.When palmitate was given 500μmol/L for 24 h,cell activity significantly decreased compared with the normal group(P<0.01),and cell survival rate significantly decreased when palmitate concentration was more than or equal to 300μmol/L for 48 h(P<0.01).Compared with the control group,palmitate intervention at 300μmol/L,400μmol/L and 500μmol/L for 24 hours could significantly reduce the glucose consumption of cells(P<0.05).Intervention at 200μmol/L,300μmol/L,400μmol/L and 500μmol/L for 48 hours significantly reduced glucose consumption(P<0.01).Compared with the blank group,glucose intervention of 40 mmol/L,50 mmol/L and 60 mmol/L for 24 hours could reduce glucose consumption of human glomerular endothelial cells(P<0.05).Compared with the blank group,the glucose consumption of human glomerular endothelial cells was significantly reduced by the intervention of 30 mmol/L,40 mmol/L,50 mmol/L and 60 mmol/L for 48 hours(P<0.01).3.After 24 h of glucose stimulation of human glomerular endothelial cells(HGREC)at different concentrations,the results showed that the levels of p-IRS/IRS and p-AKT/AKT(Ser473)decreased with glucose stimulation of 50 mmol/L and 60 mmol/L for 24 hours(P<0.01),while no significant changes were observed in other groups(P>0.05).Conclusion:The high glucose induction method could establish the insulin resistance model of HRGEC cells,which has the advantages of short modeling period,easy repetition and strong controllability,and could be used for the study of the mechanism of insulin resistance in diabetes mellitus and the screening of traditional Chinese medicine components.