高糖对人腹膜间皮细胞生成活性氧的影响

Effect of generation of reactive oxygen species in high glucose-induced human peritoneal mesothelial cells

关键词:  高糖  人腹膜间皮细胞  活性氧

Key words:  high glucose  peritoneal mesothelial cells  reactive oxygen species

摘要:

［目的］研究不同浓度的葡萄糖对人腹膜间皮细胞(human peritoneal mesothelial cells, HPMCs )生成活性氧的影响。［方法］HPMCs 体外培养并进行免疫组化鉴定，取第3代细胞用于实验。分别按照不同的实验要求将细胞分组。不同葡萄糖浓度干预组：①空白对照组；②1.5%葡萄糖组；③2.5%葡萄糖组；④4.25%葡萄糖组。不同作用时间组：①空白对照组；②2.5%葡萄糖组。两组细胞分别培养12、24、48 h。应用流式细胞仪检测细胞对氧化剂敏感的2，7-二氢二氯荧光素（2，7’-dichlorodihydrofluorescein, DCFH）发生氧化所产生的荧光量，从而间接的反映细胞内活性氧（reactive oxygen species, ROS）的水平，观察随葡萄糖浓度变化及不同作用时间对人腹膜间皮细胞产生活性氧的影响。［结果］（1）与空白对照组比较，高糖干预人腹膜间皮细胞后，其细胞内平均荧光强度明显升高（P＜0.05），并且随葡萄糖浓度升高而逐渐增高，其中4.25%葡萄糖组最高。（2）与空白对照组比较，随着时间的延长，高糖作用下细胞内平均荧光强度明显升高（P＜0.05），培养48 h其水平最高。［结论］高糖可刺激体外培养的人腹膜间皮细胞ROS的生成。

Abstract:

［Objective］To explore the effect of different glucose concentrations on generation of reactive oxygen species (ROS) of human peritoneal mesothelial cells (HPMCs).［Methods］HPMCs were cultured in vitro and identified by immunohistochemistry, and cells of the third generation were selected. were divided according to experiment required: different glucose concentrations groups:①blank control group；②1.5% glucose group；③2.5% glucose group；④4.25% glucose group. different time groups：①blank control group；②2.5% glucose group，two groups cells were cultured respectively for 12，24，48 h. The intracellular generation of ROS were assessed indirectly by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2’,7’-dichlorodihydrofluorescein (DCFH) by using flow cytometry. The level of ROS were analyzed after being intervened by glucose with different concentrations and at different time points.［Results］(1)Compared with blank control group, MFI of HPMCs which were interfered in high glucose would increase obviously (P＜0.05),and accompanying with the increased concentration of glucose, MFI of cells increased gradually, 4.25% glucose group were most high.(2) Compared with blank control group, With the extending of the time, MFI of HPMCs interfered in high glucose would increase obviously (P＜0.05), especially at the 48 h.［Conclusion］High glucose may stimulate the generation of reactive oxygen species.