持续性和波动性高糖培养对人视网膜色素上皮细胞炎性因子表达的影响

摘要

目的:观察波动性和持续性高糖对人视网膜色素上皮细胞(HRPE)炎性因子表达的影响.方法:取常规培养对数期HRPE(2×10/ml)接种于24孔板,无血清DMEM培养至细胞同步于G0/G1期,加入条件培养液继续培养,并据此分组:(1)低浓度葡萄糖(5.5mmol/L)培养对照组(N组);(2)不同渗透压对照培养组(P组),包括25mmol/L渗透压对照组 (P1组,即用 5.5mmol/L葡萄糖 和 19.5mmol/L甘露醇培养)和33mmol/L渗透压对照组(P2组,即用5.5mmol/L葡萄糖 和27.5mmol/L甘露醇培养);(3)持续性高浓度葡萄糖培养组(H组),包括25mmol/L持续高糖培养(H1组)和33mmol/L持续高糖培养(H2组).(4)波动性高浓度葡萄糖培养(F组),包括25/5.5mmol/L葡萄糖交替培养(F1组,即高糖3h,低糖2h,轮替交换,日间交换3次,25mmol/L葡萄糖过夜)和33/5.5mmol/L葡萄糖交替培养(F2组,即高糖3h,低糖2h,轮替交换,日间交换3次,33mmol/L葡萄糖过夜).各组均培养72h,并于培养24h、48h、72h检测分析HRPE培养上清液中细胞间黏附分子-1 (ICAM-1)和肿瘤坏死因子-α(TNF-α)含量变化.结果:组内比较,同组HRPE条件培养24h、48h、72h,其上清液ICAM-1、TNF-α表达量差异无统计学意义(均P>0.05).组间比较,P组ICAM-1和TNF-α水平与N组无明显差异(P>0.05),H组和F组均高于N组(P<0.01),F组较H组升高更显著(P<0.01),P1和P2、H1和H2、F1和F2各两亚组间差异均无统计学意义(P>0.05).结论:波动性高浓度葡萄糖刺激对HRPE的炎性损伤较持续性高糖更为明显,对糖尿病视网膜危害更大.%Objective: To investigate the inflammatory injury of human retinal pigment epithelial(HRPE) cells to persistent high glucose and fluctuant high glucose.Method: HRPE cells were cultured under standard conditions ,serum-starved for 24 hours and then were randomly divided into four groups: normal glucose control group (5.5mmol/L glucose, N),osmotic pressure group(19.5 mmol/L mannitol plus 5.5 mmol/L glucose, 27.5 mmol/L mannitol plus 5.5 mmol/L glucose,P1 and P2), constant high glucose group (25mmol/L glucose and 33mmol/L glucose, H1 and H2), fluctuant high glucose group (25mmol/L or 33mmol/L glucose for three hours, at an interval of two hours of 5.5mmol/L glucose, such intermittent exposure was repeated three times a day and the cells were exposed to 25mmol/L or 33mmol/L glucose in the night, F1 and F2).Concentration of tumor necrosis factor (TNF)-α and intercellular adhesion molecule-1 (ICAM-1) in every group in the supernatant were measured by use of ELISA in 24 hours,48 hours, and 72 hours according to the instructions.Results: There was no significant difference for each index in every group at different times (P>0.05).There was no significant difference for each index between the osmotic pressure group and the normal glucose group (P>0.05) at any moment.Compared with the normal glucose group, the level of ICAM-1 and TNF-a in the constant high glucose group and the fluctuant high glucose group both increased (P<0.01, respectively), the fluctuant high glucose group significantly increased than the constant high glucose group with a marked increase of ICAM-1 and TNF-α(P <0.01, respectively).There was no difference between the 25mmol/L and 33mmol/L high glucose groups,also there was no difference between the two fluctuant high glucose groups.Conclusion: Fluctuant high glucose evoked more intense inflammatory damages than constant high glucose,it was more harmful to the diabetic retinopathy.