人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧在高糖状态下的表达

Changes in inducible nitric oxide synthase and reactive oxygen species in human retinal pigment epithelial cells induced by high glucose

背景:高血糖导致的自由基损伤是糖尿病视网膜病变发病机制的中心环节。目的:观察高糖对体外培养的人视网膜色素上皮细胞的氧化损伤作用以及高糖对人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧表达的影响。方法:将培养人视网膜色素上皮细胞,分为对照组、高糖组和甘露醇组,分别用含5.5mmol/L葡萄糖,33mmol/L葡萄糖及5.5mmol/L葡萄糖和27.5mmol/L甘露醇的DMEM培养液培养。采用相差倒置显微镜观察细胞生长形态,采用免疫荧光染色研究诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达的变化,用氯甲基二氯二氢荧光素二乙酯荧光染色检测视网膜色素上皮细胞中活性氧的产生量。结果与结论:与对照组相比,应用含33mmol/L葡萄糖的DMEM培养基处理视网膜色素上皮细胞48h可见细胞胞体变薄,形态表现多样,不规则细胞增多;高糖培养的视网膜色素上皮细胞诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达增加,活性氧产生明显增多。说明高浓度葡萄糖培养可造成人视网膜色素上皮细胞氧化损伤,使细胞形态发生变化,并导致细胞中3-硝基酪氨酸产生增多。

BACKGROUND：High glucose-induced free radical damage is a central link to pathological mechanism underlying diabetic retinopathy. OBJECTIVE：To investigate the effects of high glucose on growth of human retinal pigment epithelial cells and on expression of inducible nitric oxide synthase （iNOS） and reactive oxygen species （ROS） in cells. METHODS：Human retinal pigment epithelial cells were cultured in vitro and randomly divided into three groups：control, high glucose, and mannitol. In the control, high glucose, and mannitol groups, cells were cultured with DMEM solution containing 5.5 mmol/L glucose, 33 mmol/L glucose, and 55 mmol/L glucose and 27.5 mmol/L mannitol, respectively. Cell morphology was observed by phase contrast inverted microscope. Expression of iNOS and 3-nitrotyrosine was studied by immunofluorescence staining method. Changes in ROS in the human retinal pigment epithelial cells were determined by scanning laser confocal microscope. RESULTS AND CONCLUSION： Compared with control group, at 48 hours after treatment, cell body became small with various morphologies, irregular cells increased, iNOS and 3-nitrotyrosine expression increased, and ROS expression was obviously increased, in the high glucose group. These finding suggest that high glucose can damage retinal pigment epithelial cells, leading to altered cell morphology and increased intracellular 3-nitrotyrosine level.