高糖对人视网膜色素上皮细胞Na-K-ATP酶表达的影响

Effects of high concentration of glucose on Na-K-ATPase expression of human retinal pigment epithelium cell

目的观察高糖对人视网膜色素上皮(RPE)细胞Na-K-ATP酶基因与蛋白表达的影响。方法采用ARPE-19细胞,培养4周后分为高糖组(葡萄糖浓度30mmol/L)与对照组(葡萄糖浓度7mmol/L),继续培养2周、4周,RT-PCR法检测Na-K-ATP酶α1、Na-K-ATP酶β1的mRNA表达水平的变化,Western blot法检测Na-K-ATP酶β的蛋白表达情况。结果高糖抑制Na-K-ATP酶α1与β1的mRNA表达,2周、4周时α1与β-actin光密度比值分别为0.17、0.23,对照组分别为0.37、0.36;β1与β-actin光密度比值分别为0.69、0.41,对照组分别为0.98、0.83;Na-K-ATP酶β蛋白表达水平较对照组降低,2周、4周时与β-actin的比值分别为0.51、0.25,对照组分别为0.65、0.68。结论高糖环境下人RPE细胞Na-K-ATP酶基因与蛋白表达水平降低,可能参与了黄斑水肿的发生。

Objective Diabetic macular edema is an outcome of damage of retinal inner and outer barrier. Retinal pigment epithelium（RPE） plays a critical role in retinal fluid transfer;while Na-K-ATPase is an important enzyme in the process of fluid transportation of subretinal space. Here in the experiment, we investigated the effects of high concentration of glucose on Na-K- ATPase expression in human RPE. Methods ARPE-19 cells were cultured for 4 weeks in 24 wells culture plate. The cultured cells were divided into high concentration of glucose-cultured group （ glucose addition at 30 mmol/L） or control group（ containing 7 mmol/L glucose ）. Reverse transcription polymerase chain reaction and Western-blot were used to evaluate the expression of Na- K-ATPase in cells after cultured for 2 or 4 weeks. Results High concentration of glucose downregulated the mRNA expression both Na-K-ATPase ctl and β1 subunit. The OD ratio of Na-K-ATPase ctl to β-actin was 0. 17 and 0. 23 respectively after culture for 2 or 4 weeks in high glucose group and 0. 37 and 0. 36 in normal group,and that of Na-K-ATPase β1 was 0.69 and 0.41 in high glucose group,0. 98 and 0. 83 in control group. Protein level of Na-K-ATPase β was lower in high glucose group than in normal group. The OD ratio to β-actin was 0.51 and 0. 25 respectively after cultured for 2 or 4 weeks in high glucose group and 0. 37 and 0. 36 in normal group. Conclusion The expression of mRNA and protein of Na-K-ATPase is reduced in human retinal pigment epithelium at the cultured condition of high concentration of glucose. This result suggests that high concentration of glucose may participate in the pathogenesis of diabetic macular edema.