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目的:探讨高糖条件下大鼠肾小球系膜细胞(GMCs)中内皮一氧化氮合酶/一氧化氮(eNOS/NO)的变化及可能的调节机制。方法:将大鼠GMCs常规培养在含5.5 mmol/L葡萄糖的RPMI1640培养液中,用胰蛋白酶-乙二胺四乙酸(EDTA)混合消化酶传代。用RT、实时-PCR和Western blot测定GMCs中eNOS蛋白和其mRNA的相对含量。用NO显示剂DAF-2 DA(0.5 nmol/L)标记,NO的变化用QM-6荧光计测定。结果:在GMCs中,高糖可明显增加eNOS的表达(P<0.05),并有一定的量效、时效关系,同时可促进NO产生;放线菌酮可以抑制eNOS蛋白的合成。高糖对eNOS表达的调节与eNOS mRNA的稳定性没有明显关系。结论:高糖可以上调eNOS mRNA和其蛋白的表达,并可促进NO产生。该效应可能与eNOS蛋白的合成相关,而与eNOS mRNA的稳定性没有明显关系。本实验的结果为eNOS介导的NO产生和由此所致肾小球的超滤过提供了新的思路。
Objective: To investigate the changes of endothelial nitric oxide synthase (NO) / nitric oxide (NO) and its regulatory mechanism in rat mesangial cells (GMCs) under high glucose conditions. Methods: Rat GMCs were routinely cultured in RPMI 1640 medium containing 5.5 mmol / L glucose and passaged with trypsin - EDTA digestion. The relative content of eNOS protein and its mRNA in GMCs was determined by RT, real-time-PCR and Western blot. The NO indicator DAF-2 DA (0.5 nmol / L) was used to label the change of NO with the QM-6 fluorometer. Results: In GMCs, high glucose significantly increased the expression of eNOS (P <0.05), and had some dose-effect and time-dependent effects. At the same time, it increased NO production. Cycloheximide inhibited the synthesis of eNOS protein. The regulation of eNOS expression by high glucose has no significant relationship with the stability of eNOS mRNA. Conclusion: High glucose can up-regulate the expression of eNOS mRNA and its protein, and promote the production of NO. This effect may be related to the synthesis of eNOS protein but not to the stability of eNOS mRNA. The results of this experiment provide a new idea for the eNOS-mediated NO production and the resulting ultrafiltration of glomeruli.