不同尿酸浓度对肾小球内皮细胞eNOS、ROS的合成及炎症因子表达的影响

发布时间：2018-06-15 20:09

  本文选题：尿酸 + 人肾小球内皮细胞　； 参考：《天津医科大学》2017年硕士论文

【摘要】：背景及目的:近年来随着我国人民物质生活条件的改善和生活方式的改变,高尿酸血症的患病率呈现升高趋势。高尿酸血症(Hyperuricemia)是由嘌呤代谢障碍所致的一种代谢性疾病,高尿酸血症不仅仅是痛风的病因,而且与慢性肾脏病(chronic kidney disease,CKD)、心血管疾病、高血压、代谢综合征(Metobolic syndrom)的发生密切相关。二十世纪七十年代Ross等人提出了“内皮细胞功能障碍”假说,他们提出血管内皮细胞功能障碍是血管内皮受损之后,内皮细胞合成的各种细胞因子发生改变,局部炎症反应活跃,血管通透性增加,血管的收缩/舒张失衡。血管内皮功能障碍在高血压、心血管疾病和慢性肾脏病的发生中起到了重要的作用。但是高尿酸血症对血管内皮细胞的影响及其具体致病机制尚未明确,本研究拟探讨不同尿酸浓度对肾小球内皮细胞的影响及其可能的机制,为临床上定期监测,早期预防及治疗高尿酸血症提供理论基础。方法:1、以人的肾小球内皮细胞(HRGEC)为实验对象,经CCK-8细胞毒性实验检测,选择尿酸的作用浓度。2、不同浓度(0、4、8、16 mg/dl)的尿酸作用于HRGEC 24小时,Real-timePCR法、Western blotting法分别检测eNOS、NF-κB p65、MCP-1、ICAM-1的表达,细胞免疫荧光的方法检测eNOS、NF-κB p65蛋白的表达。3、不同浓度(0、4、8、16 mg/dl)的尿酸作用于HRGEC 24小时,利用荧光探针DCFH-DA,在37℃作用于HRGEC 30分钟,倒置荧光显微镜下进行细胞内活性氧(Reactive oxygen species,ROS)检测。结果:1、与对照组相比较,4 mg/dl尿酸组HRGEC的eNOS mRNA及蛋白的表达无显著性差异(P0.05),8 mg/dl及16 mg/dl尿酸组HRGEC的eNOS mRNA及蛋白的表达显著减少(P0.05);与4 mg/dl尿酸组相比较,8 mg/dl及16 mg/dl尿酸组HRGEC的eNOS mRNA及蛋白的表达显著减少(P0.05)。2、与对照组相比较,4 mg/dl尿酸组HRGEC的ROS表达无显著性差异(P0.05),8 mg/dl及16mg/dl尿酸组HRGEC的ROS表达显著增多(P0.05);与4 mg/dl尿酸组相比较,8 mg/dl及16mg/dl尿酸组HRGEC的ROS表达显著增多(P0.05)。3、与对照组相比较,4 mg/dl尿酸组HRGEC的NF-κB p65 mRNA及蛋白的表达无显著性差异(P0.05),8 mg/dl及16 mg/dl尿酸组HRGEC的NF-κB p65mRNA及蛋白的表达显著增多(P0.05);与4 mg/dl尿酸组相比较,8 mg/dl及16 mg/dl尿酸组HRGEC的NF-κB p65 mRNA及蛋白的表达显著增多(P0.05)。4、与对照组相比较,4 mg/dl尿酸组HRGEC的MCP-1、ICAM-1 mRNA及蛋白的表达无显著性差异(P0.05),8 mg/dl及16 mg/dl尿酸组HRGEC的MCP-1、ICAM-1 mRNA及蛋白的表达显著增多(P0.05);与4 mg/dl尿酸组相比较,8 mg/dl及16 mg/dl尿酸组HRGEC的MCP-1、ICAM-1 mRNA及蛋白的表达显著增多(P0.05)。结论:1、高浓度的尿酸可能通过氧化应激途径导致肾小球内皮细胞损伤,进而造成肾脏功能损伤。2、高浓度的尿酸可能通过激活NF-κB经典炎症信号通路导致肾小球内皮细胞损伤。
[Abstract]:Background and objective: with the improvement of material living conditions and changes in lifestyle, the prevalence of hyperuricemia has increased in recent years. Hyperuricemia is a metabolic disease caused by purine metabolic disorder. Hyperuricemia is not only the cause of gout, but also is closely related to the occurrence of chronic kidney disease, cardiovascular disease, hypertension and metabolic syndrome. Ross et al. Put forward the hypothesis of "endothelial cell dysfunction" in the 1970s. They proposed that vascular endothelial cell dysfunction is caused by changes in various cytokines synthesized by endothelial cells after vascular endothelial damage. The local inflammatory reaction is active, the vascular permeability is increased, and the vasoconstriction / relaxation is out of balance. Vascular endothelial dysfunction plays an important role in the development of hypertension, cardiovascular disease and chronic kidney disease. However, the effect of hyperuricemia on vascular endothelial cells and its specific pathogenesis have not been clarified. This study aims to explore the effects of different concentrations of uric acid on glomerular endothelial cells and its possible mechanisms for regular clinical monitoring. Early prevention and treatment of hyperuricemia provide a theoretical basis. Methods the human glomerular endothelial cells (HRGECs) were used as experimental subjects. By CCK-8 cytotoxicity test, the concentration of uric acid was chosen as 0.2 and the concentration of uric acid at different concentrations was 816 mg / dl). The expression of eNOSNFB p65MCP-1 ICAM-1 was detected by real-time PCR and Western blotting. The expression of eNOS- 魏 B p65 protein was detected by cellular immunofluorescence. The uronic acid of different concentrations of eNOS- 魏 B p65 was applied to HRGEC for 24 hours. The fluorescence probe DCFH-DAwas applied to HRGECs for 30 minutes at 37 鈩，

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