Nrf-2抗氧化应激改善终末期肾病血管钙化的作用与研究

发布时间：2018-03-17 13:51

本文选题：Nrf-2　切入点：氧化应激　出处：《川北医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:氧化应激可造成血管平滑肌细胞(Rat aortic vascular smooth muscle cells,RASMCs)损伤,促RASMCs向成骨样细胞转分化,是终末期肾病(End-stage renal disease,ESRD)血管钙化的重要机制。本研究将探讨内源性抗氧化因子Nrf-2(Nuclear factor-erythroid2-relatedfactor-2)在高磷诱导血管平滑肌细胞向成骨细胞转化过程中的作用。观察其与RASMC氧化应激损伤的联系,明确Nrf-2在终末期肾病血管钙化中的作用及机制,为防治终末期肾病血管钙化提供新的靶点。方法:1.采用β-甘油磷酸处理大鼠血管平滑肌细胞构建终末期肾病血管钙化细胞模型。2.体外钙化培养72小时前,对数期生长的RASMCs主要分为以下几组:正常对照组;钙化组;Nrf-2 si RNA转染组;Nrf-2抑制剂全反式视黄酸(Retinoic Acid/Vitamine A Acid,VA,1μM or 5μM)处理组;Nrf-2激动剂莱菔硫烷(Sulforaphane,SFN,1μM or 5μM)处理组;活性氧(Reactive oxygen species,ROS)抑制剂N-乙酰基-L-半胱氨酸(N-Acetylcysteine,NAC,5 m M or 10 m M)处理组。3.冯库萨染色(Von Kossa)观察各组RASMCs内钙盐沉积情况后再测定各组RASMC中钙离子含量。4.逆转录链聚合酶反应(RT-PCR)检测各实验组细胞中的Nrf-2、纤维细胞生长因子23(fibroblast growth factor 23,FGF-23)和Klotho的m RNA表达水平。5.免疫蛋白印迹技术(Western Bloting)检测各实验组RASMCs中Nrf-2、Kelch样环氧氯丙烷相关蛋白-1(Kelch-like ECH-associated protein1,Keap-1)、骨桥蛋白(Osteopontin,OPN)和核心结合因子α1(Runt-related transcription factor 2,Runx-2)的蛋白表达情况。6.Mito-tracker Red FM孵育各组RASMCs后,激光共聚焦观测各组细胞中的线粒体损伤情况。7.检测RASMCs内ROS的含量和线粒体膜电位的变化情况。结果:1.Nrf-2 si RNA转染及Nrf-2抑制剂全反式视黄醛预处理后,并未抑制钙化细胞中钙盐的沉积,也未降低钙化细胞中钙离子的含量。而SFN激动Nrf-2及NAC抑制ROS后,抑制钙化细胞中钙盐的沉积,也降低了钙化细胞中钙离子的含量。2.Nrf-2 si RNA转染及全反式视黄醛下调Nrf-2后,降低钙化细胞中Nrf-2和Klotho的m RNA水平,增强钙化细胞中FGF-23的m RNA水平。而SFN激动Nrf-2及NAC抑制ROS后,钙化细胞中Nrf-2和Klotho的m RNA水平增加,减少钙化细胞中FGF-23的m RNA水平。3.Nrf-2 si RNA转染及全反式视黄醛下调Nrf-2后,降低钙化细胞中Nrf-2的蛋白表达,增强钙化细胞中Keap-1、OPN和Runx-2的蛋白表达。而SFN激动Nrf-2及NAC抑制ROS后,钙化细胞中Nrf-2的蛋白表达增加,Keap-1、OPN和Runx-2的蛋白表达下调。4.全反式视黄醛下调Nrf-2后,标记胞内线粒体的Mito-tracker荧光强度减弱。但是SFN或者DMF激动Nrf-2,NAC抑制ROS后,标记胞内线粒体的Mito-tracker荧光强度增强,SFN或者DMF激动Nrf-2后钙化细胞内ROS的含量减弱,细胞线粒体膜电位增加。结论:Nrf-2可调控ROS抗氧化应激,改善线粒体的氧化应激损伤,进而抑制血管平滑肌细胞成骨样变,以达到阻止终末期肾病血管钙化进展的作用,为防治终末期肾病血管钙化提供新的防治靶点与策略。
[Abstract]:Objective: oxidative stress can induce the injury of aortic vascular smooth muscle cells (RASMCs) and promote the transdifferentiation of RASMCs into osteoblast like cells. It is an important mechanism of vascular calcification in End-stage renal disease of end-stage nephropathy. This study will investigate the role of endogenous antioxidant factor Nrf-2(Nuclear factor-erythroid2-relatedfactor-2 in the process of transforming vascular smooth muscle cells into osteoblasts induced by high phosphorus. To clarify the role and mechanism of Nrf-2 in vascular calcification of end-stage nephropathy. To provide a new target for the prevention and treatment of vascular calcification in end-stage nephropathy. Methods 1. Vascular smooth muscle cells of rats treated with 尾 -glycerophosphoric acid were used to construct vascular calcification cell model of end-stage nephropathy. 72 hours before calcification in vitro, The RASMCs growing in logarithmic phase was divided into the following groups: normal control group, Nrf-2 si RNA transfection group, all trans retinoic Acid/Vitamine A acid VA1 渭 M or 5 渭 M treated group, treated with sulforaphane sulforaphane SFN 1 渭 M or 5 渭 M). Reactive oxygen speciess-ROS) inhibitor N-Acetylcysteine N-Acetylcysteine NACU (5 mm or 10 mm) treatment group .3.Von Kossa staining observed calcium deposition in RASMCs of each group, and then determined the calcium ion content. 4 reverse transcription chain polymerase in RASMC of each group. RT-PCR was used to detect the expression levels of Nrf-2, fibroblast growth factor 23FGF-23 and Klotho m RNA in each experimental group. Western blotting was used to detect Nrf-2 Kelch like ECH-associated protein 1 Keap-1, bone bridge egg. The protein expression of Osteopontinin (OPN) and the core binding factor (伪 1) Runt-related transcription factor 2 (Runx-2). 6. Mito-tracker Red FM incubated each group of RASMCs. The changes of ROS content and mitochondrial membrane potential in RASMCs were detected. Results: 1. Nrf-2si RNA transfection and Nrf-2 inhibitor all-trans retinaldehyde pretreatment. SFN did not inhibit the deposition of calcium salt in calcified cells, nor did it decrease the content of calcium in calcified cells, while SFN stimulated Nrf-2 and NAC inhibited the deposition of calcium salt in calcified cells after inhibiting ROS. After transfection of Nrf-2si RNA and down-regulation of Nrf-2 by all-trans retinaldehyde, the level of m RNA of Nrf-2 and Klotho in calcified cells was decreased, and the level of m RNA of FGF-23 in calcified cells was enhanced. SFN stimulated Nrf-2 and NAC inhibited ROS. The level of m RNA of Nrf-2 and Klotho in calcified cells increased, the m RNA level of FGF-23 in calcified cells decreased. 3. Nrf-2 si RNA transfection and all trans retinaldehyde down-regulated Nrf-2, and decreased the expression of Nrf-2 protein in calcified cells. The protein expression of Nrf-2 in calcified cells was increased after SFN stimulated Nrf-2 and NAC to inhibit ROS. The expression of Nrf-2 and Runx-2 in calcified cells was down-regulated. 4. All trans retinaldehyde down-regulated Nrf-2. The Mito-tracker fluorescence intensity of labeled mitochondria decreased, but the Mito-tracker fluorescence intensity of labeled mitochondria increased after SFN or DMF stimulated Nrf-2NAC to inhibit ROS, and the ROS content of calcified cells decreased after DMF or SFN stimulated Nrf-2. Conclusion: Nrf-2 can regulate the oxidative stress of ROS, improve the oxidative stress injury of mitochondria, and then inhibit the osteoblast-like degeneration of vascular smooth muscle cells, so as to prevent the progression of vascular calcification in end-stage nephropathy. To provide a new prevention and treatment target and strategy for the prevention and treatment of end-stage nephropathy vascular calcification.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

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