本文選題：Nrf-2　切入點：氧化應激　出處：《川北醫(yī)學院》2017年碩士論文　論文類型：學位論文

【摘要】：目的:氧化應激可造成血管平滑肌細胞(Rat aortic vascular smooth muscle cells,RASMCs)損傷,促RASMCs向成骨樣細胞轉分化,是終末期腎病(End-stage renal disease,ESRD)血管鈣化的重要機制。本研究將探討內源性抗氧化因子Nrf-2(Nuclear factor-erythroid2-relatedfactor-2)在高磷誘導血管平滑肌細胞向成骨細胞轉化過程中的作用。觀察其與RASMC氧化應激損傷的聯系,明確Nrf-2在終末期腎病血管鈣化中的作用及機制,為防治終末期腎病血管鈣化提供新的靶點。方法:1.采用β-甘油磷酸處理大鼠血管平滑肌細胞構建終末期腎病血管鈣化細胞模型。2.體外鈣化培養(yǎng)72小時前,對數期生長的RASMCs主要分為以下幾組:正常對照組;鈣化組;Nrf-2 si RNA轉染組;Nrf-2抑制劑全反式視黃酸(Retinoic Acid/Vitamine A Acid,VA,1μM or 5μM)處理組;Nrf-2激動劑萊菔硫烷(Sulforaphane,SFN,1μM or 5μM)處理組;活性氧(Reactive oxygen species,ROS)抑制劑N-乙�；�-L-半胱氨酸(N-Acetylcysteine,NAC,5 m M or 10 m M)處理組。3.馮庫薩染色(Von Kossa)觀察各組RASMCs內鈣鹽沉積情況后再測定各組RASMC中鈣離子含量。4.逆轉錄鏈聚合酶反應(RT-PCR)檢測各實驗組細胞中的Nrf-2、纖維細胞生長因子23(fibroblast growth factor 23,FGF-23)和Klotho的m RNA表達水平。5.免疫蛋白印跡技術(Western Bloting)檢測各實驗組RASMCs中Nrf-2、Kelch樣環(huán)氧氯丙烷相關蛋白-1(Kelch-like ECH-associated protein1,Keap-1)、骨橋蛋白(Osteopontin,OPN)和核心結合因子α1(Runt-related transcription factor 2,Runx-2)的蛋白表達情況。6.Mito-tracker Red FM孵育各組RASMCs后,激光共聚焦觀測各組細胞中的線粒體損傷情況。7.檢測RASMCs內ROS的含量和線粒體膜電位的變化情況。結果:1.Nrf-2 si RNA轉染及Nrf-2抑制劑全反式視黃醛預處理后,并未抑制鈣化細胞中鈣鹽的沉積,也未降低鈣化細胞中鈣離子的含量。而SFN激動Nrf-2及NAC抑制ROS后,抑制鈣化細胞中鈣鹽的沉積,也降低了鈣化細胞中鈣離子的含量。2.Nrf-2 si RNA轉染及全反式視黃醛下調Nrf-2后,降低鈣化細胞中Nrf-2和Klotho的m RNA水平,增強鈣化細胞中FGF-23的m RNA水平。而SFN激動Nrf-2及NAC抑制ROS后,鈣化細胞中Nrf-2和Klotho的m RNA水平增加,減少鈣化細胞中FGF-23的m RNA水平。3.Nrf-2 si RNA轉染及全反式視黃醛下調Nrf-2后,降低鈣化細胞中Nrf-2的蛋白表達,增強鈣化細胞中Keap-1、OPN和Runx-2的蛋白表達。而SFN激動Nrf-2及NAC抑制ROS后,鈣化細胞中Nrf-2的蛋白表達增加,Keap-1、OPN和Runx-2的蛋白表達下調。4.全反式視黃醛下調Nrf-2后,標記胞內線粒體的Mito-tracker熒光強度減弱。但是SFN或者DMF激動Nrf-2,NAC抑制ROS后,標記胞內線粒體的Mito-tracker熒光強度增強,SFN或者DMF激動Nrf-2后鈣化細胞內ROS的含量減弱,細胞線粒體膜電位增加。結論:Nrf-2可調控ROS抗氧化應激,改善線粒體的氧化應激損傷,進而抑制血管平滑肌細胞成骨樣變,以達到阻止終末期腎病血管鈣化進展的作用,為防治終末期腎病血管鈣化提供新的防治靶點與策略。
[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 (偽 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.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692.5

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相關碩士學位論文 前1條

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本文編號：1625026