氧化应激对SH-SY5Y细胞中铁调节蛋白IRP2的调控机制研究

发布时间：2018-05-18 18:24

本文选题：铁 + IRP2　；参考：《青岛大学》2017年硕士论文

【摘要】：帕金森病(Parkinson’s disease,PD)是一种常见的多发于中老年人的中枢神经系统退行性疾病,其主要病理改变为黑质多巴胺(dopamine,DA)能神经元的变性死亡以及残存神经元内路易小体的形成。目前,PD的发病机制仍不清楚。氧化应激作为自由基在体内产生的一种负面作用,被认为是导致PD的一个重要原因,其通过多种途径引起黑质DA能神经元的死亡。神经影像学检查以及尸检病理报告指出,PD病人早期铁选择性聚集在黑质,残存的DA能神经元内铁含量增高,表明铁代谢调控异常是导致PD中DA能神经元损伤的一个关键因素。细胞内铁稳态的维持,依赖于胞浆中的铁调节蛋白(iron regulatory proteins,IRPs),其通过与铁代谢相关蛋白m RNA上的铁反应元件(iron regulatory elements,IRE)结合,调节这些蛋白的表达水平,从而维持细胞内的铁稳态。IRPs分为IRP1和IRP2两种形式,尽管IRP1和IRP2在机体广泛表达,IRP1主要表达在外周组织,而IRP2主要表达在中枢神经系统。IRP2-/-小鼠在6月龄时出现神经退行性改变,黑质、小脑、海马和尾核和白质有明显的铁沉积,而IRP1-/-小鼠不出现神经系统的异常表现,说明IRP2是调控脑铁代谢稳态的关键蛋白。但是,目前关于神经系统IRP2的表达调控机制,与细胞氧化应激损伤的关系,研究极少。对于该问题的阐明,将对了解PD黑质的铁聚集机制和发病机制提供新的思路。IRP2的蛋白表达调控途径有多种,包括转录水平、转录后水平、翻译水平和翻译后修饰水平,其中翻译后蛋白修饰是维持其稳定的主要途径,而泛素化修饰在蛋白质降解过程中发挥重要的作用。靶蛋白的泛素化需要特异的泛素连接酶(ubiquitin ligase,E3),E3通过识别和结合特异的靶蛋白序列特异性地调节靶蛋白的降解代谢,经E3泛素化修饰的靶蛋白一般被26S蛋白酶体识别并降解。研究发现F-盒富含亮氨酸重复蛋白5(F-box and leucine rich repeat protein 5,FBXL5)是一种铁和氧依赖的E3泛素连接酶,在铁和氧充足时其活性和稳定性增强,催化IRP2的泛素化降解。但是,在PD中IRP2的表达变化是否与FBXL5相关尚不清楚。针对这一问题,本实验在SH-SY5Y细胞上,应用噻唑兰(methylthiazolyldiphenyl-tetrazolium bromide,MTT)观察细胞活力的变化,流式细胞术检测细胞内活性氧类物质(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,ΔΨm)的改变,实时荧光定量PCR检测FBXL5和IRP2的m RNA表达水平,免疫印迹法检测FBXL5和IRP2的蛋白表达水平,激光共聚焦显微镜技术检测细胞摄铁功能的变化,观察不同的氧化应激刺激,如H_2O_2、柠檬酸铁胺(ferric ammonium citrate,FAC)和硝普钠(sodium nitroprusside,SNP),对IRP2蛋白表达水平的影响,并初步探讨其机制。研究结果如下:1.SH-SY5Y细胞经不同浓度的H_2O_2处理24 hrs后,应用MTT方法检测细胞存活率,结果显示细胞存活率随H_2O_2浓度的升高而降低,与对照组相比,20、30、50、200、300、500和1000μmol/L H_2O_2处理组细胞存活率分别下降10.2%、12.9%、13.7%、15.3%、21.2%、33.6%和38.6%,差别具有统计学意义(P0.05)。2.200μmol/L和300μmol/L H_2O_2处理SH-SY5Y细胞24 hrs后,细胞内ROS水平分别升高了52.2%和87.3%,与对照组相比,差别具有高度统计学意义(P0.01)。细胞的ΔΨm分别降低了1.7%和8.2%,与对照组相比,300μmol/L H_2O_2处理组差别具有统计学意义(P0.05)。3.200μmol/L和300μmol/L H_2O_2处理SH-SY5Y细胞24 hrs,FBXL5的蛋白表达水平分别增高了20%和31%,IRP2的蛋白表达水平分别降低了44%和75%,与对照组相比,差别具有统计学意义(P0.05),而FBXL5和IRP2的m RNA水平无明显变化(P0.05)。应用泛素-蛋白酶体抑制剂MG132预孵育后,200μmol/L和300μmol/L H_2O_2处理组细胞的FBXL5蛋白水平无明显变化,而IRP2的蛋白水平分别升高了49%和28%,差别具有统计学意义(P0.05)。应用si-FBXL5预孵育后,200μmol/L和300μmol/L H_2O_2处理组细胞24 hrs,FBXL5蛋白水平分别降低86%和101%,而IRP2的蛋白水平分别升高39%和26%,差别具有统计学意义(P0.05)。4.200μmol/L和300μmol/L H_2O_2处理SH-SY5Y细胞24 hrs,给予100μmol/L的Fe2+灌流,应用激光共聚焦显微镜观察细胞摄铁能力的变化,结果发现细胞内的荧光强度随时间而增强,说明细胞对Fe2+的摄取能力减弱,差别具有统计学意义(P0.05)。5.SH-SY5Y细胞经不同浓度的FAC处理24 hrs后,MTT结果显示,10、30和50μg/m L FAC处理组细胞存活率无明显变化,100、300、500和1000μg/m L处理组细胞存活率随FAC浓度的升高而降低,与对照组相比,分别下降14.3%、26%、29.4%和38.5%,差别具有统计学意义(P0.05)。10μg/m L和100μg/m L FAC处理SH-SY5Y细胞24 hrs后,细胞内ROS含量分别升高63.3%和94.8%,与对照组相比,差别具有高度统计学意义(P0.01)。细胞的ΔΨm分别降低了12%和27%,与对照组相比,差别具有统计学意义(P0.05)。细胞内FBXL5的蛋白水平分别升高了21%和37%,而IRP2的蛋白水平分别降低了28%和45%,差别具有统计学意义(P0.05)。6.SH-SY5Y细胞经不同浓度的SNP处理24 hrs后,MTT结果显示,10、30、50和100μmol/L处理组细胞存活率无明显变化,300和500μmol/L组细胞存活率随SNP浓度的升高而降低,与对照组相比,分别下降21.9%和42%,差别具有统计学意义(P0.05)。100μmol/L和300μmol/L SNP处理SH-SY5Y细胞24 hrs后,细胞内ROS含量分别升高了58.6%和82.3%,与对照组相比,差别具有高度统计学意义(P0.01)。细胞的ΔΨm分别降低了43%和60%,与对照组相比,差别具有统计学意义(P0.05)。细胞内FBXL5的蛋白水平分别上升了29%和40%,IRP2的蛋白水平分别降低了13%和47%,差别具有统计学意义(P0.05)。上述实验结果表明,不同的氧化应激刺激,如H_2O_2、FAC和SNP,均可导致SH-SY5Y细胞内ROS水平升高,线粒体膜电位下降,造成细胞内的氧化应激状态,进一步引起FBXL5蛋白表达水平的升高,而IRP2蛋白表达水平降低,最终导致细胞摄铁能力的降低。而FBXL5和IRP2的m RNA水平均未变化,说明IRP2蛋白水平的降低并非由转录水平的因素影响的。而泛素-蛋白酶体途径的抑制剂MG132处理后,FBXL5蛋白表达量不变,而IRP2蛋白表达量明显升高,si-FBXL5处理后,FBXL5蛋白表达量显著降低,而IRP2蛋白表达量明显升高,说明MG132阻断了IRP2泛素化降解过程,此过程是由FBXL5介导的。本研究表明,氧化应激因素可以作用于泛素化修饰途径,从而影响IRP2的蛋白表达水平,为进一步深入揭示IRP2的中枢调控机制和在PD发病机制中的作用提供了新的实验依据。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a common degenerative disease of the central nervous system that frequently occurs in the middle and old age. The main pathological changes are the degeneration of dopamine (dopamine, DA) neurons and the formation of the Louis corpuscle in the remaining neurons. At present, the pathogenesis of PD is still unclear. Oxidative stress is taken as a self. A negative effect produced by the base in the body is considered to be an important cause of PD, which causes the death of the substantia nigra DA neurons through a variety of pathways. Neuroimaging and autopsy pathological reports indicate that early iron selective aggregation in the PD patients was in the substantia nigra, and the iron content in the remaining DA neurons was increased, indicating the regulation of iron metabolism. Abnormality is a key factor in the damage to DA neurons in PD. The maintenance of intracellular iron homeostasis depends on the iron regulatory protein (iron regulatory proteins, IRPs) in the cytoplasm, which regulates the expression level of these proteins by binding to the iron reaction element (iron regulatory elements, IRE) on the iron metabolism related protein M RNA. The iron homeostasis.IRPs is divided into two forms of IRP1 and IRP2. Although IRP1 and IRP2 are widely expressed in the body, IRP1 is mainly expressed in the peripheral tissue, while IRP2 is mainly expressed in the central nervous system of.IRP2-/- mice at 6 month old, and the substantia nigra, cerebellum, sea horse and caudal nucleus and white matter have obvious iron deposition, and IRP1-/- is small. The abnormal expression of the nervous system shows that IRP2 is the key protein to regulate the homeostasis of cerebral iron metabolism. However, few studies have been made about the relationship between the regulation mechanism of the expression of IRP2 in the nervous system and the damage of oxidative stress in cells. The clarifying of this problem will provide a new idea for understanding the mechanism of iron aggregation and pathogenesis of PD substantia nigra,.IR There are many ways to regulate the expression of protein in P2, including transcriptional level, post transcriptional level, translation level and post translation modification level, in which post-translational protein modification is the main way to maintain its stability, and ubiquitination plays an important role in the process of protein degradation. The ubiquitination of target egg white requires specific ubiquitin ligase (Ubiquiti N ligase, E3), E3 regulates the Degradation Metabolism of target proteins by identifying and combining specific target protein sequences, and the target proteins modified by E3 generally are identified and degraded by 26S proteasome. The study found that F- boxes are rich in leucine repeat protein 5 (F-box and leucine rich repeat) is a kind of iron and oxygen dependence. The activity and stability of the protein ligase, when iron and oxygen is sufficient, are enhanced to catalyze the ubiquitination of IRP2. However, it is not clear whether the expression of IRP2 in PD is related to FBXL5. In this experiment, the changes of cell vitality were observed on SH-SY5Y cells by using methylthiazolyldiphenyl-tetrazolium bromide (MTT). Flow cytometry was used to detect the levels of reactive oxygen species (ROS) and the changes of mitochondrial membrane potential (mitochondrial membrane potential, delta m). Real-time fluorescence quantitative PCR was used to detect the expression level of FBXL5 and IRP2 m, and the level of protein expression was detected by Western blot. Laser confocal microscope technique was used to detect the expression level of FBXL5 and IRP2. The changes in cell uptake were detected, and the effects of different oxidative stress stimuli such as H_2O_2, ferric ammonium citrate (FAC) and sodium nitroprusside (sodium nitroprusside, SNP) on the expression of IRP2 protein were investigated and its mechanism was preliminarily discussed. The results are as follows: 1.SH-SY5Y cells should be treated with 24 hrs by different concentrations of H_2O_2. The cell survival rate was detected by MTT method. The results showed that the cell survival rate decreased with the increase of H_2O_2 concentration. Compared with the control group, the cell survival rate of 20,30,50200300500 and 1000 mu mol/L H_2O_2 treatment group decreased by 10.2%, 12.9%, 13.7%, 15.3%, 21.2%, 33.6% and 38.6%, and the difference was statistically significant (P0.05).2.200 u mol/L and 300 u mol/L H_2. After O_2 treatment of SH-SY5Y cells 24 hrs, the intracellular ROS level increased by 52.2% and 87.3% respectively. Compared with the control group, the difference had a high statistical significance (P0.01). The cell delta m decreased by 1.7% and 8.2% respectively. Compared with the control group, the difference of the 300 mol/L H_2O_2 processing group was of unified planning significance (P0.05).3.200, mol/L and 300 micron mol/L. H-SY5Y cells 24 hrs, FBXL5 protein expression levels increased by 20% and 31%, IRP2 protein expression levels decreased by 44% and 75%, respectively, compared with the control group, the difference was statistically significant (P0.05), and FBXL5 and IRP2 m RNA level no significant changes (P0.05). Application of the ubiquitin proteasome inhibitor MG132 incubation, 200 mu mol/L and 300 micron mol/L The level of FBXL5 protein in /L H_2O_2 treatment group had no obvious change, while the protein level of IRP2 increased by 49% and 28%, respectively. The difference was statistically significant (P0.05). After si-FBXL5 preincubation, 200 mu mol/L and 300 u mol/L H_2O_2 treated group cells 24 hrs, FBXL5 protein level decreased 86% and 101%, while IRP2 protein levels increased 39%, respectively. And 26%, the difference was statistically significant (P0.05).4.200 mu mol/L and 300 mol/L H_2O_2 treatment SH-SY5Y cells 24 hrs, giving Fe2+ perfusion of 100 mu mol/L, using laser confocal microscope to observe the change of cell iron uptake. The results showed that the fluorescence intensity in the cells increased with time, indicating that the uptake ability of the cells to Fe2+ was weakened, and the difference has a difference. After P0.05.5.SH-SY5Y cells were treated with different concentrations of FAC for 24 hrs, MTT results showed that there was no obvious change in the survival rate of 10,30 and 50 g/m L FAC treatment groups. The cell survival rate of 100300500 and 1000 micron g/m L treated groups decreased with the increase of FAC concentration, and decreased by 14.3%, 26%, 29.4% and 38.5% respectively compared with the control group. P0.05.10 mu g/m L and 100 mu g/m L FAC treated SH-SY5Y cells after 24 hrs, the intracellular ROS content increased by 63.3% and 94.8% respectively. Compared with the control group, the difference had a high statistical significance (P0.01). The white level increased by 21% and 37% respectively, while the protein levels of IRP2 decreased by 28% and 45% respectively. The difference was statistically significant (P0.05).6.SH-SY5Y cells were treated with different concentrations of SNP for 24 hrs. MTT results showed that the survival rate of cells in 10,30,50 and 100 mu mol/L treatment group was not significantly changed, and the cell survival rate in 300 and 500 micron groups was increased with SNP concentration. Higher and lower, compared with the control group, the decrease was 21.9% and 42% respectively. The difference was statistically significant (P0.05).100 mol/L and 300 u mol/L SNP treated SH-SY5Y cells 24 hrs, the intracellular ROS content increased by 58.6% and 82.3%, respectively, and the difference was highly statistically significant compared with the control group (P0.01). The difference was statistically significant (P0.05). The protein levels of intracellular FBXL5 increased by 29% and 40% respectively, and the protein levels of IRP2 decreased by 13% and 47% respectively. The differences were statistically significant (P0.05). The results showed that different oxidative stress stimuli, such as H_2O_2, FAC and SNP, could lead to the increase of ROS levels in SH-SY5Y cells. The decrease of mitochondrial membrane potential causes the oxidative stress in cells, which further induces the increase of FBXL5 protein expression level, and the decrease of IRP2 protein expression level, which eventually leads to the decrease of cell iron uptake ability, while the m RNA level of FBXL5 and IRP2 is not changed, which indicates that the decrease of IRP2 protein level is not influenced by the factors of transcription level. After the treatment of the ubiquitin proteasome pathway inhibitor MG132, the expression of FBXL5 protein was unchanged, and the expression of IRP2 protein was significantly increased. After si-FBXL5 treatment, the expression of FBXL5 protein was significantly reduced, and the expression of IRP2 protein was significantly increased, indicating that MG132 blocked the ubiquitination of IRP2, which was mediated by FBXL5. This study showed that oxidation was mediated. Stress factors can affect the ubiquitination modification pathway and affect the protein expression level of IRP2. It provides a new experimental basis for further revealing the central regulatory mechanism of IRP2 and its role in the pathogenesis of PD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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