本文選題：鐵 + IRP2��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：帕金森病(Parkinson’s disease,PD)是一種常見的多發(fā)于中老年人的中樞神經(jīng)系統(tǒng)退行性疾病,其主要病理改變?yōu)楹谫|(zhì)多巴胺(dopamine,DA)能神經(jīng)元的變性死亡以及殘存神經(jīng)元內(nèi)路易小體的形成。目前,PD的發(fā)病機制仍不清楚。氧化應(yīng)激作為自由基在體內(nèi)產(chǎn)生的一種負(fù)面作用,被認(rèn)為是導(dǎo)致PD的一個重要原因,其通過多種途徑引起黑質(zhì)DA能神經(jīng)元的死亡。神經(jīng)影像學(xué)檢查以及尸檢病理報告指出,PD病人早期鐵選擇性聚集在黑質(zhì),殘存的DA能神經(jīng)元內(nèi)鐵含量增高,表明鐵代謝調(diào)控異常是導(dǎo)致PD中DA能神經(jīng)元損傷的一個關(guān)鍵因素。細(xì)胞內(nèi)鐵穩(wěn)態(tài)的維持,依賴于胞漿中的鐵調(diào)節(jié)蛋白(iron regulatory proteins,IRPs),其通過與鐵代謝相關(guān)蛋白m RNA上的鐵反應(yīng)元件(iron regulatory elements,IRE)結(jié)合,調(diào)節(jié)這些蛋白的表達(dá)水平,從而維持細(xì)胞內(nèi)的鐵穩(wěn)態(tài)。IRPs分為IRP1和IRP2兩種形式,盡管IRP1和IRP2在機體廣泛表達(dá),IRP1主要表達(dá)在外周組織,而IRP2主要表達(dá)在中樞神經(jīng)系統(tǒng)。IRP2-/-小鼠在6月齡時出現(xiàn)神經(jīng)退行性改變,黑質(zhì)、小腦、海馬和尾核和白質(zhì)有明顯的鐵沉積,而IRP1-/-小鼠不出現(xiàn)神經(jīng)系統(tǒng)的異常表現(xiàn),說明IRP2是調(diào)控腦鐵代謝穩(wěn)態(tài)的關(guān)鍵蛋白。但是,目前關(guān)于神經(jīng)系統(tǒng)IRP2的表達(dá)調(diào)控機制,與細(xì)胞氧化應(yīng)激損傷的關(guān)系,研究極少。對于該問題的闡明,將對了解PD黑質(zhì)的鐵聚集機制和發(fā)病機制提供新的思路。IRP2的蛋白表達(dá)調(diào)控途徑有多種,包括轉(zhuǎn)錄水平、轉(zhuǎn)錄后水平、翻譯水平和翻譯后修飾水平,其中翻譯后蛋白修飾是維持其穩(wěn)定的主要途徑,而泛素化修飾在蛋白質(zhì)降解過程中發(fā)揮重要的作用。靶蛋白的泛素化需要特異的泛素連接酶(ubiquitin ligase,E3),E3通過識別和結(jié)合特異的靶蛋白序列特異性地調(diào)節(jié)靶蛋白的降解代謝,經(jīng)E3泛素化修飾的靶蛋白一般被26S蛋白酶體識別并降解。研究發(fā)現(xiàn)F-盒富含亮氨酸重復(fù)蛋白5(F-box and leucine rich repeat protein 5,FBXL5)是一種鐵和氧依賴的E3泛素連接酶,在鐵和氧充足時其活性和穩(wěn)定性增強,催化IRP2的泛素化降解。但是,在PD中IRP2的表達(dá)變化是否與FBXL5相關(guān)尚不清楚。針對這一問題,本實驗在SH-SY5Y細(xì)胞上,應(yīng)用噻唑蘭(methylthiazolyldiphenyl-tetrazolium bromide,MTT)觀察細(xì)胞活力的變化,流式細(xì)胞術(shù)檢測細(xì)胞內(nèi)活性氧類物質(zhì)(reactive oxygen species,ROS)水平和線粒體膜電位(mitochondrial membrane potential,ΔΨm)的改變,實時熒光定量PCR檢測FBXL5和IRP2的m RNA表達(dá)水平,免疫印跡法檢測FBXL5和IRP2的蛋白表達(dá)水平,激光共聚焦顯微鏡技術(shù)檢測細(xì)胞攝鐵功能的變化,觀察不同的氧化應(yīng)激刺激,如H_2O_2、檸檬酸鐵胺(ferric ammonium citrate,FAC)和硝普鈉(sodium nitroprusside,SNP),對IRP2蛋白表達(dá)水平的影響,并初步探討其機制。研究結(jié)果如下:1.SH-SY5Y細(xì)胞經(jīng)不同濃度的H_2O_2處理24 hrs后,應(yīng)用MTT方法檢測細(xì)胞存活率,結(jié)果顯示細(xì)胞存活率隨H_2O_2濃度的升高而降低,與對照組相比,20、30、50、200、300、500和1000μmol/L H_2O_2處理組細(xì)胞存活率分別下降10.2%、12.9%、13.7%、15.3%、21.2%、33.6%和38.6%,差別具有統(tǒng)計學(xué)意義(P0.05)。2.200μmol/L和300μmol/L H_2O_2處理SH-SY5Y細(xì)胞24 hrs后,細(xì)胞內(nèi)ROS水平分別升高了52.2%和87.3%,與對照組相比,差別具有高度統(tǒng)計學(xué)意義(P0.01)。細(xì)胞的ΔΨm分別降低了1.7%和8.2%,與對照組相比,300μmol/L H_2O_2處理組差別具有統(tǒng)計學(xué)意義(P0.05)。3.200μmol/L和300μmol/L H_2O_2處理SH-SY5Y細(xì)胞24 hrs,FBXL5的蛋白表達(dá)水平分別增高了20%和31%,IRP2的蛋白表達(dá)水平分別降低了44%和75%,與對照組相比,差別具有統(tǒng)計學(xué)意義(P0.05),而FBXL5和IRP2的m RNA水平無明顯變化(P0.05)。應(yīng)用泛素-蛋白酶體抑制劑MG132預(yù)孵育后,200μmol/L和300μmol/L H_2O_2處理組細(xì)胞的FBXL5蛋白水平無明顯變化,而IRP2的蛋白水平分別升高了49%和28%,差別具有統(tǒng)計學(xué)意義(P0.05)。應(yīng)用si-FBXL5預(yù)孵育后,200μmol/L和300μmol/L H_2O_2處理組細(xì)胞24 hrs,FBXL5蛋白水平分別降低86%和101%,而IRP2的蛋白水平分別升高39%和26%,差別具有統(tǒng)計學(xué)意義(P0.05)。4.200μmol/L和300μmol/L H_2O_2處理SH-SY5Y細(xì)胞24 hrs,給予100μmol/L的Fe2+灌流,應(yīng)用激光共聚焦顯微鏡觀察細(xì)胞攝鐵能力的變化,結(jié)果發(fā)現(xiàn)細(xì)胞內(nèi)的熒光強度隨時間而增強,說明細(xì)胞對Fe2+的攝取能力減弱,差別具有統(tǒng)計學(xué)意義(P0.05)。5.SH-SY5Y細(xì)胞經(jīng)不同濃度的FAC處理24 hrs后,MTT結(jié)果顯示,10、30和50μg/m L FAC處理組細(xì)胞存活率無明顯變化,100、300、500和1000μg/m L處理組細(xì)胞存活率隨FAC濃度的升高而降低,與對照組相比,分別下降14.3%、26%、29.4%和38.5%,差別具有統(tǒng)計學(xué)意義(P0.05)。10μg/m L和100μg/m L FAC處理SH-SY5Y細(xì)胞24 hrs后,細(xì)胞內(nèi)ROS含量分別升高63.3%和94.8%,與對照組相比,差別具有高度統(tǒng)計學(xué)意義(P0.01)。細(xì)胞的ΔΨm分別降低了12%和27%,與對照組相比,差別具有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞內(nèi)FBXL5的蛋白水平分別升高了21%和37%,而IRP2的蛋白水平分別降低了28%和45%,差別具有統(tǒng)計學(xué)意義(P0.05)。6.SH-SY5Y細(xì)胞經(jīng)不同濃度的SNP處理24 hrs后,MTT結(jié)果顯示,10、30、50和100μmol/L處理組細(xì)胞存活率無明顯變化,300和500μmol/L組細(xì)胞存活率隨SNP濃度的升高而降低,與對照組相比,分別下降21.9%和42%,差別具有統(tǒng)計學(xué)意義(P0.05)。100μmol/L和300μmol/L SNP處理SH-SY5Y細(xì)胞24 hrs后,細(xì)胞內(nèi)ROS含量分別升高了58.6%和82.3%,與對照組相比,差別具有高度統(tǒng)計學(xué)意義(P0.01)。細(xì)胞的ΔΨm分別降低了43%和60%,與對照組相比,差別具有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞內(nèi)FBXL5的蛋白水平分別上升了29%和40%,IRP2的蛋白水平分別降低了13%和47%,差別具有統(tǒng)計學(xué)意義(P0.05)。上述實驗結(jié)果表明,不同的氧化應(yīng)激刺激,如H_2O_2、FAC和SNP,均可導(dǎo)致SH-SY5Y細(xì)胞內(nèi)ROS水平升高,線粒體膜電位下降,造成細(xì)胞內(nèi)的氧化應(yīng)激狀態(tài),進(jìn)一步引起FBXL5蛋白表達(dá)水平的升高,而IRP2蛋白表達(dá)水平降低,最終導(dǎo)致細(xì)胞攝鐵能力的降低。而FBXL5和IRP2的m RNA水平均未變化,說明IRP2蛋白水平的降低并非由轉(zhuǎn)錄水平的因素影響的。而泛素-蛋白酶體途徑的抑制劑MG132處理后,FBXL5蛋白表達(dá)量不變,而IRP2蛋白表達(dá)量明顯升高,si-FBXL5處理后,FBXL5蛋白表達(dá)量顯著降低,而IRP2蛋白表達(dá)量明顯升高,說明MG132阻斷了IRP2泛素化降解過程,此過程是由FBXL5介導(dǎo)的。本研究表明,氧化應(yīng)激因素可以作用于泛素化修飾途徑,從而影響IRP2的蛋白表達(dá)水平,為進(jìn)一步深入揭示IRP2的中樞調(diào)控機制和在PD發(fā)病機制中的作用提供了新的實驗依據(jù)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R742.5

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本文編號：1906713