本文選題：氧化應(yīng)激　切入點(diǎn)：年齡相關(guān)性黃斑變性　出處：《第四軍醫(yī)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：年齡相關(guān)性黃斑變性（age-related macular degeneration，AMD）是一種嚴(yán)重?fù)p害老年人視力的進(jìn)行性疾病，通常雙眼先后發(fā)病，多發(fā)生于50歲以上老年人。主要表現(xiàn)為視物變形、中心暗點(diǎn)、對比度下降以及視野縮小等，，早期眼底多表現(xiàn)為色素紊亂和玻璃膜疣生成，一般不引起視力變化；晚期眼底新生血管生成，引起視力下降。迄今為止，關(guān)于AMD發(fā)病機(jī)制進(jìn)行了大量的基礎(chǔ)以及臨床研究，但發(fā)病機(jī)制并不十分清楚，多數(shù)情況下認(rèn)為其是一種多因素疾病，受到環(huán)境、個(gè)體因素等多方面影響，同時(shí)與基因相關(guān)，因此也缺乏有效治療方法。近年來，關(guān)于氧化應(yīng)激在AMD發(fā)病過程中的作用日益研究深入。 氧化應(yīng)激是指機(jī)體在遭受各種有害刺激時(shí)，體內(nèi)高活性分子如活性氧自由基(reactive oxygen species，ROS)和活性氮自由基(reactive nitrogenspecies，RNS)產(chǎn)生過多，氧化程度超出氧化物的清除，氧化系統(tǒng)和抗氧化系統(tǒng)失衡，從而導(dǎo)致組織損傷。正常情況下機(jī)體內(nèi)活性氧的產(chǎn)生和清除處于動態(tài)平衡狀態(tài)，對機(jī)體無有害影響。而多種內(nèi)源性及外源性有害刺激可以打破這種平衡，致活性氧大量生成，超過抗氧化系統(tǒng)的清除能力，機(jī)體即形成氧化應(yīng)激狀態(tài)，引起DNA氧化損傷和蛋白質(zhì)的表達(dá)異常，產(chǎn)生細(xì)胞毒效應(yīng)并最終對機(jī)體造成不可逆損害。細(xì)胞凋亡在機(jī)體生長發(fā)育和衰老等許多病理生理過程中起重要作用。觸發(fā)凋亡的因素包括衰老、病毒感染和氧化性損傷等。研究發(fā)現(xiàn)，活性氧所致的氧化應(yīng)激是造成細(xì)胞凋亡的重要環(huán)節(jié)。 視網(wǎng)膜光感受器外節(jié)膜盤富含多不飽和脂肪酸，極易受到氧化損傷的攻擊，而后啟動細(xì)胞溶解鏈發(fā)生。AMD早期，視網(wǎng)膜色素上皮（retinal pigmentepithelium，RPE）細(xì)胞隨年齡增長負(fù)擔(dān)加重，RPE吞噬溶酶體過程中發(fā)生光感受器碎片酶的降解，降解物為未完全消化的不能溶解聚集物形式，稱作脂質(zhì)，脂質(zhì)由不同分子混合而成，包括視網(wǎng)膜誘導(dǎo)復(fù)合物，其中一些具有光誘導(dǎo)性質(zhì)，可增強(qiáng)氧化應(yīng)激損傷，脂質(zhì)堆積在細(xì)胞內(nèi)，干擾RPE細(xì)胞的代謝，造成代謝產(chǎn)物堆積于Bruch膜上，導(dǎo)致氧彌散障礙，誘發(fā)新生血管；而脈絡(luò)膜新生血管（choroidal neovascularization，CNV）中血管內(nèi)皮生長因子（vascular endothelial growth factor,VEGF）主要由RPE產(chǎn)生，參與RPE與脈絡(luò)膜毛細(xì)血管之間的旁分泌信號轉(zhuǎn)導(dǎo)。RPE位于神經(jīng)視網(wǎng)膜和脈絡(luò)膜之間，通過提供營養(yǎng)和代謝廢物來滋養(yǎng)視網(wǎng)膜，成為血視網(wǎng)膜屏障的外層，控制著視網(wǎng)膜下間隙的化學(xué)成分，其頂面細(xì)胞膜與細(xì)胞外基質(zhì)相連，在早期胚胎發(fā)育過程中，RPE自發(fā)轉(zhuǎn)分化為神經(jīng)視網(wǎng)膜組織，在其鄰近的光感受器發(fā)育和維持中起重要作用，光照條件下RPE可產(chǎn)生大量活性氧。 視網(wǎng)膜本身具有對抗氧化應(yīng)激損傷機(jī)制，可有效中和ROS損傷�？寡趸冈诖诉^程中發(fā)揮重要作用，包括超氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽過氧化物酶（glutathione peroxidase, GSH）和谷胱甘肽轉(zhuǎn)硫酶（glutathione S-transferase, GST）等。 基于以上研究，本實(shí)驗(yàn)收集AMD患者臨床血液標(biāo)本，進(jìn)行體內(nèi)抗氧化酶水平的檢測，同時(shí)，在細(xì)胞水平利用H_2O_2模擬體內(nèi)氧化應(yīng)激狀態(tài)，研究抗氧化酶水平的表達(dá)變化情況，觀察H_2O_2對RPE細(xì)胞損傷程度的影響，分析RPE細(xì)胞相關(guān)保護(hù)作用，并對其可能機(jī)制進(jìn)行了初步探討。 其主要結(jié)果如下： 一AMD患者血漿內(nèi)抗氧化酶變化：AMD患者組GST水平（76.96U/L）顯著高于對照組（62.74U/L），而GSH水平（61.38mgGSH/L）低于對照組（81.33mgGSH/L）；SOD在AMD患者組和正常對照組水平分別為64.47U/L和62.53U/L。兩組相比GST和GSH活力具有統(tǒng)計(jì)學(xué)意義（Z=㧟2.082，P=0.037；Z=㧟2.900，P=0.004），兩組間SOD水平差異無統(tǒng)計(jì)學(xué)意義（Z=㧟0.090，P=0.725）。低水平的GSH是AMD患病危險(xiǎn)因素。AMD患者血漿中GST水平升高，提示AMD患者處于氧化應(yīng)激狀態(tài)，GSH水平降低使AMD患者更易受到氧化應(yīng)激損傷的攻擊。 二氧化應(yīng)激狀態(tài)下RPE形態(tài)學(xué)變化：熒光顯微鏡下正常RPE細(xì)胞核呈均勻彌散熒光。經(jīng)過H_2O_2處理后，可觀察到RPE細(xì)胞膜完整，細(xì)胞核濃聚深染，核內(nèi)熒光致密，由勻質(zhì)狀態(tài)固縮成高凝集的點(diǎn)狀結(jié)構(gòu)，出現(xiàn)典型的凋亡形態(tài)。經(jīng)過Vit C預(yù)處理的RPE細(xì)胞，再經(jīng)H_2O_2刺激24h后，核固縮數(shù)目減少，染色質(zhì)邊聚和核碎片減少。 三RPE細(xì)胞活力變化：不同濃度H_2O_2預(yù)處理RPE細(xì)胞24h后，細(xì)胞活性明顯下降，其中，400μmol/L以上濃度H_2O_2刺激后細(xì)胞活力與對照組相比具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；不同濃度Vit C處理RPE細(xì)胞后24h，細(xì)胞活力明顯增加，各濃度組與對照組相比均具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；不同濃度VitC預(yù)處理RPE細(xì)胞4h后，用單濃度H_2O_2繼續(xù)處理RPE細(xì)胞24h，與對照組相比，經(jīng)過150μmol/L以上濃度Vit C處理的細(xì)胞其活性較未處理細(xì)胞活性明顯升高，具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；經(jīng)單濃度Vit C處理RPE細(xì)胞4h后，再對RPE細(xì)胞行不同濃度H_2O_2刺激24h，與對照組相比，經(jīng)過不同濃度Vit C處理的RPE細(xì)胞活性升高明顯。 四氧化應(yīng)激狀態(tài)下RPE細(xì)胞內(nèi)抗氧化酶水平變化：H_2O_2預(yù)處理RPE細(xì)胞24h后，胞內(nèi)SOD、GST和GSH水平均下降；維生素C(vitamine C,Vit C)處理RPE細(xì)胞后胞內(nèi)SOD、GST和GSH水平均升高；經(jīng)Vit C預(yù)處理后再接受H_2O_2刺激的RPE細(xì)胞胞內(nèi)SOD、GST和GSH水平相較單純H_2O_2刺激后水平升高。 五Vit C保護(hù)作用機(jī)制：Western blot結(jié)果顯示，Vit C和H_2O_2處理細(xì)胞后顯著增加了phospho-ERK1/ERK2水平，而PD98059（ERK1/ERK2抑制劑）則抑制phospho-ERK1/ERK2的上調(diào)。細(xì)胞活力檢測結(jié)果顯示PD98059明顯抑制了Vit C保護(hù)RPE細(xì)胞免受H_2O_2刺激的能力，說明Vit C通過phospho-ERK1/ERK2通路對RPE細(xì)胞起保護(hù)作用。 總之，通過本實(shí)驗(yàn)研究證明，AMD患者血漿中抗氧化酶GST水平升高，患者處于氧化應(yīng)激狀態(tài)，GSH水平降低，降低的GSH水平使AMD患者更易于受到氧化應(yīng)激損傷攻擊，低水平的GSH是AMD發(fā)病的危險(xiǎn)因素；細(xì)胞水平證實(shí)氧化應(yīng)激狀態(tài)下胞內(nèi)三種抗氧化酶活性均下降，經(jīng)過氧化劑Vit C處理的細(xì)胞其胞內(nèi)抗氧化酶活性升高，可有效保護(hù)RPE細(xì)胞免受氧化應(yīng)激損傷引起的凋亡。同時(shí)，GSH活力在血漿中降低而在RPE細(xì)胞胞內(nèi)升高，推測在氧化應(yīng)激狀態(tài)下，GSH僅在眼局部發(fā)揮作用，這一結(jié)果需要更多大量的研究來證實(shí)。在上述實(shí)驗(yàn)研究的基礎(chǔ)上，進(jìn)一步的研究重點(diǎn)在于更多樣本量的觀察研究，包括尋找其可能的保護(hù)機(jī)制及原因，為臨床治療AMD提供新的方法與方向。
[Abstract]:Age related macular degeneration (age-related macular, degeneration, AMD) is a serious injury in the elderly eye disease, usually has eyes disease, occurs in the elderly over the age of 50. Mainly for metamorphopsia, central scotoma, reduced contrast and narrow vision, fundus manifestation of early generation pigment disorders and drusen, generally do not cause the changes of visual acuity; advanced fundus neovascularization, lead to loss of vision. So far, about the pathogenesis of AMD for a large number of basic and clinical research, but the pathogenesis is not very clear, in most cases it is a multifactorial disease, affected by the environment, many individual factors at the same time, effects of genetically related, therefore it lacks effective treatment. In recent years, the role of oxidative stress in the pathogenesis of AMD is studied.
Oxidative stress refers to the body subjected to a variety of harmful stimuli in vivo, highly active molecules such as reactive oxygen free radicals (reactive oxygen, species, ROS) and reactive nitrogen free radicals (reactive nitrogenspecies, RNS) produces too much beyond the oxidation degree of oxide removal, oxidation system and antioxidant system imbalance, leading to tissue damage. Clear and in a state of dynamic balance of active oxygen in the normal circumstances, no harmful effects on the body. And a variety of endogenous and exogenous noxious stimuli can break this balance, causing a large number of active oxygen generation, more than the antioxidant system scavenging ability of the body to form the state of oxidative stress, induced oxidative damage and protein expression of DNA anomaly. Have a cytotoxic effect and eventually cause irreversible damage to the body. Apoptosis in body growth and senescence of many pathophysiological process which plays an important role in The factors that trigger apoptosis include aging, viral infection and oxidative damage. It is found that oxidative stress induced by reactive oxygen species is an important part of apoptosis.
Photoreceptor outer membrane disc is rich in polyunsaturated fatty acids, susceptible to oxidative damage attacks, then start the chain of cell lysis.AMD early, retinal pigment epithelial cells (retinal pigmentepithelium, RPE) with age increasing the burden of RPE degradation of photoreceptor enzyme fragments phagocytic lysosome in the process of degradation is not complete digestion of insoluble aggregates, called lipid, lipid by different molecular mixture, including the retina induced by complex, some of which have light induced properties, can enhance the oxidative stress injury, lipid accumulation in cells, RPE interference cell metabolism, resulting in accumulation of metabolites in the Bruch membrane, leading to oxygen diffusion disorder, induced neovascularization; and choroidal neovascularization (choroidal neovascularization, CNV) in the vascular endothelial growth factor (vascular endothelial growth factor, and VEGF) To be produced by RPE, in between RPE and choriocapillary paracrine signal transduction of.RPE in neural retina and choroid, by providing nutrition and metabolic wastes to nourish the retina, become the outer blood retinal barrier, to control the chemical composition of the subretinal space, the top surface of the cell membrane and the extracellular matrix in. Early embryogenesis, RPE spontaneously transdifferentiate into neural retinal tissue, play an important role in the development and maintenance of the adjacent photoreceptors, the light conditions RPE can produce large amounts of reactive oxygen species.
The retina itself has a mechanism against oxidative damage, can effectively neutralize the injury. ROS play an important role in the process of antioxidant enzymes, including superoxide dismutase (superoxide dismutase SOD), glutathione peroxidase (glutathione peroxidase GSH) and glutathione S-transferase (glutathione S-transferase, GST).
Based on the above research, we collected the blood samples of patients with AMD, detection, in vivo antioxidant levels at the same time, at the cellular level using the H_2O_2 simulation of oxidative stress, expression of antioxidant enzyme levels, the effect of H_2O_2 on RPE cell damage, analysis of protective effect of RPE cells, and the possible the mechanism was discussed.
The main results are as follows:
The changes of antioxidant enzyme AMD in the plasma of AMD patients with GST level (76.96U/L) was significantly higher than the control group (62.74U/L), while the level of GSH (61.38mgGSH/L) lower than that of the control group (81.33mgGSH/L); SOD patients in AMD group and normal control group respectively 64.47U/L and 62.53U/L. two were compared with GST and GSH activity were statistically significant (Z=? 2.082, P=0.037; Z=? 2.900, P=0.004), there was no significant difference in SOD levels between the two groups (Z=? 0.090, P=0.725). Low levels of GSH are elevated plasma GST levels in patients with.AMD risk factors of AMD, suggesting that AMD patients in the state of oxidative stress, the decrease of GSH level in the patients with AMD more easily under oxidative stress attack.
Changes of RPE morphology stress two oxidation under the fluorescent microscope, normal RPE nuclei were uniformly dispersed fluorescence. After treatment with H_2O_2 can be observed in RPE cell membrane integrity, cell nucleus concentrated anachromasis nucleus by fluorescence, dense, homogeneous condensation into dot structure high agglutination, appeared typical apoptotic morphology after. Vit C pretreatment of RPE cells stimulated by H_2O_2, then 24h, karyopyknosis number decreased, chromatin condensation and nuclear fragmentation.
Changes in three RPE cell viability: different concentrations of H_2O_2 pretreated RPE cells after 24h cell activity decreased significantly, the cell viability was statistically significant compared with the control group of more than 400 mol/L concentration after H_2O_2 stimulation (P < 0.05); different concentrations of Vit C in RPE cells after treatment with 24h, significantly increased the cell viability, each concentration group and the control group were statistically significant compared (P < 0.05); different concentrations of VitC pretreated RPE cells after 4h, with a single concentration of H_2O_2 treated RPE cells to 24h, compared with the control group, after more than 150 mol/L concentration of Vit cells treated with C activity compared with the untreated cells were significantly increased, with statistical significance (P < 0.05); the concentration of Vit C single RPE cells treated with 4h, then to RPE cells were stimulated by different concentration of H_2O_2 24h, compared with the control group, the activity of RPE cells with different concentrations of Vit and C treatment increased significantly.
Antioxidant levels of RPE cells in oxidative stress four H_2O_2 pretreatment of RPE cells after 24h, intracellular SOD, GST and GSH levels were decreased; vitamin C (vitamine C, Vit C) SOD in cells after treatment of RPE cells, GST and GSH levels were elevated by Vit; after receiving C pretreatment H_2O_2 stimulated RPE cells SOD, GST and GSH levels compared to elevated levels only after H_2O_2 stimulation.
Five Vit C protection mechanism: Western blot results showed that Vit, C and H_2O_2 cells after treatment significantly increased the level of phospho-ERK1/ERK2, PD98059 (ERK1/ERK2 inhibitor) inhibited the upregulation of phospho-ERK1/ERK2. Cell viability assay indicated that PD98059 significantly inhibited the Vit C protect RPE cell from the ability of H_2O_2 to stimulate Vit C protective effect on RPE cells through the phospho-ERK1/ERK2 pathway.
In short, through this experiment, antioxidant enzymes increased plasma GST levels in patients with AMD, patients in the state of oxidative stress, the decrease of GSH level, reduce the level of GSH AMD were more susceptible to oxidative stress damage attacks, low levels of GSH are the risk factors of AMD; the cell level confirmed that oxidative stress in cells three anti oxidase activity decreased after Vit cells treated with C oxidant antioxidant enzyme activity in the cells increased, which can effectively protect RPE cells from apoptosis induced by oxidative stress. At the same time, the GSH activity decreased in plasma increased in RPE cells, presumably under the state of oxidative stress, GSH only in the eyes of local play a role in this result need more studies to confirm. Based on the above experimental research, further research is focused on more observations of the sample size, including the search for its possible The protection mechanism and causes provide new methods and directions for the clinical treatment of AMD.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.5

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