氧化应激在年龄相关性黄斑变性中的作用

发布时间：2018-03-17 11:10

本文选题：氧化应激　切入点：年龄相关性黄斑变性　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：年龄相关性黄斑变性（age-related macular degeneration，AMD）是一种严重损害老年人视力的进行性疾病，通常双眼先后发病，多发生于50岁以上老年人。主要表现为视物变形、中心暗点、对比度下降以及视野缩小等，，早期眼底多表现为色素紊乱和玻璃膜疣生成，一般不引起视力变化；晚期眼底新生血管生成，引起视力下降。迄今为止，关于AMD发病机制进行了大量的基础以及临床研究，但发病机制并不十分清楚，多数情况下认为其是一种多因素疾病，受到环境、个体因素等多方面影响，同时与基因相关，因此也缺乏有效治疗方法。近年来，关于氧化应激在AMD发病过程中的作用日益研究深入。氧化应激是指机体在遭受各种有害刺激时，体内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)产生过多，氧化程度超出氧化物的清除，氧化系统和抗氧化系统失衡，从而导致组织损伤。正常情况下机体内活性氧的产生和清除处于动态平衡状态，对机体无有害影响。而多种内源性及外源性有害刺激可以打破这种平衡，致活性氧大量生成，超过抗氧化系统的清除能力，机体即形成氧化应激状态，引起DNA氧化损伤和蛋白质的表达异常，产生细胞毒效应并最终对机体造成不可逆损害。细胞凋亡在机体生长发育和衰老等许多病理生理过程中起重要作用。触发凋亡的因素包括衰老、病毒感染和氧化性损伤等。研究发现，活性氧所致的氧化应激是造成细胞凋亡的重要环节。视网膜光感受器外节膜盘富含多不饱和脂肪酸，极易受到氧化损伤的攻击，而后启动细胞溶解链发生。AMD早期，视网膜色素上皮（retinal pigmentepithelium，RPE）细胞随年龄增长负担加重，RPE吞噬溶酶体过程中发生光感受器碎片酶的降解，降解物为未完全消化的不能溶解聚集物形式，称作脂质，脂质由不同分子混合而成，包括视网膜诱导复合物，其中一些具有光诱导性质，可增强氧化应激损伤，脂质堆积在细胞内，干扰RPE细胞的代谢，造成代谢产物堆积于Bruch膜上，导致氧弥散障碍，诱发新生血管；而脉络膜新生血管（choroidal neovascularization，CNV）中血管内皮生长因子（vascular endothelial growth factor,VEGF）主要由RPE产生，参与RPE与脉络膜毛细血管之间的旁分泌信号转导。RPE位于神经视网膜和脉络膜之间，通过提供营养和代谢废物来滋养视网膜，成为血视网膜屏障的外层，控制着视网膜下间隙的化学成分，其顶面细胞膜与细胞外基质相连，在早期胚胎发育过程中，RPE自发转分化为神经视网膜组织，在其邻近的光感受器发育和维持中起重要作用，光照条件下RPE可产生大量活性氧。视网膜本身具有对抗氧化应激损伤机制，可有效中和ROS损伤。抗氧化酶在此过程中发挥重要作用，包括超氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase, GSH）和谷胱甘肽转硫酶（glutathione S-transferase, GST）等。基于以上研究，本实验收集AMD患者临床血液标本，进行体内抗氧化酶水平的检测，同时，在细胞水平利用H_2O_2模拟体内氧化应激状态，研究抗氧化酶水平的表达变化情况，观察H_2O_2对RPE细胞损伤程度的影响，分析RPE细胞相关保护作用，并对其可能机制进行了初步探讨。其主要结果如下：一AMD患者血浆内抗氧化酶变化：AMD患者组GST水平（76.96U/L）显著高于对照组（62.74U/L），而GSH水平（61.38mgGSH/L）低于对照组（81.33mgGSH/L）；SOD在AMD患者组和正常对照组水平分别为64.47U/L和62.53U/L。两组相比GST和GSH活力具有统计学意义（Z=㧟2.082，P=0.037；Z=㧟2.900，P=0.004），两组间SOD水平差异无统计学意义（Z=㧟0.090，P=0.725）。低水平的GSH是AMD患病危险因素。AMD患者血浆中GST水平升高，提示AMD患者处于氧化应激状态，GSH水平降低使AMD患者更易受到氧化应激损伤的攻击。二氧化应激状态下RPE形态学变化：荧光显微镜下正常RPE细胞核呈均匀弥散荧光。经过H_2O_2处理后，可观察到RPE细胞膜完整，细胞核浓聚深染，核内荧光致密，由匀质状态固缩成高凝集的点状结构，出现典型的凋亡形态。经过Vit C预处理的RPE细胞，再经H_2O_2刺激24h后，核固缩数目减少，染色质边聚和核碎片减少。三RPE细胞活力变化：不同浓度H_2O_2预处理RPE细胞24h后，细胞活性明显下降，其中，400μmol/L以上浓度H_2O_2刺激后细胞活力与对照组相比具有统计学意义（P＜0.05）；不同浓度Vit C处理RPE细胞后24h，细胞活力明显增加，各浓度组与对照组相比均具有统计学意义（P＜0.05）；不同浓度VitC预处理RPE细胞4h后，用单浓度H_2O_2继续处理RPE细胞24h，与对照组相比，经过150μmol/L以上浓度Vit C处理的细胞其活性较未处理细胞活性明显升高，具有统计学意义（P＜0.05）；经单浓度Vit C处理RPE细胞4h后，再对RPE细胞行不同浓度H_2O_2刺激24h，与对照组相比，经过不同浓度Vit C处理的RPE细胞活性升高明显。四氧化应激状态下RPE细胞内抗氧化酶水平变化：H_2O_2预处理RPE细胞24h后，胞内SOD、GST和GSH水平均下降；维生素C(vitamine C,Vit C)处理RPE细胞后胞内SOD、GST和GSH水平均升高；经Vit C预处理后再接受H_2O_2刺激的RPE细胞胞内SOD、GST和GSH水平相较单纯H_2O_2刺激后水平升高。五Vit C保护作用机制：Western blot结果显示，Vit C和H_2O_2处理细胞后显著增加了phospho-ERK1/ERK2水平，而PD98059（ERK1/ERK2抑制剂）则抑制phospho-ERK1/ERK2的上调。细胞活力检测结果显示PD98059明显抑制了Vit C保护RPE细胞免受H_2O_2刺激的能力，说明Vit C通过phospho-ERK1/ERK2通路对RPE细胞起保护作用。总之，通过本实验研究证明，AMD患者血浆中抗氧化酶GST水平升高，患者处于氧化应激状态，GSH水平降低，降低的GSH水平使AMD患者更易于受到氧化应激损伤攻击，低水平的GSH是AMD发病的危险因素；细胞水平证实氧化应激状态下胞内三种抗氧化酶活性均下降，经过氧化剂Vit C处理的细胞其胞内抗氧化酶活性升高，可有效保护RPE细胞免受氧化应激损伤引起的凋亡。同时，GSH活力在血浆中降低而在RPE细胞胞内升高，推测在氧化应激状态下，GSH仅在眼局部发挥作用，这一结果需要更多大量的研究来证实。在上述实验研究的基础上，进一步的研究重点在于更多样本量的观察研究，包括寻找其可能的保护机制及原因，为临床治疗AMD提供新的方法与方向。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.5

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