【作者】 李大伟；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2013， 博士

【摘要】 帕金森病（Parkinson’s disease，PD）是一种常见的中枢神经系统变性疾病，以黑质多巴胺能神经元慢性进行性缺失为主要病理特征，最终导致纹状体内多巴胺神经递质耗竭。尽管PD发病机理仍未阐明，但大量研究证实氧化应激在本病发病中起主要作用。活性氧物质和线粒体相互作用触发了线粒体死亡途径，这种方式被认为是PD中氧化应激介导细胞凋亡的主要方式。1-甲基-4-苯基吡啶（1-methyl-4-phenylpyridiniumion, MPP~+）诱导PC12细胞是体外研究PD可靠的细胞模型。PC12细胞是大鼠肾上腺嗜铬细胞瘤细胞，能够产生多巴胺，并具有多巴胺转运系统，可作为细胞模型研究多巴胺能神经元的变性。1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP）是一种神经毒素，可选择性地损害多巴胺能神经元并在人类及哺乳动物中引起类似PD的病理改变及临床症状。MPTP为脂溶性物质，极易透过血脑屏障，在星形胶质细胞内由B型单胺氧化酶催化生成其代谢产物MPP~+，MPP~+具有神经毒性，可选择性地引起多巴胺能神经元变性死亡。MPP~+引起多巴胺能神经元变性的确切机制尚未完全阐明，可能是活性氧物质生成及氧化应激触发了多巴胺能神经元的变性死亡。目前，PD治疗主要以多巴胺替代治疗为主，此类药物虽能缓解PD症状，但并不能控制多巴胺能神经元持续变性。如何有效地抑制多巴胺能神经元持续变性已成为本病治疗的焦点。研究已证实神经系统变性疾病与氧化应激密切相关，减弱氧化应激可产生神经保护作用。硫辛酸又名1,2-二硫戊环-3-戊酸，通过清除活性氧物质及螯合重金属而表现出较强的抗氧化活性。这些作用均有利于改善与氧化应激有关的疾病包括PD。此外，亦有报道证实硫辛酸在一系列细胞及组织中具有抗炎及增加谷胱甘肽(glutathiose，GSH)形成的作用，这对PD亦有积极影响。硫辛酸作为抗氧化应激药物已应用于临床与氧化应激相关疾病的治疗，且治疗剂量副作用很小。为验证α-硫辛酸（α-lipoic acid，LA）是否对MPP~+诱导PC12细胞损伤具有防护作用，我们采用MTT实验测定细胞活力，应用AO/EB染色从凋亡角度分析LA防护MPP~+诱导的PC12细胞凋亡，并通过2’,7’-二氯荧光黄双乙酸盐（2’,7’-dichlorofluorescein-diacetate，DCFH-DA）使用流式细胞仪验证LA对MPP~+诱导PC12细胞活性氧簇（Reactive oxygen species,ROS）生成的影响。接着，我们采用蛋白质组学方法分析LA防护MPP~+诱导PC12细胞损伤的蛋白表达水平变化。结果显示：单独加入LA对细胞活力无明显影响；500μM MPP~+作用72h，PC12细胞活力明显下降；提前1h加入0.01μM LA与损害组相比细胞活力明显增加。凋亡染色亦证实0.01μM LA可显著减少由MPP~+诱导的细胞凋亡，进一步证实了LA可对抗MPP~+的神经毒性作用。结果还显示MPP~+单独作用PC12细胞组2’,7’-二氯荧光黄（2’,7’-dichlorofluorescein，DCF）荧光密度明显增强，提前加入LA可有效抑DCF荧光强度，说明LA可有效抑制MPP~+诱导PC12细胞的ROS生成。应用蛋白质组学方法，成功的鉴定出4个表达具有明显差异的蛋白，分别是增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）、膜联蛋白A2(annexin A2)、鸟嘌呤核苷酸结合蛋白(i)α亚单位(guanine nucleotide-bingdingprotein Gi subunit alpha,Giα)、O-唾液酸糖蛋白内肽酶（O-sialoglycoproteinendopeptidase,OSGEP），从分子水平探讨了LA对MPP~+诱导PC12细胞凋亡作用的保护机制，为PD有效的药物治疗提供了理论基础。更多还原

【Abstract】 Parkinson’s disease (PD) is a common neurodegenerative disorder characterizedby the selective and progressive loss of dopaminergic neurons in the substantia nigra,leading to a depletion of the dopamine neurotransmitter in the striatum. While theunderlying mechanisms by which nigrostriatal dopaminergic neuron degeneration arenot completely understood, accumulating data have suggested that oxidative stressplays a crucial role in dopaminergic cell death in PD. The interaction of ROS withmitochondria triggers sequential events in mitochondrial cell death pathway, which isthought to be responsible for oxidative stress-mediated the cell death in PD. PC12cells treated with MPP~+provide a reliable model for insight into the pathogenesis ofPD. PC12cell is a rat pheochromocytoma cell line which has been shown to producedopamine (DA), and possess a DA uptake system, indicating that these cells can beused as cell culture model for studying the dopaminergic neuron degeneration.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin which selecti-vely kills dopaminergic neurons and causes irreversible parkinsonism-like symptomsin humans and primates. MPTP is lipophilic molecule and can rapidly crossblood-brain barrier. Once crossing the barrier, it is oxidized in the brain to its toxicmetabolite MPP~+by type B monoamine oxidase (MAO-B) and finally lead todopaminergic neuron death. The exact mechanism for this toxic action of MPP~+,although incompletely understood, seems to involve ROS generation and oxidativedamage, which triggering sequential events in apoptosis in dopaminergic neuronalcells.Currently, the main therapeutic approaches for treating PD are limited to thereplacement of dopamine in the brain either by levodopa or by dopamine agonists.Although these drugs can relieve the symptoms of Parkinson’s disease, thedegeneration of dopaminergic neurons is not prevented and still slowly progressive.Promising new concepts in therapy of PD should prevent or reverse the progressivedopaminergic neuron degeneration. Since neurodegenerative diseases are mainlyrelated to oxidative stress, attenuation of oxidative stress levels may result inneuroprotection in these disorders. Lipoic acid, also known as1,2-dithiolane-3-pentanoic acid or thioctic acid, exhibits effective antioxidative activities byscavenging ROS, and inhibits free radical formation by chelating various metal types, indicating that it can exert beneficial effects on various disorders correlated withoxidative stress including PD. In addition, Lipoic acid is also reported to haveanti-inflammatory property and increase intracellular glutathione (GSH) formation ina range of cell types and tissues，which may be of additional benefit in neurode-generative conditions. Importantly, Lipoic acid serving as effective antioxidant hasbeen in common clinical used in several diseases that are associated with increasedproduction of free radicals, and administrated in moderate doses with no evidence ofserious side effects.To determine the ability of α-Lipoic acid (LA) to prevent the cytotoxicity ofMPP~+in PC12cells, cell viability was assayed by MTT、AO/EB assay. Were alsoundertook to determine whether LA protects PC12cells from MPP~+-inducedapoptosis. To investigate the effects LA on ROS levels in MPP~+-treated PC12cells,we evaluated the levels of ROS by flow cytometry with2’,7’-dichlorofluorescein-diacetate (DCFH-DA). Then we investigated the changes of proteins involved in thepathogenesis of PD and the protection of LA in PD model by proteomics analysis.The measurements show that LA administered alone did not induce changes in thecell viability, while the administration of MPP~+greatly reduced the cell viability incomparison with control cells.0.01μM LA showed a significant protective effect onthe cells. Similarly results show that LA administered strongly prevented the numberof apoptotic cells induced by MPP~+, confirming the anti-apoptotic activities of LAagainst the toxicity of MPP~+. Our results also show that exposure of PC12cells toMPP~+alone induced a significant increase of2’,7’-dichlorofluorescein (DCF) fluore-scence levels, which was prevented by a pre-treatment with LA, indicating apreventive role of LA in ROS formation in PC12cells treated by MPP~+. The result ofproteomics analysis show that four protein spots were differentially expressed inlevels. They are proliferating cell nuclear antigen (PCNA), annexin A2, guaninenucleotide-bingding protein Gi subunit alpha(Giα) and O-sialoglycoprotein endopep-tidase (OSGEP). In the present study, we investigate the underlying molecularmechanisms by which LAprotect PC12cells against MPP~+-induced apoptosis，whichmay provide potential effective strategies for PD treatment.更多还原