【作者】 施建婷；

【导师】 赵政；

【作者基本信息】 华东师范大学 ， 基因组学， 2009， 硕士

【摘要】 钙离子对神经元发挥基本功能起关键作用,包括对轴突生长和突触发生的调节、突触传递与可塑性以及细胞存活,但是过高的胞内钙水平对细胞是有害的。在人的衰老过程中,尤其在神经变性疾病病理过程中,细胞内钙调节系统失常可导致突触机能障碍、神经可塑性受损和产生氧化应激,从而导致神经元变性和凋亡。氧化应激可导致机体能量代谢异常和疾病相关蛋白的聚集,这反过来又影响钙稳态,它能通过钙离子通道的开启引发胞外钙内流和内质网的钙释放。年龄依赖性的神经元机能衰退与细胞膜上、内质网和线粒体中钙调相关蛋白的改变有着紧密的联系。衰老对神经元钙调节的损害程度与遗传因素和环境因素有关。深入了解衰老过程中氧化应激和钙稳态失调的细胞和分子机制,对人们最终找到治疗诸如阿尔茨海默、帕金森等神经退行性疾病的新方法有重要意义。1.钙稳态变化与氧化应激作用关系的研究为了研究氧化应激对钙稳态的影响,我们使用SH-SY5Y细胞及H₂O₂建立氧化应激模型,用Fluo-4钙离子荧光染料和Flexstation转液式荧光检测系统检测钙流的变化。结果发现H₂O₂可以诱导SH-SY5Y细胞钙离子内流以及内钙库钙离子的释放,钙流的大小与H₂O₂的浓度成正比;这种由氧化应激诱导的胞内钙稳态变化可在抗氧化剂L-抗坏血酸（维生素C）存存时得以缓解和改善。另外,对本所在研的姜黄素（CCM）及其一个结构衍生物（CCM-2）的研究表明,它们可以在保护细胞减轻氧化应激引起的损伤的同时,显著抑制H₂O₂诱导的钙流变化;而EUK4010则对氧化应激引起的钙流没有明显的抑制作用。2.PS敲除AD模型小鼠氧化应激状态研究早老素1（PS1）和早老素2（PS2）前脑条件性双敲除的阿尔茨海默病模型鼠,没有Aβ沉积,但却表现出认知能力减退、大脑萎缩、脑室扩大等神经退行性病变。氧化应激一直被认为在Aβ沉积的AD病理过程中起着重要作用,但是其在PS功能缺失诱导的AD中的机理尚未阐明,而PS蛋白在细胞内质网形成钙离子泄漏通道,同时钙离子水平的升高也可以间接产生氧化应激,所以为了了解氧化应激机制在PS功能缺失导致的AD模型中的病理作用,我们研究了4、8、12、16月龄的PS cDKO、PS1 cKO、PS2 KO小鼠与同年龄同性别的正常小鼠大脑皮层、海马和血浆中iPF_2α-Ⅲ（脂质过氧化指标）水平。结果表明PS敲除小鼠皮层、海马和血浆中iPF_2α-Ⅲ水平有显著升高,且变化呈现性别差异和年龄依赖性,PS cDKO小鼠iPF_2α-Ⅲ增高水平与单敲小鼠iPF_2α-Ⅲ增高水平显著相关,提示PS1、PS2都参与了氧化应激的产生。另外,在对12月龄的小鼠皮层和海马的α亚型钙/钙调蛋白依赖性蛋白激酶Ⅱ（α-CaMKII）的免疫印迹分析中发现PS cDKO小鼠的α-CaMKII表达有明显上调,其诱因可能与该基因型小鼠体内钙稳态失调有关,此结果与AD致病机理中的钙离子假说相一致。以上结果提示氧化应激,钙稳态失调以及它们的作用关系在PS功能缺失AD的病理变化过程中扮演者重要的角色。3.EUK4010对PS cDKO小鼠脂质过氧化的抑制作用研究EUK4010作为一种中药提取物中筛选出的天然酮类单体化合物。本研究所前期的研究发现该化合物对Aβ诱导的神经元凋亡有明显的抑制作用。为进一步了解EUK4010是否与PS cDKO小鼠神经变性的氧化应激病理学机理有关,本研究测定分析了喂食6个月EUK4010的6月龄cDKO小鼠大脑皮层和血浆中iPF_2α-Ⅲ水平,发现EUK4010对cDKO小鼠体内氧化应激状态无显著改善作用,提示其抑制Aβ诱导的神经元凋亡的作用可能与氧化应激通路无关。更多还原

【Abstract】 Calcium(Ca²⁺)plays critical role in fundamental functions of neurons,from the regulation of neurite outgrowth,synaptogenesis,synaptic transmission and plasticity to cell survival.Although it is essential for neurons,excessively high level of intracellular Ca²⁺is toxic to the cell.During aging,and especially in neurodegenerative disorders,cellular Ca²⁺homeostasis is interrupted,which leads to synaptic dysfunction,impaired plasticity and oxidative stress.All these factors contribute to neuronal degeneration and apoptosis.Oxidative stress,resulting in metabolic disorder of energy and aggregation of disease-related proteins,adversely affect Ca²⁺homeostasis in return.It causes Ca²⁺fluxes into the cytoplasm from the extracelluar environment and from the endoplasmic reticulum through the Ca²⁺ channels.Alterations of Ca²⁺-regulating proteins in the plasma membrane, endoplasmic reticulum,and mitochondria are involved in age-related neurodegeneration.The harm of aging on neuronal Ca²⁺regulation are subject to genetic and environmental factors that may cause or affect the risk of neurodegenerative disease.A good understanding of the cellular and molecular mechanisms of oxidative stress and cellular Ca²⁺dyshomeostasis during aging may be helpful to find novel therapies in neurological disorders such as Alzheimer’s and Parkinson’s diseases.1.The Relationship of Calcium Homeostasis and Oxidative Stress in VitroTo investigate the influence of oxidative stress on the calcium homeostasis,we used a model of oxidative stress on SH-SY5Y cells with H₂O₂.Fluo-4 calcium indicator and Flexstation were used to detect intracellular calcium flux.As a result, H₂O₂ caused Ca²⁺influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum（ER）,depending on the concentration of H₂O₂.And L-ascorbic acid was proved of being effective to reduce the Ca²⁺influx caused by H₂O₂.Synthesized curcumin（CCM）and one of its derivatives（CCM-2）were studied as well.The results showed that these compounds could significantly protect the cells from intracellular Ca²⁺increase caused by H₂O₂,while another natural compound, EUK4010,was demonstrated to have no such effect on Ca²⁺homeostasis.2.Oxidative Stress in PS Knockout Mouse Model of ADForebrain-specific and conditional presenilin-1（PS1）and presenilin-2（PS2） double knock-out mouse exhibits some usual hallmarks of Alzheimer’s disease（AD）, such as progressive memory dysfunction,forebrain degeneration and ventricle enlargemen,without the accumulation of Aβ.Oxidative stress mechanism has been long implicated predominantly in amyloidosis-mediated AD pathologies;however,its role in the pathogenic mechanism of loss-of-function of PS in AD remains unclear.It is believed that presenilins form ER Ca²⁺leak channels and elevated Ca²⁺levels in the cytoplasm can also indirectly cause oxidative stress.To identify and understand oxidative imbalance as an important mechanism of AD in response to PS loss-of-function,we examined oxidative stress status,using F₂-isoprostanes(iPF_2α-Ⅲ) as the marker of lipid peroxidation in vivo,in both brain tissues and circulation of 4-, 8-,12-,and 16-month PS cDKO,PS1 cKO,PS2 KO and the age-,gender-matched wide-type control mice（WT）.Enhanced lipid peroxidation was occurred in a genderand age-related manner in PS KO mice,and the enhancement of iPF_2α-Ⅲin PS cDKO mice are dependent on both PS1 and PS2 deficiency.Western blot analysis of a-CaMKII revealed a significant increase of this protein in the brain of 12-month-old PS cDKO mice,implicating an involvement of calcium dyshomeostasis in the PS loss-of-function mechanism.In conclusion,the interaction of oxidative stree and calcium dyshomeostasis might have important role in response to the loss-of-function of PS in AD pathogenesis.3.The Effect of EUK4010 on Lipid Peroxidation in PS cDKO miceEUK4010 is a natural compound isolated from a herb,which has been used frequently in China for the treatment of cardiovascular diseases.EUK4010 has been identified to exhibit an inhibitory effect on beta-amyloid（Aβ）-induced loss of neuronal cell viability.To investigate if there is an anti-oxidative mechanism for EUK4010 in protecting neurons from neurodegeneration in PS cDKO mice,we administrated the 6-month-old PS cDKO mice with EUK4010 for 6 months and iPF_2α-Ⅲwas detected in both cerebral cortex and circulation after treatment.The results showed that there was no significant difference in production of iPF_2α-Ⅲbetween the EUK4010-treated animals and their controls,suggesting that neuronal pharmacological protecting role of EUK4010 might be not oxidative stress-related.更多还原