【作者】 李灵敏；

【导师】 张策；

【作者基本信息】 山西医科大学 ， 神经生理学， 2006， 博士

【摘要】 阿尔茨海默病（Alzheimer disease，AD）是一种常见的以进行性记忆减退、认知障碍为主要临床表现的中枢神经系统退行性疾病，病理改变以大脑皮层和海马等脑区的细胞外老年斑形成、细胞内出现神经纤维缠结、神经突触的丢失和神经元大量死亡为特征。关于AD的发病机制说法各异，但均无定论，因而缺乏有效的预防与治疗手段。越来越多的研究表明，β-淀粉样蛋白（β-amyloid protein，Aβ）在脑组织中的过度沉积可能与AD的发病密切相关，是AD形成和发展的关键因素。20世纪90年代初，Yankner等首先观察到较高浓度的Aβ_1-42能引起神经元死亡；之后的研究又证实Aβ_25～35，一个小的Aβ片段具有与完整肽链相类似的毒性作用，因而认为Aβ_25～35可能是Aβ发挥毒性作用的必需结构或活性中心。最近，本实验室和其他学者的工作证实，Aβ_31-35一个更小的肽段，也具有与完整肽链和Aβ_25～35相似的毒性作用，提示这个五肽可能是Aβ中更短的关键性序列结构，在Aβ发挥毒性作用的过程中具有更重要的意义。Stadelmann等（1998）证实了AD发病过程中神经元死亡的主要形式是凋亡，之后的离体实验证实Aβ包括Aβ_1-42、Aβ_25-35及Aβ_31-35都可引起神经元凋亡，因而探讨能抑制Aβ所致的神经元凋亡就成为预防及治疗AD的重要途径。 Humanin（HN）是Hashimoto（2001）报道的存在于AD患者脑组织中具有神经保护作用的多肽类物质。实验证实，HN能有效抑制多种FAD基因和Aβ衍生物诱发的神经元的死亡，并且证实HN可抑制Aβ_1-42引起的原代培养神经元的死亡，但这种抑制机制未见相关报道。由于Aβ_31-35与Aβ_1-42具有相似的毒性作用，那么HN是否也能抑制Aβ_31-35的神经毒性以及作用机制如何?本实验从以下三方面进行观察和分析：1、观察HN对Aβ_31-35致神经元凋亡的影响；2、对HN拮抗Aβ_31-35致凋亡作用的机制进行研究分析；3、观察HN对Aβ_31-35引起的神经元[C_a²⁺]_ⅰ升高的影响。 第一部分 Humanin拮抗Aβ_31-35致凋亡作用的观察 文献报道，HN能够抑制Aβ_1-42的毒性作用。本实验室及其他学者的研究结果显示，Aβ_31-35与Aβ完整肽链具有相似的毒性作用。那么HN是否也能抑制Aβ_31-35的毒性作用?本部分采用原代培养的大鼠大脑皮层神经元观察HN对Aβ_31-35致凋亡作用的影响。 方法：选用≤3天的Wistar大鼠，分离皮层神经元，阿糖胞苷去除胶质细胞纯化神经元，MAP₂鉴定神经元。在加入Aβ_31-35（25μM）同时或提前不同时间（8小时、16小时）加入不同浓度（5μM、10μM、20μM）的HN，再加入Aβ_31-35（25μM）继续培养24小时后，采用电镜观察神经元凋亡的形态学改变；采用流式细胞术、TUNEL法、Hochest 33342染色定更多还原

【Abstract】 Alzheimer’s disease is the most prevalent neurodegenerative disease, characterized by the presence of extensive extracellular amyloid plaques , intracellular neurofibrillary tangles, and synaptic loss, neuronal death in cerebral cortex and hippocampus, along with progressive impairment of cognition and emotional activities. Despite some clinical trials with encouraging results, no curative therapy for this disease has so far been developed. Increasing evidences shown that P-amyloid（Ap） deposition is the leading cause for AD. β-amyloid are major protein component of the amyloid plaques and is involved in the pathogenesis of AD. Subsequent studies have demonstrated that apoptosis is a major form of neuronal death in AD which could be induced by Aβ_1-42、 Aβ_25-35 as well as Aβ_31-35 .Humanin is a recently identified peptide that suppresses cell death initiated by Alzheimer’s disease-related insults in neuronal cells including Aβ_1-42 .induced neuronal death （Hashimoto ,2001）. AβP31-35, an smaller amyloid peptide fragment reported possesses the similar toxicity as full length of Aβ and fragment of Aβ_25-35 do. Thus the part I of this study is to examine whether HN protects neurons from Aβ_31-35-induced apoptosis. The part II is to explore mechanisms of HN against Aβ_31-35-induced apoptosis or to determine if extrinsic and/or intrinsic pathway are involved in neuroprotection of HN. The part III is to observe the effects of HN on elevation of intracellular calcium concentration induced by Aβ_31-35.PART I HN protects cultured neurons against apoptosis induced by Aβ_31-35In the present study we investigated the effects of I IN on neuronal apoptosis induced by Aβ_31-35 in cultured cortical neurons in rats by morphological analysis, flow-cytometric assays, TUNEL examinations and HO 33342 staining .The results showed that: 1. labeling culture cells with MAP₂ （microtubuie-associatedprotein-2, marker protein of neurons）, showed that 90% of culture cells are MAP₂ positive cells namely the neuronal cells 2. Apoptotic neurons induced by Aβ_31-35 presented a typical apoptotic morphology by morphological analysis: the cytoplasm concentrated , the chromatin aggregated peripherally into dense masses and laid against the nuclear membrane , the shape of nucleus became irregular, and finally form nuclear fragments known as apoptotic bodies. 3. We examined whether HN inhibited apoptotic events induced by AβP_31-35（25μM） by flow cytometric assay , TUNEL and HO 33342 staining .The results obtained by flow cytometric assay showed that 1)更多还原