老年痴呆发病机制的研究:APP17肽对β-淀粉样肽导致神经元毒性作用的影响

背景老年斑是老年性痴呆(Alzheimer'sDisease,AD)的主要病理特征之一,以β-淀粉样肽(β-Amyloid,Aβ)沉积为核心,Aβ的神经元毒性定位于Aβ25-35.Aβ前体蛋白(β-amyloidprotein precursor,APP)中319-335肽段即APP17肽(APP17-mer peptide),具有神经营养和神经保护作用.目的通过观察APP17肽对Aβ引起神经毒性作用的影响,进一步证实此肽的神经营养作用,并为阐明AD的发病机制和临床治疗提供理论依据.设计标准对照实验研究.地点和材料本研究的地点为首都医科大学宣武医院神经生化研究室.SY5Y细胞株由瑞典卡罗琳斯卡研究所赠送,APP17肽和Aβ25-35由本室用固相法合成,高效液相纯化.方法固相法合成APP17肽、Aβ25-35,高效液相纯化,以人神经母细胞瘤株SY5Y为细胞模型,分为正常对照组、Aβ25-3510μmol/L损伤组和Aβ25-35 10μmol/L加APP17肽10 μmol/L保护组.以细胞计数、噻唑蓝代谢率、乳酸脱氢酶漏出率、细胞轴突长度、胞体面积和细胞内游离钙离子(Ca2+)浓度为观察指标.结果与正常对照组相比,Aβ25-35损伤组轴突长度(35.74±16.25)和胞体面积(495.92±87.68)均缩小,细胞计数接种后第6天[(8.39±1.31)×107L-1]减少,噻唑蓝代谢率降低,乳酸脱氢酶漏出率升高,细胞内游离钙离子浓度升高,而加入APP17肽保护后,可使上述指标恢复或接近正常.结论APP17肽具有神经营养和神经保护作用,可减轻Aβ引起的神经元损伤.

Effect of APP 17-mer peptide on the neurotoxicity of beta-amyloid:a study on the pathogenesis of senile dementia

BACKGROUND: Senile plaque is one of the main pathological features in Alzheimer' s disease(AD) .beta-amyloid(Aβ) deposition forms the core of senile plaques, and Aβ25-35 is now recognized as a neurotoxic segment.Aβprotein precursor(APP) 319-335 segment(APP 17-mer peptide) has neurotrophic and neuro-protective effects.OBJECTIVE: To illustrate the effect of APP 17-mer peptide on the neurotoxicity of Aβ.DESIGN: A standard controlled study.SETTING and MATERIALS: The study was made in the Department of Neurobiochemisty, Xuanwu Hospital of Capital University of Medical Sciences.SY5Y cell line is a gift of Karolinska Institute in Sweden.APP 17-mer peptide and Aβ25 -35 were synthesized by means of solid phase method, and purified by high performance liquid chromatography (HPLC).METHODS: APP 17-mer peptide and Aβ25-35 were synthesized by solid phase method and purified by HPLC.Human neuroblastoma cells SYSY were grouped into normal control group, Aβ25-35 damaged group and Aβ25-35 + APP 17-mer peptide for neuroprotection group.Cell count, MTT metabolic rate,lactate dehydrogenase(LDH) leakage rate, axonal length, area of cell body,and concentration of intracellular free calcium ion were used as indicators.RESULTS: Compared with the normal control group, Aβ25-35 reduced thecell count[on day 6 after inoculation, (8.39 ± 1.31) × 107 L-1] and MTT metabolic rate, increased LDH leakage rate, diminished the axonal length (35.74 ± 16.25 ) and the area of cell body (495.92 ± 87.68 ), and increased the concentration of intracellular free calcium ion, while the addition of APP 17-mer peptide normalized the foregoing changes.CONCLUSION: APP 17-mer peptide has neurotrophic and neuroprotective effect in alleviating the neurotoxicity of Aβ.