Novel Experimental Model in vitro for Amyloid beta Production and Molecular Biological Analysis of Amyloidogenesis

β淀粉样蛋白产生的新型体外实验模型和淀粉样蛋白生成的分子生物学分析

• 批准号: 07457211
• 负责人: FUKATSU Ryo
• 金额: $ 3.46万
• 依托单位: SAPPORO MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457211/
• 关键词: Alzheimer's disease model; amyloid precursor protein; amyloid beta protein; proteolytic processing; in vitro; myocyte; chloroquine; acidic compartment; アルツハイマー病; 培養細胞系; エンドソーム; リソソーム経路; Alzheimer病; APP

Production of amyloid beta protein (Abeta), which characteristically deposits in Alzheimer's disease (AD) affected brain, by proteolytic processing of amyloid precursor protein (APP) is a key issue in the pathogenesis of AD.We describe here a novel experimental model in vitro, in which Abeta is cleaved from APP in cultured human myocyte after chloroquine (CQN) treatment.1. Pathological studies on cultured of human myocyte with/without CQN treatment.1) Histopathological studies of cultured myocytes : Vacuoles appeared in perinuclear area of the cultured myocytes 12 hours after CQN treatment, and increase in number until up to 24 hours. Modified Gomori method stained red granular materials around these vacuoles, and inside the vacuoles.2) Immunopathological studies of APP and Abeta localization : CQN induced vacuoles were stained with anti-APP,and anti-Abeta antibodies. A number of these vacuoles were also labeled with antibodies against specific protein to intracellular compartments, ra … More b 7 and cathepsin D for lysosomes, only a few of these vacuoles were stained with anti-garactosyltransferase. But no immunoreactivities were observed with anti-ERGIC 53, specific for endoplasmic reticulum.2. Biochemical studies on proteolytic processing of APP : Western blot analyzes demonstrated there were many amyloidogenic or non-amyloidogenic APP fragments in cell fractions obtained from cultured myocytes with CQN treatment. 4 kDa fragment, apparently Abeta, become remarkable after CQN treatment.3. Molecular biological studies of APP gene expression : mRNA of APP gene was determined sequentially by Northern ELISA.The mRNA began to increase 3 hours after CQN treatment, and showed a peak 9 hours after CQN treatment, 2.5-fold increase compared to mRNA of myocyte without CQN treatment.This novel experimental model provides in vitro model to study mechanism underlying proteolytic process of APP into Abeta production. Our data indicate that acidic compartments, lysosomal pathway, may play an important role in the generation of amyloidogenic fragments and Abeta. Less

淀粉样前体蛋白（amyloid precursor protein，APP）在阿尔茨海默病（Alzheimer's disease，AD）脑组织中的沉积是AD发病机制中的一个关键问题。我们在此描述了一种新的体外实验模型，在该模型中，氯喹（chloroquine，CQN）处理后，培养的人心肌细胞中的APP裂解出淀粉样β蛋白（amyloid beta protein，Abeta）。CQN处理前后培养的人心肌细胞的病理学研究1）培养的心肌细胞的组织学研究：CQN处理后12h，培养的心肌细胞核周区出现小泡，并持续到24h。改良Gomori法将空泡周围和空泡内部的红色颗粒物质染色。2）APP和Abeta定位的免疫病理学研究：用抗APP和抗Abeta抗体对CQN诱导的空泡进行染色。这些空泡中的一部分也被细胞内区室的特异性蛋白抗体标记， ...更多信息 B 7和组织蛋白酶D的溶酶体，只有少数这些空泡染色的抗半乳糖基转移酶。但与内质网特异性抗ERGIC 53抗体无免疫反应. APP蛋白水解过程的生化研究：Western blot分析表明，在CQN处理的培养心肌细胞中获得的细胞组分中存在许多淀粉样或非淀粉样APP片段。CQN治疗后，4kDa片段，明显的Abeta，变得显著. APP基因表达的分子生物学研究：采用北方ELISA法检测APP基因mRNA的表达，CQN处理后3h，APP基因mRNA表达开始增加，9h达高峰，是未处理前的2.5倍，为研究APP蛋白水解转化为A β的机制提供了体外模型。我们的数据表明，酸性腔室，溶酶体途径，可能在淀粉样蛋白生成片段和A β的产生中发挥重要作用。少

项目成果

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