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

淀粉样蛋白（Abeta）是阿尔茨海默病（AD）患者大脑中特有的淀粉样蛋白沉积，通过淀粉样前体蛋白（APP）的蛋白水解加工而产生的淀粉样蛋白（Abeta）是AD发病机制中的关键问题。我们在此描述了一种新的体外实验模型，在体外培养的人肌细胞中，经过氯喹（CQN）处理后，Abeta从APP中分离出来。加/不加CQN对人心肌细胞培养的病理研究。1)培养的肌细胞的组织病理学研究：CQN处理后12小时，培养的肌细胞核周出现空泡，直至24小时，空泡数量增加。改进的Gomori法染色液泡周围和液泡内部的红色颗粒物质。2) APP和Abeta定位的免疫病理研究：CQN诱导空泡染色抗APP和抗Abeta抗体。许多液泡还被标记有针对细胞内区室的特定蛋白的抗体，而溶酶体的b7和组织蛋白酶D更多，只有少数液泡被标记有抗乳糖基转移酶。但内质网特异性抗ergic 53未见免疫反应。APP蛋白水解过程的生化研究：Western blot分析显示，在CQN处理的培养肌细胞的细胞组分中，存在许多淀粉样或非淀粉样的APP片段。4 kDa片段，明显是Abeta，在CQN治疗后变得显著。APP基因表达的分子生物学研究：应用Northern ELISA法对APP基因mRNA进行序列检测。心肌细胞mRNA在CQN处理后3 h开始升高，在CQN处理后9 h达到峰值，较未处理心肌细胞mRNA增加2.5倍。该实验模型为研究APP蛋白水解生成β的机制提供了体外模型。我们的数据表明，酸性区室，溶酶体途径，可能在淀粉样蛋白片段和β的产生中起重要作用。少

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