Molecular biological studies on a role of Aβ production and degrading enzyme in experimental model.

实验模型中 Aβ 产生和降解酶作用的分子生物学研究。

• 批准号: 15591239
• 负责人: TSUZUKI Kayo
• 金额: $ 1.92万
• 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 (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591239/
• 关键词: Alzheimer's disease; amyloid β protein; BACE; neprilysin; amyloid precursor protein; BACE gene; Chloroquine rat; myocyte; アミロイドβタンパク; ノックアウト; アミロイドβ蛋白; PCR; APP遺伝子; 抗原賦活化処理

Deposition of amyloid β protein (Aβ) in the brain is an invariant neuropathological feature of Alzheimer's disease (AD). Aβ is generated by two cleavages of amyloid precursor protein (APP). The initial cleavage by BACE is followed by γ-secretase cleavage of the C-terminal APP fragment. Once formed, Aβ is mainly degraded small regulatory peptides by neprilysin (NEP). The generative process of AD is linked to a shift in the balance between Aβ production and degradation. We investigated the possible functional significance of BACE and NEP in cultured myocyte systems under chloroquine (CQN) as an experimental model to study APP processing for Aβ production.1.Human myocyte (CCL136) was transfected with mutation APPcDNA. The APPmRNA level reached the maximum after 12 hours CQN treatment. Microvacuoles were appeared in perinuclear area 12 hours after CQN treatment, and increased in number until up to 24 hours. Red granular materials around these vacuoles, and inside the vacuoles were stained … More modified Gomori-trichrome.2.Perinuclear and heterogeneous materials surrounding vacuoles containing stained granular bodies reacted with anti Aβ antibody. Double labeling with anti Aβ and anti BACE antibodies, demonstrated that Aβ and BACE were co-localized in some of the vacuoles.3.4,12,20,28,60,70 kDa bands were detected using anti Aβ antibody, 12,35,40,70 kDa bands were detected using anti BACE antibody, 70,80 kDa bands were detected using anti NEP antibody by Western blotting. 4 kDa band, apparently Aβ, became remarkable after CQN treatment.4.BACEmRNA levels were reduced 50% in the siRNA treated myocyte cells relative to control. Double immunostaining of BACEmRNA knockdown myocyte with anti Aβ and anti BACE antibodies, immunoreactivities were co-localized in some of the microvacuoles, but apparently decreased in number5.NEPmRNA vector was tranfected into myocyte using Lipofectamine. The recombinant myocyte which increased of NEPmRNA level is not provided at present.CQN myocyte system is a good experimental model to study the balance between the generation and degradation of Aβ appears altered. Less

淀粉样蛋白(Aβ，Aβ)在脑内的沉积是阿尔茨海默病(AD)的一个恒定的神经病理特征。β是由淀粉样前体蛋白(APP)的两次切割产生的。在BACE的初始切割之后，C-末端APP片段被γ-分泌酶切割。一旦形成，Aβ主要是由Neprilysin(NEP)降解的小调节肽。AD的发生过程与Aβ产生和降解之间的平衡变化有关。以氯喹作用下培养的心肌细胞系为实验模型，研究APP对Aβ合成的影响。1.将突变的APP基因导入人心肌细胞系CCL136。CQN作用12h后，APPmRNA水平达到最大值。CQN处理后12小时，核周区出现微空泡，并逐渐增多，直至24小时。这些液泡周围的红色颗粒物质，以及液泡内部被染色的…2.含有染色颗粒的空泡周围的异质物质与抗Aβ抗体反应。抗Aβ抗体检测到3.4、12、20、28、60、70 kDa条带，抗β抗体检测到12、35、40、70 kDa条带，Western blotting检测到70、80 kDa条带。4.小干扰β处理组大鼠心肌细胞BACEmRNA表达水平较对照组降低了50%。用抗Aβ和抗BACE抗体对BACEm RNA敲除心肌细胞进行双重免疫染色，免疫反应在部分微泡中共存，但在5号微泡中明显减少。CQN肌细胞系是研究A-β生成与降解平衡出现改变的良好实验模型。较少