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

β淀粉样蛋白（Aβ）在大脑中的沉积是阿尔茨海默病（AD）不变的神经病理特征。Aβ是由淀粉样蛋白前体蛋白（APP）的两次裂解产生的。BACE首先进行裂解，然后γ-分泌酶对c端APP片段进行裂解。一旦形成，Aβ主要是由NEP降解的小调节肽。AD的生成过程与a β产生和降解之间平衡的转变有关。我们研究了BACE和NEP在氯喹（CQN）作用下培养的肌细胞系统中可能的功能意义，作为研究APP对Aβ产生的作用的实验模型。用突变的APPcDNA转染人心肌细胞（CCL136）。在CQN处理12 h后，APPmRNA水平达到最大值。CQN处理12小时后，核周出现微空泡，并在24小时内增多。液泡周围和液泡内部的红色颗粒物质被染色。含有染色颗粒体的液泡周围的核周和异质物质与抗Aβ抗体反应。用抗Aβ和抗BACE抗体进行双标记，发现Aβ和BACE在部分空泡中共定位。用抗Aβ抗体检测3.4、12、20、28、60、70 kDa条带，用抗BACE抗体检测12、35、40、70 kDa条带，用抗NEP抗体进行Western blotting检测70、80 kDa条带。4. CQN治疗后，kDa条带显著增加，其中以Aβ条带明显。与对照组相比，siRNA处理的心肌细胞中BACEmRNA水平降低了50%。用抗Aβ抗体和抗BACE抗体对BACEmRNA敲除的肌细胞进行双重免疫染色，免疫反应在部分微泡中共定位，但在5个微泡中明显降低。用Lipofectamine将NEPmRNA载体转染到肌细胞中。目前还没有发现NEPmRNA水平升高的重组肌细胞。CQN肌细胞系统是研究a β生成与降解平衡发生改变的良好实验模型。少

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