STUDY OF THE MECHANISM OF NEURONAL CELL DAMAGE IN DEMENTIA FOR EARLY DIAGOSIS

在痴呆如阿尔茨海默病（AD）中，在脑中观察到由于神经元细胞凋亡引起的形态学变化。据报道，痴呆中的细胞损伤是由线粒体和微粒体的功能障碍引起的。由于肽A的积累，细胞内Ca^<2+>升高参与了AD的病理生理学。AD通常在中度晚期被诊断。早发现，早治疗。在这项研究中，我们的目的是调查分子标记代表AD。我们首先分析了培养细胞（人神经母细胞瘤NB-1和大鼠神经胶质瘤C6细胞）分泌的蛋白质，这些蛋白质由A23187引起的Ca^2+损伤诱导。然后，我们分析了痴呆患者血浆中的蛋白质。由于血脑屏障严格限制通过物理（紧密连接）和代谢（酶）屏障进入大脑的运输，并且相对疏水的材料倾向于穿过屏障，因此我们分离了脂质体（PC：PS=9：1）结合蛋白。膜联蛋白A5、A2和胎球蛋白在2D凝胶上用LC-MS分离后被鉴定。由于膜联蛋白A2在血细胞中高度表达，我们将重点放在膜联蛋白A5上。在下一步中，使用夹心ELISA测量AD、其他痴呆患者和对照者的血浆膜联蛋白A5水平。AD患者血浆Annexin A5水平为3.92±2.08 ng/ml，对照组为0.87±0.21 ng/ml。血浆Annexin A5水平随着长谷川痴呆量表（HDS-R）评分的降低而升高。另一方面，血管性痴呆患者血浆膜联蛋白A5水平有升高趋势（0.87±1.29 ng/ml），但与对照组相比无显著性差异。膜联蛋白A5在AD病因学中的生理作用有待进一步阐明。

In dementia such as Alzheimer's disease (AD) morphological change due to neuronal cell apoptosis is observed in the brain. The cell damage in dementia has been reported to be caused by functional disorder of mitochondria and microsomes. Intracellular elevation of Ca^<2+> is involved in the pathophysiology of AD due to accumulation of peptide A. AD is often diagnosed at a moderately advanced stage. Early detection is required for early treatment. In this study we aimed to investigate molecular markers representing AD. We first analyzed secreted proteins from culture cells (human neuroblastoma NB-1 and rat glioma C6 cells), induced by Ca^<2+>-damage with A23187. Then, we analyzed the protein in blood plasma of dementia patients. As the blood-brain barrier strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers and relatively hydrophobic materials tends to go through the barrier, we isolated the lipid liposome (PC : PS=9 : 1) binding proteins. Annexiss A5, A2 and fetuin were identified after separation on 2D-gel with LC-MS. As annexin A2 is highly expressed in blood cells, we focused on annexin A5. In the next step, blood plasma level of annexin A5 was measured using sandwich ELISA with AD, other dementia patients and controls. The level of plasma annexin A5 was 3.92±2.08 ng/ml in AD, while that in control was 0.87±0.21 ng/ml. The plasma annexin A5 level tended to increase with decreasing level of Hasegawa dementia scale (HDS-R) score. On the other hand, plasma annexin A5 level tended to be high (0.87±1.29 ng/ml) in vascular dementia patients, although not significantly higher than in controls. The physiological role of annexin A5 in the etiology of AD is next to be clarified.