Title Page
초록
Contents
INTRODUCTION 10
MATERIALS AND METHODS 15
1. Materials 15
2. Cell cultures 16
3. Cellular Fractionation 17
4. Two polyacrylamide gel electrophoresis and immunoblot analysis 18
5. Preparation of Aβ1-42 oligomer solution (이미지참조) 19
6. Morphological observation of the oligomers by bio-atomic force microscopy 20
7. Thioflavin T fluorescence assay 21
8. Measurement of cell viability 22
9. Measurement of Intracellular ROS 23
10. Immunocytochemistry - Detection of co-localization of PrPc and Aβ1-42 oligomer(이미지참조) 24
11. Immunocytochemistry - Discrimination of apoptotic cells 25
12. Statistical analysis 26
RESULTS 27
1. The cells containing different PrPc concentration were employed in this study 27
2. AFM confirmation 28
3. Different concentrations and incubation times optimize Aβ1-42 oligomerization(이미지참조) 29
4. Aβ1-42 oligomer including dimer, trimer, and tetramer and Aβ1-42 monomer revealed by Tricine-PAGE and western blot analysis.(이미지참조) 31
5. Aβ1-42 oligomer and PrPc partially co-localize on the cell surface(이미지참조) 33
6. Apoptosis induced by Aβ1-42 oligomer considerably occurred in comparison to necrotic and healthy cells(이미지참조) 36
7. The L2-2B1 cell expressing many PrPc are more susceptibly reduced by cytotoxicity of Aβ1-42 oligomers.(이미지참조) 38
8. Extracellular Aβ1-42 oligomer triggers the change of intracellular ROS(이미지참조) 40
9. Aβ1-42 oligomer impair Ca2+ homeostasis and lead it into cytosol(이미지참조) 41
10. Analysis for expression level of Apoptotic proteins 42
Discussions 47
REFFERENCES 55
ABSTRACT 67
Fig. 1. Western blot analysis of PrPc expression in PrPc-deficient, PrPc-over-expressed and parental N2a cells. PrPc expression was determined in 35ug of proteins from thetotal cell lysates by using T2HRP antibody. The band depicts total PrPc... 27
Fig. 2. AFM topographical images of (A) monomeric beta-amyloid 1-42, (B) oliomeric beta-amyloid 1-42, (C) distilled water (as control). Left side is original images of AFM and right side is 3-dimensional images rendered by software. Oligomers were... 28
Fig. 3. Confirmation of oligomerization with ThT staining. (A) 0,0.32, 0.625, 1.25, 2.5, 5, 10 uM of Aβ1-42 oligomers were evaluated with ThT (100uM) to confirm the degree of aggregation. (B) Oligomers incubated for 0 (also indicated to monomer state), 1, 2,...(이미지참조) 30
Fig. 4. Coomasie staining and western blot comparison of synthetic Aβ1-42 oligomer species. 5uM of Aβ1-42 oligomers were collected and separated by 16.5% Tris-Tricine gel. A gelwas stained with coomasie blue G-250 (A) and another gel was...(이미지참조) 33
Fig. 5. Immunocytochemical localization of PrPc and Aβ1-42 oligomerin Aβ1-42 oligomer treated PrPc-over-expressed L2-2B1 cells. Neuronal markers, PrPc and Aβ1-42 oligomer were shown to green and red fluorescence. They were incubated with mouse...(이미지참조) 34
Fig. 6. Localization of Hoechst 33342, Annexin V and EtD-III dyes attached in apoptotic cell. Neuronal apoptosis is illustrated here with representative microscopic fields of reverse-phase micrographs of three-color fluorescence cell viability... 37
Fig. 7. Cytotoxic effect of Aβ1-42 oligomer was determined by MTT assay. Various doses of Aβ1-42 oligomer were treated into N2a, HpL 3-4, L2-2B1 cells. HpL 3-4 shows the protective effect against Aβ1-42 oligomer induced cytotoxicity whereas it is more...(이미지참조) 39
Fig. 8. Measurements of ROS with DCFH-DA were compared in N2a, HpL 3-4, L2-2B1 cell with 0, 0.1, 1, 10, 20uM of Aβ1-42 oligomer.The data were subtracted from untreated control cell and each value represents the mean±S.D.P 〈0.005 compared...(이미지참조) 41
Fig. 9. Effect of Aβ1-42 oligomer [0.1, 1, 10uM] on basal [Ca2+]i in cytosol. N2a, HpL 3-4, L2-2B1 cellscultured in 96-well plate were treated dose-dependently. The fluorescence ofIndo 1-AM loaded cell were measured, and the [Ca2+]i was calculated as...(이미지참조) 42
Fig. 10. Relative expression levels of CaM (A), CalA (B), Bcl-2 (C), Bax (D) and Full length caspase-8 (E) in N2a, HpL 3-4, L2-2B1 neuronal cells treated with 20uM of Aβ1-42 oligomer.The Aβ1-42 oligomer had affect on elevation of Bcl-2, Bax and...(이미지참조) 45