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title page
Abstract
Contents
List of Abbreviation 9
Introduction 10
Experimental procedure 27
1. Materials 27
2. Preparation of chitosan oligosaccharides using an UF membrane bioreactor 27
3. Sample preparation 29
4. Cell culture 29
5. Cell Viability (MTT assay) 30
6. Acetyl cholinesterase (AChE) inhibitory activity 30
7. Cellular ROS determination by Dichlorofluorescin diacetate (DCFH-DA) 32
8. Fluorescence microscopy analysis 32
9. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay 32
10. Cell cycle analysis by flow cytometry 33
11. RNA extraction 34
12. Real-time polymerase chain reaction (Real-time PCR) 34
13. Western Blot assay 37
14. Measurement of total protein concentration 37
15. Statistical analysis 37
Results and Discussion 38
1. pH of COS and culture media 38
2. Effect of COS on viability of pc-12 cells (rat adrenal pheochromocytoma) 38
3. COS inhibited AChE activity induced by Aβ25-35(이미지 참조) 41
4. Effect of COS on viability of pc-12 cells affected by Aβ25-35(이미지 참조) 44
5. Effect of COS on intracellular ROS level induced by Aβ25-35 in pc-12 cells.(이미지참조) 44
6. Morphological analysis of pc-12 cells affected by Aβ25-35-induced apoptosis(이미지참조) 47
7. Confirm of apoptotic cell death by TUNEL assay 47
8. Determination of apoptosis by cell cycle 50
9. COS inhibit apoptosis via reduction of p53 expression level 53
10. COS inhibit target genes in apoptosis pathway that affected by Aβ25-35(이미지참조) 56
Summary 67
References 69
Acknowledgment 77
Table 1. The sequences of the primer for real-time PCR 36
Table 2. pH of sample solution 39
Figure 1. These images represent a cross-section of the brain as seen from the front. The cross-section on the left represents a brain from a normal individual and the one on the right represents a brain with Alzheimer's disease. In the early stages of Alzheimer's disease,... 12
Figure 2. The Aβ peptide is derived via proteolysis from a larger precursor molecule called the amyloid precursor protein (APP). APP can undergo proteolytic processing by one of two pathways. Most is processed through the non-amyloidogenic pathway,... 13
Figure 3. Putative Amyloid Cascade. This hypothesis of the amyloid cascade, which progresses from the generation of the Aβ peptide from the amyloid precursor protein, through multiple secondary steps, to cell death, forms the foundation for... 14
Figure 4. Activation and inhibition of apoptosis. Several mechanisms have been identified in mammalian cells for the induction of apoptosis. These mechanisms include factors that lead to perturbation of the mitochondria leading to leakage of cytochrome c or... 18
Figure 5. p53 signaling pathway. P53 can cause growth arrest of the cell at a checkpoint to allow for DNA damage repair or can cause the cell to undergo apoptosis if the damage cannot be repaired. 19
Figure 6. Caspase Cascade. Apoptosis or programmed cell death is triggered by a variety of stimuli, including cell surface receptors, the mitochondrial response to stress. The caspases comprise a class of cystein proteases, many members of which are involved... 20
Figure 7. Oxidative damage due to the endogenous production of reactive oxygen species by mitochondria can lead to cell death via apoptosis. Two such factors, cytochrome c and apoptosis inducing factor (AIF), begin a cascade of proteolytic activity that ultimately... 21
Figrue 8. Structures of Chitin and Chitosan 24
Figure 9. Schematic diagram of the dual reactor system developed for continuous production of chitooligosaccharide (COS). Adapted from Jeon and Kim (2000) 29
Figure 10. Ellman's assay for determination of AChE activity 31
Figure 11. Pc-12 cells (rat adrenal pheochromocytoma) were treated with COS at the indicated concentrations. Cell viability was determined by the MTT assay after 24 hours of treatment with COS. Blank : the group that was not treated with COS. 40
Figure 12. Measurement of AChE activity stimulated by Aβ25-35 in pc-12 cells. Pc-12 cells were treated with 25 μM Aβ25-35 and incubated for 24 h. AChE activity was evaluated by Ellman assay.(이미지참조) 42
Figure 13. Western blot assay of AChE protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ (25μM) for 24 h, cell extracts were resolved by SDS-PAGE.... 43
Figure 14. Pc-12 cells (rat adrenal pheochromocytoma) were treated with COS at the indicated concentrations and 25 μM Aβ25-35. Cell viability was determined by the MTT assay after 24 hours of treatment with COS....(이미지참조) 45
Figure 15. Cellular radical scavenging activity of COS. Pc-12 cells were labeled with non-toxic fluorescence dye, DCFH-DA, and treated with different concentrations of COS. Fluorescence intensities of DCF due to oxidation of DCFH by cellular ROS... 46
Figure 16. Morphological analysis of pc-12 treated with 1000 μg and Aβ (25 μM) for 24 h. Cells treated with COS were visualized with fluorescent microscope and compared with blank (non-treated cell) and control (only treated 25 μM Aβ). 48
Figure 17. TUNEL stained cells after 24 h of treatment. Pc-12 cells were treated with 1000 μl of COS and Aβ (25 μM) after 24 h TUNEL assay was employed to identify apoptotic cells having free 3'-OH of cleaved DNA.... 49
Figure 18. Cells cycle analysis of pc-12 cells in the presence of COS. Cells were treated with indicated concentrations of COS and Aβ (25 μM) and harvested after 24 h. Propidium iodide staining was used to quantify cells undergoing apoptosis and to... 51
Figure 19. Quantification of apoptosis in pc-12 cells treated with COS after PI staining. Cells were treated with different concentrations of COS and Aβ (25 μM) for 24 h. Propidium iodide staining was used to quantify cells undergoing apoptosis and to analyze the cell cycle.... 52
Figure 20. p53 mRNA expression in COS treated pc-12 cells. Different concentrations of COS treated pc-12 cells were incubated for 24 h and total RNA was extracted as described in method section.... 54
Figure 21. Western blot assay of p53 protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ (24μM) for 24 h, cell extracts were resolved by SDS- PAGE.... 55
Figure 22. Western blot assay of MDM2 and p21 protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ(24μM) for 24 h, cell extracts were resolved by SDS-PAGE.... 58
Figure 23. Bcl-2 mRNA expression level treated pc-12 cells. Different concentrations of COS treated pc-12 cells were incubated for 24 h and total RNA was extracted as described in method section.... 59
Figure 24. Bax mRNA expression level in COS treated pc-12 cells. Different concentrations of COS treated pc-12 cells were incubated for 24 h and total RNA was extracted as described in method section.... 60
Figure 25. Western blot assay of Bax and Bcl-2 protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ(25μM) for 24 h, cell extracts were resolved by SDS-PAGE.... 61
Figure 26. Western blot assay of Apaf-1 and cytochrome C protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ (25μM) for 24 h, cell extracts were resolved by SDS-PAGE.... 62
Figure 27. Caspase-9 mRNA expression level in COS treated pc-12 cells. Different concentrations of COS treated pc-12 cells were incubated for 24 h and total RNA was extracted as described in method section.... 64
Figure 28. Caspase-3 mRNA expression in COS treated pc-12 cells. Different concentrations of COS treated pc-12 cells were incubated for 24 h and total RNA was extracted as described in method section.... 65
Figure 29. Western blot assay of caspase-9, caspase-3 and caspase-8 protein expression in pc-12 cells treated with COS. After treatment of COS and Aβ (25μM) for 24 h, cell extracts were resolved by SDS-PAGE.... 66
Figure 30. The proposed signaling cascade in pc-12 cells affected by COS. Aβ25-35 induced apoptosis via p53 signaling cascade and mitochondria pathway. When COS was treated into the pc-12 cell apoptotic signaling of this cascade was inhibited. 68
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