Title Page
Contents
LIST OF ABBREVATIONS 13
ABSTRACT 15
I. INTRODUCTION 17
A. Inflammation 17
1. Inflammatory response 18
2. Acute Inflammation 20
3. Chronic inflammation 21
4. The inflammatory Pathway 22
5. Macrophages 23
6. Inflammatory cytokines 25
7. Resolution of Inflammation 27
B. Atopic dermatitis 28
1. Atopy as atopic march 29
2. Epidemiology of AD 30
3. Skin barrier dysfunction in AD 32
4. Mechanism of AD development 32
5. Immune modulation of AD skin 35
C. Circadian rhythms and genes 36
1. Properties and functions of circadian rhythms 36
2. Transcription- translational oscillator loop of circadian genes synthesis 39
D. Circadian rhythms role in Immune system and Inflammation 40
1. Circadian rhythms involvement in Immune system 40
2. Circadian rhythms influence in inflammation through circadian genes 42
3. Anti-inflammatory circadian gene BMAL-1 46
4. Circadian gene Per2 46
5. Influence of circadian gene Per2 in inflammation 47
6. Clock gene Per2 relation with inflammatory diseases 48
II. CHAPTER-1. The role of circadian gene Per2 in Atopic dermatitis animal model and relation with BMAL-1, GATA3 50
A. Aim of the study 50
B. Materials and Methods 52
1. Animals 52
2. Genotyping 53
3. Reagents 54
4. Agarose Gel-electrophoresis 54
5. Induction of AD 55
6. Observation test 56
7. Isolation of splenocytes 56
8. Real-time polymerase chain reaction (qPCR) 58
9. Measurement of IgE 60
10. Cell surface staining and Fluorescence activated cell sorting (FACS) analysis 61
C. RESULTS 62
D. DISCUSSION 93
III. CHAPTER-2. The Relation between Circadian gene and inflammatory genes in vitro 97
A. Aim of Study 97
B. Materials and Methods 99
1. Reagents 99
2. Cell culture and treatment 99
3. Transfection with Per2 siRNA and treatment 99
4. Nitric-oxide Assay 100
5. Quantitative real time polymerase chain reaction 100
6. Enzyme linked immunosorbent assay 101
C. Results 102
D. DISCUSSION 113
IV. CONCLUSION 115
V. References[원문불량;p.116] 116
Table 1. This table is showing classification factors that are involving epidemiology... 31
Table 2. Primers used for Genotyping and their PCR temperature conditions 53
Table 3. qRT-PCR primer sequence for the genes and conditions used in this study 59
Fig 1. Schematic presentation of mechanism of AD development through JAK-... 34
Fig 2. Schematic explanation of the complex network of the central nervous system,... 37
Fig 3. Coordination of environmental cues and peripheral organs by the... 39
Fig 4. This schematic presentation explains the influence of Circadian genes to... 45
Fig 5. This schematic diagram explaining the relation of circadian gene with T-cell... 49
Fig 6. The scheme of induction of the Atopic dermatitis animal model 55
Fig 7. Agarose gel electrophoresis of the RT-PCR results of the Per2-/- DNA. Total...[이미지참조] 62
Fig 8. DNCB induced Atopic dermatitis development difference showing between... 63
Fig 9. Dorsal skin lesions observation of all groups of mice. Per2+/+ DNCB...[이미지참조] 64
Fig 10. After Sacrifice of all groups mice isolated the spleen and evaluated by size,... 67
Fig 11. Quantitative real-time PCR measuring relative for measure mRNA... 70
Fig 12. GATA3 expression was measured by qRT-PCR. Per2+/+ DNCB group...[이미지참조] 72
Fig 13. Th2 cytokines and TNF-α expression in spleen was measured by qRT-PCR.... 74
Fig 14. Th1 and Th2 cytokine levels were measured by qRT-PCR. A. Th2 cytokine... 77
Fig 15. Th1/Th2 cells of Spleen of Per2+/+, Per2-/- control and DNCB groups by...[이미지참조] 79
Fig 16. Quantitative real-time PCR measuring relative for measure mRNA... 82
Fig 17. GATA3 expression was measured by qRT-PCR. Per2+/+ DNCB group...[이미지참조] 84
Fig 18. Th2 cytokines and TNF-α expression in skin was measured by qRT-PCR.... 87
Fig 19. Th1 and Th2 cytokine levels were measured by qRT-PCR. A. Th2 cytokine... 90
Fig 20. Serum IgE levels were analysed by ELISA. Per2+/+ DNCB group mice have...[이미지참조] 92
Fig 21. This schematic presentation explaining our study circadian gene Per2 role... 96
Fig 22. Quantitative real-time PCR measured relative mRNA expression level... 102
Fig 23. Per2 and BMAL-1 expressions were measured by qRT-PCR. Total... 105
Fig 24. RAW 264.7 and SiRAW 264.7cells were stimulated with LPS for 12 hours... 106
Fig 25. RAW 264.7 and SiRAW 264.7 cells were stimulated with LPS for 12 hours.... 109
Fig 26. RAW 264.7 and Si RAW 264.7 cells were stimulated with LPS for 12 hours... 112