Title Page
Abstract
Contents
PART I. OSTEOCLAST ACTIVITY REGULATION BY LOW LEVEL LIGHT THERAPY (LLLT) 20
I. INTRODUCTION 20
1. Background 20
2. Osteoclastogenesis 21
2.1. Origins and commitment of osteoclast 21
2.2. Differentiation of osteoclast 21
2.3. Fusion of osteoclast 23
2.4. Bone resorption of osteoclast 25
3. Low Level Light Therapy (LLLT) 26
4. Study objectives 27
II. MATERIALS AND METHODS 28
1. Cell culture 28
1.1. Whole bone marrow cells (BMCs) 28
1.2. Bone marrow-derived monocyte/macrophage precursor cells (BMMs) 29
1.3. RAW264.7 cells 29
2. The light source and Irradiation 30
3. Cell Viability Assay 32
4. TRAP Staining 32
5. TRAP Solution Assay 33
6. Actin Ring Formation Assay 33
7. Resorption Assay 34
8. RT-PCR analysis 34
9. Microarray analysis 36
10. Real-time quantitative PCR (qRT-PCR) analysis 37
11. Statistical Analysis 37
III. RESULTS 39
1. Characterization of the light source 39
2. The optimal wavelength and irradiation schedule to inhibit ROC formation 39
3. The optimal fluence rate to inhibit ROC formation 42
4. Inhibition of ROC formation in BMC, BMM, and RAW264.7 48
5. Inhibition of actin ring formation and bone resorption 51
6. LLLT effect on osteoclast specific genes during their differentiation 52
7. Overall gene expression profiling in BMM irradiated by 635㎚ light 55
8. Genes highly up-regulated by RANKL and down-regulated by LLLT 57
9. Genes highly down-regulated by RANKL and up-regulated by LLLT 60
10. Effect of LLLT on osteoclast specific marker genes 62
11. Validation of microarray analysis results using real time RT-PCR 62
IV. DISCUSSION 68
V. REFERENCES 81
PART II. OSTEOCLAST ACTIVITY REGULATION BY IMMUNOSUPPRESSANTS 90
I. INTRODUCTION 90
1. Background 90
2. Immunosuppressants 92
2.1. FK506 (Tacrolimus) 92
2.2. FK520 (ascomycin) 94
3. Study objectives 95
II. MATERIALS AND METHODS 96
1. Cell culture 96
1.1. Whole bone marrow cells (BMCs) 96
1.2. Bone marrow-derived monocyte/macrophage precursor cells (BMMs) 97
1.3. RAW264.7 cells 97
2. Chemical compounds and treatment 98
3. Cell Viability Assay 98
4. TRAP Staining 99
5. TRAP Solution Assay 99
6. Microarray analysis 100
7. Real-time quantitative PCR (qRT-PCR) analysis 100
8. Statistical Analysis 101
III. RESULTS 104
1. Inhibitory effect of FK506 and FK520 on ROC formation by dose dependent manner 104
2. Effect of FK506 and FK520 on osteoclast specific marker genes 105
3. Inhibitory effect of FK506 and FK520 on ROC formation in BMC, BMM, and RAW264.7 117
4. Inhibitory effect of FK506 and FK520 on ROC formation by time dependent manner 118
5. Overall gene expression profiling in RAW264.7 cells treated with FK506 or FK520 126
6. Osteoclast associated-genes in FK520 or FK506 treated-groups 131
6.1. Osteoclast associated genes affected by 1nM FK520 134
6.2. Osteoclast associated genes affected by 10nM FK520 in the pool of 1nM and 10nM FK520 treated groups 134
6.3. Osteoclast associated genes affected by both 1nM FK520 and 10nM FK520 137
6.4. Osteoclast associated genes affected by 1nM FK506 137
6.5. Osteoclast associated genes affected by 10nM FK520 in the pool of 1nM FK506 and 10nM FK520 treated groups 140
6.6. Osteoclast associated genes affected by both 1nM FK506 and 10nM FK520 140
7. Validation of microarray analysis results by qRT-PCR 143
IV. DISCUSSION 146
V. REFERENCES 157
국문초록 165
PART I. OSTEOCLAST ACTIVITY REGULATION BY LOW LEVEL LIGHT THERAPY (LLLT) 14
Table 1. Primer sequences and conditions for RT-PCR 35
Table 2. Primer sequences and conditions for qRT-PCR 38
Table 3. The fluence rates by currents and wavelengths (n=3) 41
Table 4. List of genes highly up-regulated by RANKL and down-regulated by LLLT 59
Table 5. List of genes highly down-regulated by RANKL and up-regulated by LLLT 61
Table 6. List of osteoclast specific marker genes affected by LLLT 63
PART II. OSTEOCLAST ACTIVITY REGULATION BY IMMUNOSUPPRESSANTS 14
Table 1. Primer sequences of osteoclast specific marker genes for qRT-PCR 102
Table 2. Primer sequences used for qRT-PCR validation of miroarray data 103
Table 3. List of osteoclast associated genes affected only by 1nM FK520 (12) 135
Table 4. List of osteoclast associated genes affected only by 10nM FK520 (21) 136
Table 5. List of osteoclast associated genes affected by both 1nM and 10nM FK520 (6) 138
Table 6. List of osteoclast associated genes affected only by 1nM FK506 (5) 139
Table 7. List of osteoclast associated genes affected by 10nM FK520 in the pool of 1nM FK506 and 10nM FK520 treated groups (20) 141
Table 8. List of osteoclast associated genes affected by both 1nM FK506 and 10nM FK520 (7) 142
PART I. OSTEOCLAST ACTIVITY REGULATION BY LOW LEVEL LIGHT THERAPY (LLLT) 16
Figure 1. Essential molecules for commitment, differentiation, fusion, and resorption of osteoclast. 22
Figure 2. Signaling cascades during osteoclastogenesis. 24
Figure 3. Custom-made LED arrays for each wavelength 31
Figure 4. Relative emission of LED arrays 40
Figure 5. Effects of LED irradiation on round-shaped osteoclast (ROC) formation derived from RAW264.7 under different wavelengths at 30㎽/㎠ 43
Figure 6. Effects of LED irradiation on cell viability and ROC formation in RAW264.7 under different wavelengths at 30㎽/㎠ for... 44
Figure 7. Effects of LED irradiation on ROC formation derived from RAW264.7 under different wavelengths at 2㎽/㎠ 45
Figure 8. Effects of LED irradiation on cell viability and ROC formation in RAW264.7 under different wavelengths at 2㎽/㎠ for... 46
Figure 9. Cell viability, total TRAP activity, and ROC formation induced from RAW 264.7 under different fluence rates 47
Figure 10. Effect of LED irradiation on osteoclast formation of BMC, BMM, and RAW264.7 49
Figure 11. Effect of LED irradiation on osteoclast formation of BMC, BMM, and RAW264.7 50
Figure 12. Effect of LLLT on actin ring formation in RAW264.7 cells 52
Figure 13. Effect of LLLT on ROC formation with actin ring in RAW264.7 cells 53
Figure 14. Effect of LLLT on pit formation in RAW264.7 cells 54
Figure 15. Effect of LLLT on gene expression levels during osteoclastogenesis (2㎽/㎠, 72hrs, 635㎚) in three different cell types derived from mice 56
Figure 16. Pair-wise scatter plot between vehicle and R, vehicle and R + L, or R and R + L 58
Figure 17. Validation of microarray analysis results using real time RT-PCR (qRT-PCR) 65
PART II. OSTEOCLAST ACTIVITY REGULATION BY IMMUNOSUPPRESSANTS 16
Figure 1. Chemical structure of cyclosporine A (CsA). Wikipedia 91
Figure 2. Chemical structures of FK506 (tacrolimus; left) and FK520 (ascomycin; right). 93
Figure 3. Inhibitory effect of FK506 and FK520 on ROC formation by a dose dependent manner. 105
Figure 4. Inhibitory effect of FK520 and FK506 on cell viability, total TRAP activity, and ROC formation by a dose dependent manner. 106
Figure 5. Effects of FK506 and FK520 on gene expression levels during osteoclast differentiation in RAW264.7 cells. 109
Figure 6. Inhibitory effect of 1nM FK506 and 10nM FK520 on ROC formation in BMC, BMM, and RAW264.7 cells. 119
Figure 7. Inhibitory effect of 10nM FK520 and 1nM FK506 on cell viability, total TRAP activity, and ROC formation in BMC, BMM, and RAW264.7 cells. 120
Figure 8. 1nM FK506 and 10nM FK520 incubation time schedule for RAW264.7 cells with RANKL (100ng/㎖). 122
Figure 9. Inhibitory effect of 1nM FK506 on round shaped-osteoclast (ROC) by a time dependent manner. 123
Figure 10. Inhibitory effect of 1nM FK506 on cell viability, total TRAP amount, and ROC formation by a time dependent manner. 124
Figure 11. Inhibitory effect of 10nM FK520 on ROC formation by a time dependent manner. 127
Figure 12. Inhibitory effect of 10nM FK520 on cell viability, total TRAP amount, and ROC formation by a time dependent manner. 128
Figure 13. Pair-wise scatter plot between control and 1nM FK506, control and 1nM FK520, and control and 10nM FK520. 130
Figure 14. Cluster image showing the differential expression profiles between... 132
Figure 15. Venn diagram presenting the number of differentially expressed genes. 133
Figure 16. Validation of microarray analysis results using real time RT-PCR (qRT-PCR). 144