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

 골다공증과 골연화증과 같은 골 질환의 치료방법은 파골세포의 활성을 줄이는데 목표를 두고 있다. 본 연구는 RANKL로 유도된 파골세포의 분화 억제를 위한 최적의 조건 확립을 위하여 다양한 파장과 플루언스율을 적용한 저출력광선요법 (LLLT)을 사용하였다. MTT 분석을 통하여 세포 생존율을 확인하였으며, TRAP에 반응하는 전체 파골세포의 양은 TRAP 용액 분석을 통하여, 둥근 모양 파골세포 (ROC)의 수는 TRAP염색을 통하여 측정하였다. ROC 형성에 대한 LLLT의 억제효과는 마우스에서 유래된 RAW264.7 외에 골수세포 (BMC)와 골수유래 대식세포 (BMM)에서도 측정하였다. ROC가 액틴링을 가지는지를 확인하기 위하여 로다민 팔로이딘으로 염색하였으며, 상아질 절편을 사용하여 ROC의 골 흡수 능력을 확인하였다. 이외에도, RT-PCR을 사용하여 파골세포 특이 표지 유전자들의 발현 정도를 확인하였으며, 마이크로어레이를 통하여 유전자 발현 프로파일링을 확인하였다. 형태적 분석에서, 액틴링을 가진 파골세포의 형성은 세 가지 모든 세포 형태에서 635nm LED 어레이를 사용하였을 때 현저하게 억제됨을 확인하였다. 대조군에서의 액틴링은 대부분 파골세포의 가장자리에서 관찰되었다. 반면, 실험군에서의 파골세포는 형태가 틀어진 패치모양의 액틴링이 관찰되었으며, 형태가 틀어지지 않은 액틴링을 가지는 파골세포의 수가 현저하게 줄어드는 양상을 보였다. RT-PCR 분석에서는, 골 흡수 관련 유전자인 intergrin β3, calcitonin receptor, c-src의 발현 수준이 실험군에서 현저하게 감소되었다. RT-PCR과 마이크로어레이의 분석에서, 발현양상이 현저한 차이를 보이는 유전자들이 동일하지는 않았지만, 이들 유전자들은 대부분 골 흡수 및 파골세포 성숙에 관련되어 있는 것들이었다.

본 연구는 또한 파골세포 활성을 조절하기 위하여 면역억제제인 FK506과 FK520을 사용하였다. 위에 설명하였던 세 가지 다른 세포유형에서, ROC 형성을 억제하기 위한 가장 효과적이면서도 최소한의 농도는 FK506은 1nM, FK520은 10nM로 확인되었다. 파골세포 특이 마커 유전자들 중, calcitonin receptor와 intergrin β3의 유전자 발현이 두 종류의 면역억제제에 의해서 현저하게 감소됨을 확인하였다. 또한 마이크로어레이를 이용한 유전자 발현 프로파일링에서는 Rg12가 두 면역억제제에 의해서 현저하게 감소되는 양상을 보였다. 한편, Runx2의 유전자 발현 수준이 1nM의 FK506에 의해서 현저하게 감소되었지만 10nM의 FK520에 의해서는 유의한 차이가 나타나지 않는 것으로 나타났다. 종합적으로 볼 때, 본 연구 결과는 LLLT 또는 낮은 농도의 면역억제제가 골 질환 치료에 중요한 역할을 할 수 있을 것으로 판단되며, 이 치료법들이 동물실험에서도 골 밀도조절에 효과적인지 확인해 보아야 할 것이다.