Title Page
Contents
ABBREVIATIONS 12
ABSTRACT 14
CHAPTER Ⅰ. LITERATURE REVIEW 16
1. Background of Adiponectin 17
1.1. Adipose tissue in bone marrow 17
1.2. Adiponectin structure and signaling 20
2. Bone Marrow-derived Mesenchymal Stem Cells(BMSCs) 24
2.1. Mesenchymal stem cells in bone marrow microenvironment 24
2.2. The migration of bone marrow-derived mesenchymal stem cells in the tumor microenvironment 28
2.3. Adiponectin and bone marrow-derived mesenchymal stem cells 32
3. The Aim of This Study 34
CHAPTER Ⅱ. The enhanced migratory ability of APN KO BMSCs via the regulation of hypoxia-inducible factor 1α 35
1. INTRODUCTION 36
2. MATERIALS AND METHODS 38
2.1. Animals 38
2.2. Colony-forming unit-fibroblast (CFU-F) assay 38
2.3. Bone marrow-derived Mesenchymal Stem Cells (BMSCs) isolation and culture 39
2.4. RT-PCR and real-time PCR analysis 39
2.5. Western blotting 44
2.6. Transwell migration assay 44
2.7. Flow cytometry analysis 45
2.8. CHIP-qPCR 45
2.9. Production and purification of recombinant APN 48
2.10. Antibodies and chemicals 48
2.11. Statistical analysis 49
3. RESULTS 50
3.1. APN deficiency increases the stemness, proliferation, and migration of BMSCs 50
3.2. HIF1α expression in APN KO BMSCs is associated with activation of STAT3 54
3.3. SCF increases HIF1α expression via STAT3 60
3.4. APN deficiency induces HIF1α accumulation by GSK3β inhibition 63
3.5. High expression of CD44 is associated with an increase in migration of APN KO BMSCs 67
4. DISCUSSION 71
CHAPTER Ⅲ. APN KO BMSCs with enhanced migratory ability inhibit EL-4 lymphoma growth 73
1. INTRODUCTION 74
2. MATERIALS AND METHODS 76
2.1. Bone marrow-derived Mesenchymal Stem Cells (BMSCs) isolation and culture 76
2.2. EL-4 bearing mouse models 76
2.3. Transwell migration assay 77
2.4. Chemokine panel (CCL8) and ELISA (APN and CCL8) 80
2.5. Western blotting 80
2.6. Preparation of cells 81
2.7. Immunohistochemistry 81
2.8. RT-PCR and real-time PCR analysis 82
2.9. Flow cytometry analysis 82
2.10. Antibodies and chemicals 83
2.11. Statistical analysis 83
3. RESULTS 84
3.1. Binding CD44 and HA is associated with the enhanced migration ability of APN KO BMSCs 84
3.2. CCL8 secreted from APN KO BMSCs recruits CD8⁺ T cells into the tumor mass 94
3.3. Enhanced migration of BMSCs from old APN KO mice suppresses the growth of EL-4 tumors 103
4. DISCUSSION 109
REFERENCES 113
ABSTRACT IN KOREAN 127
Table 1. List of primers for RT-PCR 41
Table 2. List of primers for real-time PCR 42
Table 3. List of CHIP-qPCR primers 47
Table 4. List of CCL8-specific siRNA sequences 79
Figure 1. Schematic representation illustrating the functional roles of BMAs during the aging process 19
Figure 2. Representative scheme of APN structure and receptors 22
Figure 3. Overview of the functional analysis of APN receptors 23
Figure 4. Scheme of the oxygen experience of MSCs depending on the in vivo niche 26
Figure 5. Schematic representation of the phenomena of MSCs under short- and long-term hypoxia exposure 27
Figure 6. Proposed schematic diagram of the migration of MSCs toward the tumor and injury site 30
Figure 7. Schematic diagram of the dual role of MSCs in the crosstalk with tumor tissue 31
Figure 8. Schematic representation of the active effect of APN from bone marrow adipocyte on the osteodifferentiation of BMSCs 33
Figure 9. The absence of APN in BMSCs promotes the population and proliferation 51
Figure 10. The deficiency of APN increases the stemness of BMSCs 52
Figure 11. The deficiency of APN facilitates the migratory ability of BMSCs 53
Figure 12. The mRNA and protein levels of HIF1α are increased in APN KO BMSCs 55
Figure 13. The increased HIF1α promotes the migration in APN KO BMSCs 56
Figure 14. The activation of STAT3 is responsible for HIF1α increase in APN KO BMSCs 57
Figure 15. The activation of STAT3 directly regulates the HIF1α promoter 59
Figure 16. Scf expression is increased in the APN KO BMSCs 61
Figure 17. HIF1α stimulates SCF expression 62
Figure 18. The absence of APN inhibits the activation of GSK3β 64
Figure 19. APN-induced GSK3β inactivation inhibits STAT3/HIFα axis 65
Figure 20. The activation of SCF/STAT3/HIF1α axis in APN KO BMSCs enhances CD44 expression 68
Figure 21. High expression of CD44 is associated with increase in migration of APN KO BMSCs 70
Figure 22. The migration of APN KO BMSCs is enhanced toward the EL-4 lymphoma 86
Figure 23. Inhibition of CD44 in EL-4 suppresses the migration of APN KO BMSCs 88
Figure 24. The growth of EL-4 lymphoma is suppressed in APN KO mice 90
Figure 25. BMSCs within EL-4 tumor stimulates the infiltration of CD8⁺ T cells 93
Figure 26. CD8⁺ T cells are more recruited into EL-4 tumor site 98
Figure 27. The CCL8 expression regulated by HIF1α is increased in APN KO BMSCs 100
Figure 28. CCL8 secreted from APN KO BMSCs recruits CD8⁺ T cells 101
Figure 29. The population of BMSCs is more increased in BMSC from old APN KO mice than young APN KO mice 105
Figure 30. The old APN KO BMSCs have more migratory ability than the young APN KO BMSCs 107
Figure 31. Old APN KO mice shows the most highly retarded EL-4 tumor growth via increase in infiltration of BMSCs and CD8⁺ T cells 108
Figure 32. Graphical summary 112