Title Page
Contents
ABSTRACT 12
I. INTRODUCTION 16
1. Pregnancy 16
2. Parturition 18
3. Preterm 20
4. Melatonin 22
5. Macrophage 25
II. MATERIALS AND METHODS 28
1. Animals 28
2. Establishment of a mouse model of preterm birth 28
3. Macrophage depletion using clodronate liposome 29
4. Cell culture 29
5. Isolation of bone marrow-derived macrophage 30
6. RNA preparation, RT-PCR, and real-time RT-PCR 31
7. Protein extraction and Western blotting 32
8. Flow cytometry 32
9. Immunocytochemistry 33
10. Analysis of cytokine array 34
11. Gene ontology enrichment analysis 35
12. Statistical analysis 35
III. RESULTS 43
1. Inflammation in the uterus provokes the inflammation-induced preterm birth (IPB) in mice 43
2. The population of various immune cells fluctuate before the IPB 50
3. M2 macrophage is reduced in the uterus in mice and women with the preterm birth 58
4. Partial depletion of uterine macrophage induced the preterm birth 64
5. Systemic administration of melatonin prevents the preterm birth, the adverse prenatal outcomes, and the production of pro-inflammatory cytokines in the IPB 68
6. Melatonin only restores the balance of M1/M2 ratio in the uterus of the mice with the IPB 75
7. Melatonin reduces the expression of factors to induce muscle contraction in macrophage 84
8. Melatonin suppresses the expression of contractile factors in the uterus of the mice with the IPB 92
9. Melatonin inhibits the M1 macrophage via p38 MAPK-Yap1 signaling 95
10. LPS do not disrupt the balance of M1/M2 ratio in macrophage-specific Yap1-knockout mice, resulting in term labor 100
IV. DISCUSSION 107
V. CONCLUSION 116
References 119
ABSTRACT IN KOREAN 144
Table 1. primer sequences for genotyping 36
Table 2. primer sequences for RT-PCR and real-time RT-PCR 37
Table 3. List of primary antibodies 39
Table 4. List of secondary antibodies 41
Table 5. List of antibodies for flow cytometry 42
Figure 1. Establishment of a murine model of the inflammation-induced... 46
Figure 2. The increased expression of pro-inflammatory cytokines... 49
Figure 3. The population of various immune cells in the maternal-fetal... 53
Figure 4. The fluctuated population of immune cells in the MFI before... 57
Figure 5. Comparison of gene ontology in women with the term or... 61
Figure 6. Reduction of the M2 macrophage in the mice with the IPB 63
Figure 7. Partial depletion of uterine macrophage resulting in the... 67
Figure 8. Prevention of the IPB after administration of melatonin 70
Figure 9. The anti-inflammatory effect of melatonin in the MFI of the... 74
Figure 10. Melatonin-induced repolarization of macrophage 79
Figure 11. Restoration for the population of the total, M1 and M2... 83
Figure 12. The decrease of contractile character in the uterine smooth... 88
Figure 13. Analyses of cytokine arrays with conditioned medium of... 91
Figure 14. The decreased expression of factors related to muscle.. 94
Figure 15. Inhibition of the phosphorylation of the factors related M1... 99
Figure 16. Decreased susceptibility to lipopolysaccharide (LPS) in... 104
Figure 17. Maintenance of the M1/M2 ratio after LPS injection in... 106
Figure 18. A schematic diagram illustrating a concept for the... 118