Title Page
Contents
ABSTRACT 12
GENERAL INTRODUCTION 15
I. Ovary 15
II. Placenta-derived mesenchymal stem cells 22
CHAPTER I. Effect of PD-MSCs on ovariectomized rat model 27
I. INTRODUCTION 28
II. MATERIALS & METHODS 33
1. Animals 33
2. Ovariectomized Rat Model Establishment 33
3. Cell Culture of PD-MSCs and Transplantation into an Ovariectomized Rat Model 34
4. Hormone Enzyme-linked immunosorbent assay (ELISA) Assay 37
5. Genomic DNA Isolation 37
6. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Analysis 38
7. Protein Isolation and Western Blot Analysis 39
8. Ovarian Follicle Counting Using Hematoxylin and Eosin (H&E) Staining 41
9. PKH67-Labeled PD-MSCs Tracking 41
10. Immunofluorescence Staining 42
11. Immunohistochemistry 42
12. Statistical Analysis 43
III. RESULTS 44
1. Transplanted PD-MSCs engraft into the damaged ovary of an ovariectomized rat model 44
2. PD-MSC transplantation reduced ROS accumulation in an ovariectomized rat model 46
3. PD-MSC transplantation enhanced the antioxidant effect in an ovariectomized rat model 48
4. PD-MSC transplantation attenuate the effect of apoptosis in an ovariectomized rat model 50
5. PD-MSC transplantation improve the hormone levels in an ovariectomized rat model 52
6. PD-MSC transplantation improve the gene expression related to folliculogenesis in an ovariectomized rat model 54
7. PD-MSC transplantation improved follicular development in an ovariectomized rat model 56
IV. DISCUSSIONS 58
V. CONCLUSION 61
CHAPTER II. Effect of human placenta - derived mesenchymal stem cells on thioacetamide - injured rat model 63
I. INTRODUCTION 64
II. MATERIALS AND METHODS 69
1. Construction of ovarian failure model by TAA injection 69
2. Cell culture of PD-MSCs and PKH 67 labeling 71
3. Ovarian explant culture for Ex-vivo 71
4. RNA isolation and qRT-PCR 72
5. Protein isolation and Western Blot 73
6. Enzyme-linked immunosorbent assay (ELISA) 74
7. Histological analysis 75
8. Immunohistochemistry 75
9. Immunofluorescence staining 75
10. Mito SOX and Mito Tracker staining 76
11. Statistical analysis 77
III. RESULTS 78
1. Transplanted PD-MSCs engrafted into damaged ovarian TAA-injured rat model 78
2. PD-MSC transplantation reduced inflammation factors in TAA-injured rat model 80
3. PD-MSC transplantation induced attenuation of ROS levels and higher antioxidant effects in TAA-injured rat model. 82
4. PD-MSC transplantation regulated mitochondrial dynamics in TAA-injured rat model 86
5. PD-MSC transplantation regulated mitochondrial biogenesis in TAA-injured rat model 88
6. PD-MSC transplantion promoted proliferation factors in TAA-injured rat model 90
7. PD-MSC transplantation promoted proliferation factors in TAA-injured rat model 92
8. PD-MSC transplantation promoted autophagy factors in TAA-injured rat model 94
9. PD-MSC transplantation improved the hormone levels in TAA-injured rat model 96
10. PD-MSCs improve the follicular development in TAA-injured rat model 98
11. PD-MSCs promote the folliculogenesis in TAA-injured rat model 100
12. PD-MSC cocultivation improved the estrogen and stem cell factor levels in supernatant of TAA-treated ovary (Ex-vivo) 102
13. PD-MSCs improve the ovary function in TAA-treated ovary (Ex-vivo) 104
14. PD-MSCs regulate autophagy pathway in TAA-treated ovary (Ex-vivo) 106
IV. DISCUSSIONS 108
V. CONCLUSION 111
VI. FINAL CONCLUSION 113
REFERENCES 115
ABSTRACT IN KOREAN 123
Figure 1. Describe the internal structure of human ovary. 16
Figure 2. Describe the menstrual cycle 20
Figure 3. Summary of PD-MSCs advantages. 24
Figure 4. Characterization of PDMSCs. 25
Figure 5. Immunomodulatory effects of MSCs derived from... 26
Figure 6. Reactive oxygen species in ovary. 30
Figure 7. Scheme of ovariectomized rat model construction. 36
Figure 8. Placenta-derived mesenchymal stem cells (PD-MSCs)... 45
Figure 9. Effect of PD-MSC transplantation on ROS... 47
Figure 10. Effect of PD-MSC transplantation on antioxidants in... 49
Figure 11. Effect of PD-MSC transplantation on apoptosis in the... 51
Figure 12. Effect of PD-MSC transplantation on hormone levels... 53
Figure 13. Effect of PD-MSC transplantation on follicular... 55
Figure 14. Effect of PD-MSCs transplantation on follicular... 57
Figure 15. The recovery pathway of ovarian dysfunction according to... 62
Figure 16. Simplified model for the formation of thioacetamide metabolites. 66
Figure 17. Scheme of TAA-injured rat model construction. 70
Figure 18. Effect of PD-MSCs on engraftment activity in... 79
Figure 19. Effect of PD-MSC transplantation on... 81
Figure 20. Effect of PD-MSC transplantation on ROS... 84
Figure 21. Effect of PD-MSC transplantation on antioxidants in... 85
Figure 22. Effect of PD-MSC transplantation on mitochondrial... 87
Figure 23. Effect of PD-MSC transplantation on mitochondrial... 89
Figure 24. Effect of PD-MSC transplantation on apoptosis in... 91
Figure 25. Effect of PD-MSC transplantation on proliferation in... 93
Figure 26. Effect of PD-MSC transplantation on autophagy in... 95
Figure 27. Effect of PD-MSC transplantation on hormone levels... 97
Figure 28. Effect of PD-MSCs on follicular development in... 99
Figure 29. Effect of PD-MSCs on folliculogenesis in... 101
Figure 30. Effect of PD-MSC cocultivation on necrosis, E2... 103
Figure 31. Effect of PD-MSC cocultivation on folliculogenesis in... 105
Figure 32. Effect of PD-MSC cocultivation on autophagy in... 107
Figure 33. The recovery pathway of ovarian dysfunction... 112
Figure 34. The recovery pathway of ovarian dysfunction... 114