Title Page

Contents

Abstract 13

INTRODUCTION 15

1. Ovary 15

2. Ovarian dysfunction 24

3. Human placenta-derived mesenchymal stem cells 28

4. Generation of functional PD-MSCs (PD-MSCsPRL⁻¹)[이미지참조] 33

Ⅱ. MATERIALS AND METHODS 36

1. Cell culture and gene transfection 36

2. Construction of the OVX rat model and transplantation of stem cells 37

3. RNA isolation and quantitative real-time polymerase chain reaction 39

4. Ovarian explant ex-vivo culture 41

5. Nuclear fraction 41

6. Protein isolation and western blotting 42

7. ELISA 45

8. H&E staning for follicle counting 45

9. Immunohistochemistry staining 48

10. Immunofluorescence staining 48

11. HUVECs tube formation assay 49

12. HUVECs permeability dextran assay for vascular function 50

13. Statistical analysis 50

Ⅲ. RESULTS 51

1. Comparison of cytokine expressions between Naȉve PD-MSCs and PD-MSCsPRL⁻¹.[이미지참조] 51

2. Effect of PD-MSCsPRL⁻¹ on vascular function in ovary of OVX rat model.[이미지참조] 60

3. Effect of PD-MSCsPRL⁻¹ on ovary function in ovary of OVX rat model.[이미지참조] 74

4. Therapeutic mechanism of PD-MSCsPRL⁻¹ on ovary function in ovary of OVX rat model via PDGF signaling pathway.[이미지참조] 80

Ⅳ. DISCUSSIONS 90

Ⅴ. CONCLUSION 95

REFERENCES 97

ABSTRACT IN KOREAN 107

Table 1. Comparison of ovarian structure in normal and abnormal ovary. 26

Table 2. Summary on preclinical modeling on premature ovarian failure. 27

Table 3. Summary on preclinical study of mesenchymal stem cell therapy on premature ovarian failure. 32

Table 4. List of primer used for qRT-PCR analysis 40

Table 5. List of primary antibody used for Western blot analysis 44

Table 6. Follicle counts in ovary tissue depend on transplantation. 79

Figure 1. The structure of the human ovary. 16

Figure 2. The stage of ovarian follicular development. 17

Figure 3. The structure between follicles and blood vessel in an ovarian. 20

Figure 4. Characteristic of vascular dysfunction in ovarian. 21

Figure 5. The blood vessels affect follicular development. 22

Figure 6. The stucture and role of pericyte. 23

Figure 7. The features of placenta-derived mesenchymal stem cells (PD-MSCs). 31

Figure 8. Advantage of enhanced MSCs through gene modification. 35

Figure 9. Scheme of ovariectomized rat model construction. 38

Figure 10. The classification of follicular stages for follicle counting analysis. 47

Figure 11. The cytokine assay of PD-MSCs and PD-MSCsPRL⁻¹.[이미지참조] 52

Figure 12. Secretion of PDGF-BB cytokine in Naȉve PD-MSCs and PRL-1 overexpressed PD-MSCs. 53

Figure 13. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on PDGF activity in OVX rat model. [이미지참조] 57

Figure 14. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on PDGF expression in OVX rat model.[이미지참조] 58

Figure 15. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on PDGF downstream in OVX rat model. [이미지참조] 59

Figure 16. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on artery structure in OVX rat model.[이미지참조] 61

Figure 17. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on VEGF expression in OVX rat model.[이미지참조] 63

Figure 18. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on angiogenesis in OVX rat model.[이미지참조] 64

Figure 19. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on pericyte recruitment via PDGF in OVX rat model.[이미지참조] 68

Figure 20. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on autophagy in OVX rat model.[이미지참조] 69

Figure 21. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on vascular permeability in OVX rat model.[이미지참조] 70

Figure 22. Scheme of in-vitro co-cultivation. 72

Figure 23. Effect of PD-MSCs and PD-MSCsPRL⁻¹ co-cultivation on vascular function in 5-FU treated HUVECs.[이미지참조] 73

Figure 24. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on sex hormone levels in OVX rat model.[이미지참조] 76

Figure 25. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on follicular development in OVX rat model.[이미지참조] 77

Figure 26. Effect of PD-MSCs and PD-MSCsPRL⁻¹ transplantation on follicular development in OVX rat model.[이미지참조] 78

Figure 27. Scheme of ex-vivo co-cultivation treated with siPRL-1. 83

Figure 28. Effect of PD-MSCs and PD-MSCsPRL⁻¹ on PDGF expression in siPRL-1 injected ovary.[이미지참조] 84

Figure 29. Effect of PD-MSCs and PD-MSCsPRL⁻¹ on follicular development in siPRL-1 injected ovary.[이미지참조] 85

Figure 30. Scheme of ex-vivo co-cultivation treated with PDGFR inhibitor / PDGF recombinant. 88

Figure 31. Effect of PD-MSCs and PD-MSCsPRL⁻¹ co-cultivation on PDGF expression in PDGFR inhibitor/PDGF-BB recombinant treated ovary.[이미지참조] 89

Figure 32. The recovery pathway of vascular dysfunction in an ovarian according to PD-MSCsPRL⁻¹ transplantation.[이미지참조] 96