Title Page
Contents
ABSTRACT 15
GENERAL INTRODUCTION 18
CHAPTER I. Establishment of function-enhanced mesenchymal stem cells from bone marrow and placenta using lentiviral and nonviral gene delivery systems 22
I. INTRODUCTION 23
II. MATERIALS & METHODS 29
1. Plasmid and lentiviral vector construction 29
2. Cell culture and gene transfection 29
3. Reverse transcription and quantitative PCR analysis 30
4. Western blotting 33
5. FACS analysis 33
6. Multi-differentiation 34
7. Teratoma formation 35
8. XF mitostress assay 35
9. Lactate assay 36
10. Statistical analysis 37
III. RESULTS 38
1. Establishment of BM- and PD-MSCsPRL-1 using lentiviral and nonviral AMAXA gene delivery systems.[이미지참조] 38
2. Characterizations 41
3. Differentiation 44
4. Teratoma formation 47
5. Cellular metabolism 50
IV. DISCUSSIONS 53
CHAPTER II. Epithelial-to-mesenchymal transition mechanism by PD-MSCsPRL-1 using a nonviral system in a cirrhotic rat model with bile duct ligation[이미지참조] 57
I. INTRODUCTION 58
II. MATERIALS AND METHODS 61
1. Cell culture 61
2. RNA interference and inhibitor 61
3. Animal models and MSC transplantation 62
4. Isolation of primary hepatocyte 63
5. Histopathological analysis and blood chemistry 63
6. Immunohistochemistry & immunofluorescence 64
7. Enzyme-linked immunosorbent assay (ELISA) 65
8. Western blotting 65
9. quantitative PCR analysis 66
10. Nuclear-cytoplasmic fraction 66
11. Statistical analysis 67
III. RESULTS 69
1. Increased engraftment into injured liver tissues by transplantation of PD-MSCsPRL-1 using a nonviral gene delivery system in a rat model with bile duct ligation (BDL)[이미지참조] 69
2. Significantly anti-fibrotic effects by PD-MSCsPRL-1 in a cirrhotic rat model[이미지참조] 72
3. Hepatic functions by PD-MSCsPRL-1 in a BDL rat model[이미지참조] 74
4. Anti-apoptotic effects by PD-MSCsPRL-1 in a BDL-injured rat model[이미지참조] 76
5. Increased proliferative activity by PD-MSCPRL-1 transplantation in a BDL rat model[이미지참조] 78
6. PD-MSCsPRL-1 decrease mRNA expression of EMT markers in BDL-injured rat liver[이미지참조] 80
7. PD-MSCsPRL-1 decrease protein levels of EMT markers in BDL-injured rat liver[이미지참조] 82
8. PD-MSCPRL-1 co-cultivation declines expression of EMT markers in primary hepatocyte[이미지참조] 85
9. PD-MSCsPRL-1 increase MET markers in a cirrhotic rat liver[이미지참조] 87
10. PD-MSCPRL-1 transplantation induces MET markers in cirrhotic rat liver through BMP7/SMAD1/5 signaling[이미지참조] 89
11. PD-MSCsPRL-1 induce MET markers in primary hepatocyte[이미지참조] 91
12. PD-MSCsPRL-1 enhance MET phenotype regulating BMP7/SMAD1/5 pathway in primary hepatocyte[이미지참조] 93
13. PRL-1 regulates SMAD family through BMP7 95
IV. DISCUSSIONS 98
CHAPTER III. Hepatic metabolism by PD-MSCsPRL-1 using a nonviral system in a cirrhotic rat model with bile duct ligation[이미지참조] 102
I. INTRODUCTION 103
II. MATERIALS AND METHODS 107
1. Cell culture 107
2. RNA interference and inhibitor 107
3. Animal models and MSC transplantation 108
4. Isolation of primary hepatocyte 108
5. Histological staining and blood chemistry 109
6. Immunofluorescence 110
7. Glucose uptake assay 110
8. Western blotting 111
9. Quantitative PCR analysis 111
10. Cell proliferation assay (CCK-8) 112
11. Enzyme-linked immunosorbent assay (ELISA) 112
12. Fluorescence resonance energy transfer (FRET) biosensor 113
13. Statistical analysis 113
III. RESULTS 115
1. PD-MSCsPRL-1 are dominant under metabolic states compared to naïve[이미지참조] 115
2. PD-MSCPRL-1 transplantation restores insulin levels and glycogen storage[이미지참조] 117
3. PD-MSCPRL-1 co-culture regulates glucose uptake in hepatocyte[이미지참조] 119
4. PD-MSCPRL-1 transplantation improves hepatic glycolysis[이미지참조] 121
5. PD-MSCPRL-1 co-culture increases glycolysis by activating ChREBP expression in hepatocyte[이미지참조] 124
6. Administration of PD-MSCsPRL-1 is responsible for fatty acid oxidation[이미지참조] 126
7. PD-MSCsPRL-1 stimulate activation of AMPK in BDL-injured rat liver[이미지참조] 129
8. recombinant PRL-1 activates AMPK expression 131
9. PRL-1 regulates glucose metabolism through AMPK activation in hepatocyte 133
IV. DISCUSSIONS 135
V. CONCLUSION 140
REFERENCES 142
ABSTRACT IN KOREAN 152
Table 1. Primer sequences using RT-PCR for stemness markers 31
Table 2. Primer sequences using quantitative RT-PCR for differentiation 32
Table 3. Previous reports about improved effects by function-enhanced... 56
Table 4. Primer sequences using quantitative RT-PCR for EMT markers 68
Table 5. Primer sequences using quantitative RT-PCR for metabolism 114
Figure 1. Placenta-derived mesenchymal stem cells (PD-MSCs). 26
Figure 2. Advantages of function-enhanced MSCs. 27
Figure 3. Role of PRLs signaling pathway. 28
Figure 4. Generation of PRL-1 function-enhanced in BM-MSCs using... 39
Figure 5. Generation of PRL-1 function-enhanced in PD-MSCs using... 40
Figure 6. Characterizations of BM-MSCsPRL-1 using lentiviral and...[이미지참조] 42
Figure 7. Characterizations of PD-MSCsPRL-1 using lentiviral and...[이미지참조] 43
Figure 8. Differentiation in BM-MSCsPRL-1 using lentiviral and nonviral...[이미지참조] 45
Figure 9. Differentiation in PD-MSCsPRL-1 using lentiviral and nonviral...[이미지참조] 46
Figure 10. Teratoma formation in BM-MSCsPRL-1 using lentiviral and...[이미지참조] 48
Figure 11. Teratoma formation in PD-MSCsPRL-1 using lentiviral and...[이미지참조] 49
Figure 12. Cellular metabolic states of BM-MSCsPRL-1 using lentiviral and...[이미지참조] 51
Figure 13. Cellular metabolic states of PD-MSCsPRL-1 using lentiviral and...[이미지참조] 52
Figure 14. in vivo Schematic diagram by PD-MSCsPRL-1 using a nonviral...[이미지참조] 70
Figure 15. Increased engraftment into injured liver tissues by... 71
Figure 16. In vivo nonviral PD-MSCsPRL-1 alleviate liver fibrosis in a rat...[이미지참조] 73
Figure 17. Improved hepatic functions by PD-MSCsPRL-1 in a rat model...[이미지참조] 75
Figure 18. Anti-apoptotic effects by PD-MSCsPRL-1 in a BDL-injured rat model.[이미지참조] 77
Figure 19. Increased proliferative activity by PD-MSCPRL-1 transplantation...[이미지참조] 79
Figure 20. PD-MSCsPRL-1 decrease mRNA expression of EMT markers in...[이미지참조] 81
Figure 21. PD-MSCsPRL-1 decrease protein levels of EMT markers in...[이미지참조] 83
Figure 22. Schematic diagram of PD-MSCPRL-1 co-culture with primary hepatocyte.[이미지참조] 84
Figure 23. PD-MSCPRL-1 co-cultivation declines expression of EMT...[이미지참조] 86
Figure 24. PD-MSCsPRL-1 increase MET markers in a cirrhotic rat liver.[이미지참조] 88
Figure 25. PD-MSCPRL-1 transplantation induces MET markers in cirrhotic...[이미지참조] 90
Figure 26. PD-MSCsPRL-1 induce MET markers in primary hepatocyte.[이미지참조] 92
Figure 27. PD-MSCsPRL-1 enhance MET phenotype regulating...[이미지참조] 94
Figure 28. PRL-1 regulates BMP7 96
Figure 29. PRL-1 regulates SMAD family. 97
Figure 30. PD-MSCsPRL-1 are dominant under metabolic states compared...[이미지참조] 116
Figure 31. PD-MSCPRL-1 transplantation restores insulin levels and...[이미지참조] 118
Figure 32. PD-MSCPRL-1 co-culture regulates glucose uptake in hepatocyte.[이미지참조] 120
Figure 33. PD-MSCPRL-1 transplantation improves hepatic glycolysis.[이미지참조] 122
Figure 34. PD-MSCsPRL-1 activate ChREBP expression.[이미지참조] 123
Figure 35. PD-MSCPRL-1 co-culture increases glycolysis by activating...[이미지참조] 125
Figure 36. Administration of PD-MSCsPRL-1 decreases lipid accumulation in...[이미지참조] 127
Figure 37. Administration of PD-MSCsPRL-1 is responsible for fatty acid oxidation.[이미지참조] 128
Figure 38. PD-MSCsPRL-1 stimulate activation of AMPK in BDL-injured rat liver.[이미지참조] 130
Figure 39. recombinant PRL-1 activates AMPK expression. 132
Figure 40. PRL-1 regulates glucose metabolism through AMPK activation... 134
Figure 41. Summarized diagram of therapeutic mechanisms by... 141