Title Page

Contents

Abstract 12

Ⅰ. INTRODUCTION 14

1. Liver 14

2. Mechanism of liver diseases 15

3. ER stress in liver diseases 19

4. Perturbation of ER to mitochondria calcium status in liver diseases 21

5. Correlation between calcium and RTK 25

6. Status of liver disease treatment 26

7. Stem cell therapy 27

Ⅱ. MATERIALS AND METHODS 31

1. Cell culture 31

2. Animal models and transplantation of PD-MSCs 32

3. Isolation of Primary hepatocyte 33

4. Liver histology and serum biochemistry 36

5. Immunofluorescent staining 36

6. Immunohistochemical staining 37

7. Quantitative real-time PCR 38

8. Western blotting 40

9. Calcium influx using a fluorescence resonance energy transfer (FRET) biosensor 42

10. Phospho-RTK proteome profiler array 42

11. Statistical analysis 43

Ⅲ. RESULTS 44

1. Effect of PD-MSCs PRL⁻¹ on ER stress in a rat model of BDL and hepatocytes[이미지참조] 44

2. Effect of PD-MSCs PRL⁻¹ on calcium channels in a rat model of BDL and hepatocytes[이미지참조] 50

3. Effect of PD-MSCs PRL⁻¹ on calcium influx in rat hepatocytes[이미지참조] 58

4. Effect of PD-MSCs PRL⁻¹ on EGFR-PI3K-CaM calcium signaling in BDL-injured rat liver[이미지참조] 61

5. Hepatic regenerative effects of PD-MSCs PRL⁻¹ in a rat model of BDL[이미지참조] 65

Ⅳ. DISCUSSION 69

Ⅴ. CONCLUSION 75

REFERENCES 77

ABSTRACT IN KOREAN 85

Table 1. Survival rate of BDL-injured rat model. 35

Table 2. Primer sequences using quantitative RT-PCR for calcium channels and calcium signaling. 39

Table 3. A list of antibody used for Western blot analysis 41

Figure 1. Liver structure. 17

Figure 2. Mechanism of liver diseases. 18

Figure 3. ER stress mechanism in liver diseases. 20

Figure 4. Perturbation of ER calcium channels in hepatocytes. 23

Figure 5. Perturbation of MT calcium channels in hepatocytes 24

Figure 6. Effect of Placenta-derived mesenchymal stem cells (PD-MSCs). 29

Figure 7. Role of PRLs signaling pathway 30

Figure 8. Schematic diagram of BDL-injured rat modeling. 34

Figure 9. Immunohistochemistry detection of CHOP in BDL-injured rat model. 46

Figure 10. In vivo protein levels of PERK, p/t-eIF2α, ATF4, and CHOP by Western blot analysis. 47

Figure 11. Schematic diagram of ER stress on calcium depletion and o in vitro) experimental outline. 48

Figure 12. In vitro protein levels of PERK, p/t-eIF2α, ATF4, and CHOP by Western blot analysis. 49

Figure 13. In vivo protein levels of IP3R, SERCA2b, VDAC1, and MCU by Western blot analysis. 52

Figure 14. mRNA expression levels of IP3R, VDAC1, and MCU by qRT-PCR in BDL-injured rat model. 53

Figure 15. In vitro protein levels of IP3R, VDAC1, and MCU by Western blot analysis. 54

Figure 16. mRNA expression levels of IP3R, VDAC1, and MCU by qRT-PCR in rat hepatocytes treated with LCA. 55

Figure 17. mRNA expression levels of IP3R, VDAC1, and MCU by qRT-PCR in rat hepatocytes treated with LCA and recombinant... 56

Figure 18. ER calcium channel SERCA2b co-localized with ER tracker in BDL-injured rat model. 57

Figure 19. mRNA expression levels of SERCA sub-units in BDL-injured rat model. 59

Figure 20. Calcium FRET biosensor in rat hepatocytes. 60

Figure 21. PRL-1 binding EGFR receptor by Dot blot assay. 62

Figure 22. PRL-1 regulating EGFR-PI3K-CaM calcium signal pathway in BDL-injured rat model and rat hepatocytes. 63

Figure 23. PD-MSCPRL⁻¹ co-cultivation declines expression of CaM in BDL-injured rat model.[이미지참조] 64

Figure 24. PD-MSCPRL⁻¹ transplantation alleviates liver fibrosis in BDL-injured rat model.[이미지참조] 66

Figure 25. PD-MSC PRL⁻¹ transplantation improves hepatic function in BDL-injured rat model.[이미지참조] 67

Figure 26. PD-MSCs PRL⁻¹ transplantation increase proliferative activity in BDL-injured rat model.[이미지참조] 68

Figure 27. Summarized diagram of therapeutic mechanisms by PD-MSCs PRL⁻¹ in BDL-injured rat model.[이미지참조] 76