간세포성장인자 발현 중간엽줄기세포의 간섬유화 치료
Therapeutic Effect of Hepatocyte Growth Factor-Secreting Mesenchymal Stem Cells in Established Rat Liver Fibrosis Model
- 주제(키워드) liver fibrosis , therapy , stem cells , hepatocyte growth factor , cell tracking
- 발행기관 아주대학교
- 지도교수 이재호
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000009540
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Purpose: Bone marrow-derived stem cells (BMSC) have been reported to be beneficial for the treatment of liver fibrosis in several studies. However, improvement in its efficacy is needed for this cell therapy to be considered as a practical therapy against liver fibrosis. Here, we investigated whether cell therapy using genetically engineered mesenchymal stem cells (MSC) to overexpress hepatocyte growth factor (HGF) resulted in a better therapeutic effect than MSC alone. The underlying mechanisms for the therapeutic effect and the bio-distribution of transplanted cells were also investigated. Materials & Methods: Liver fibrosis was induced by the intraperitoneal injection of dimethylnitrosamine. Adenovirus with HGF cDNA was infected to MSC to make HGF-overexpressing MSC (MSC/HGF). Normal or fibrotic rats were intra-splenically injected with the cells, and sacrificed 12 days after. Cell tracking was performed by using MSC radio-labelled with 111Indium. Results: Evidently, tissue fibrosis measured histochemically was reduced after MSC delivery, and even more by MSC/HGF, corroborating with elevated HGF levels in portal vein. Importantly, collagen levels from liver extract measured by Sirius red dye revealed the actual decrease after MSC/HGF delivery, suggesting the recovery of the fibrosis. HGF mRNA transcripts in liver and HGF protein in portal vein were significantly elevated by delivery of MSC/HGF. Furthermore, serum parameters showing the liver function were improved in MSC/HGF group, suggesting that transplantation with MSC/HGF resulted in not only the reduction of liver fibrosis but also the improvement of hepatocyte function. As for the underlying mechanisms, mRNA levels of the fibrogenic cytokines, PDGF-bb and TGF-β1, were significantly decreased after MSC/HGF delivery. Subsequent to the decrease of collagen, expressions of MMP-9, MMP-13, MT1-MMP and uPA were augmented by MSC/HGF, whereas TIMP-1 expression was reduced. It was of note that, even without immunosuppressant, a considerable amount of xeno-transplanted cells could survive for 12 days in both MSC and MSC/HGF groups. Both cells were found mainly in the spleen and the liver and additionally, in the lung of fibrosis group from 0 day and 2 day. Notably, xeno-transplanted cells were stably maintained only in fibrotic group. Conclusion: Cell therapy with MSC/HGF resulted in a better therapeutic effect than MSC alone probably through various anti-fibrotic activities of HGF and relatively stable maintenance of MSC. Thus, MSC/HGF would be a promising candidate as a cell therapy against liver fibrosis.
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TABLE OF CONTENTS
Title ⅰ
Abstract ⅱ
TABLE OF CONTENTS ⅳ
LIST OF FIGURES ⅵ
Ⅰ. INTRODUCTION 1
A. Liver fibrosis 1
B. Using stem cells for the treatment of liver fibrosis 2
C. Differentiation of BMSC in liver fibrosis; friend or foe? 3
D. The alternative explanation: therapeutic cytokine production 4
E. The purpose of this study 5
Ⅱ. MATERIALS AND METHODS 6
A. Preparation of HGF overexpressing human MSC 6
B. Animal grouping based on collagen content and cell transplantation 7
C. Biochemical analysis of serum 8
D. Liver histology and immunohistochemsitry 9
E. Hydroxyproline content assay 10
F. Western blotting 10
G. Reverse transcription- polymerase chain reaction (RT-PCR) 11
H. ELISA assay for human HGF 12
I. Gelatin zymography 13
J. Indium labeling 13
K. -camera monitoring 14
L. Statistical analysis 14
Ⅲ. RESULTS 16
Part. 1. Therapeutic effect of MSC/HGF in established rat liver fibrosis model 16
A. Preparation of MSC/HGF 16
B. Enhanced resolution of fibrosis by MSC/HGF 16
C. Attenuation of liver damage and enhancement of liver function by MSC/HGF 19
Part. 2. Mechanism of HGF therapeutic effects 21
A. Characterization of transplanted cells and determination of human HGF in vivo 21
B. Decrease of fibrogenic cytokine expression and inactivation of Ito cells by MSC/HGF 22
C. Effect on the extracellular matrix remodeling molecules in fibrosis resolution 24
Part. 3. In vivo cell tracking 26
A. Isotope labeling efficiency and radioactivity per cells or rat 26
B. Considerable survival of cells in vivo 27
C. Bio-distribution of the transplanted cells 27
Ⅳ. DISCUSSION 29
Ⅴ. CONCLUSION 34
BIBLIOGRAPHY 59
국문요약 65
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LIST OF FIGURES
Fig.1. Major phenomena that affect liver fibrosis 35
Fig.2. Anti-fibrosis effect of hepatocyte growth factor 36
Fig.3. Secretion of functional HGF by MSC/HGF 37
Fig.4. Fibrosis resolution 39
Fig.5. Recovery of serum parameters related with liver injury and function by MSC/HGF 42
Fig.6. Maintenance of transplanted cells 44
Fig.7. Decreased expression of fibrogenic cytokines by MSC/HGF 46
Fig.8. Enhanced inactivation of Ito cells by MSC/HGF 48
Fig.9. Effects on extracellular matrix remodeling molecules in the liver 50
Fig.10. Structure of tropolone and 111In(tropolone)3 53
Fig.11. Radiolabeling and transplantation of the cells 54
Fig.12. Whole-body images of rats transplanted with 111In–MSC or 111In-MSC/HGF 55
Fig.13. Survival of cells in whole body 56
Fig.14. Biodistribution of the transplanted cells over time 57
Fig.15. Schematic diagram showing the effects of MSC/HGF on rat liver fibrosis 58
