Therapeutic Potential of Mesenchymal Stem Cells Overexpressing Human Foxa2 Gene in Regeneration of Damaged Liver Tissues
- 주제(키워드) mesenchymal stem cell , hepatocyte , Foxa2 gene , stem cell therapy
- 발행기관 고려대학교 대학원
- 지도교수 고용
- 발행년도 2012
- 학위수여년월 2012. 8
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 원문페이지 121 p
- 실제URI http://www.dcollection.net/handler/korea/000000034896
- 본문언어 영어
- 제출원본 000045717843
초록/요약
Several transcription factors and growth factors have been reported to play important roles in hepatocyte differentiation and regulation of hepatogenic gene expression. Especially, a transcription factor Foxa2 (hepatocyte nuclear factor 3┑) is known to control liver development during embryogenesis and to regulate gene expressions of adult liver cells. Based on the preliminary study that transient overexpression of Foxa2 up-regulates a number of liver-specific genes in rat mesenchymal stem cells (rMSCs), this study was conducted to assess the functional roles of Foxa2 in regulating cellular viability and liver-specific functions of rMSCs containing hFoxa2 gene after cell transplantation. Rat MSCs were isolated, characterized, and induced to hepatocytes, which expressed liver-specific markers. Then, rMSCs were transfected with human Foxa2 gene in pIRES-EGFP vector, induced into hepatogenic linage and observed expressions of several liver-specific genes, such as Foxa2, ┒-fetoprotein (AFP), CK-18, hepatocyte nuclear factor-1┒ (HNF-1┒), hepatocyte growth factor (HGF) and c-Met. Among transplanted different cell groups, rMSCs overexpressing hFoxa2 were most significantly effective in treating rat liver fibrosis, based on data from RT-PCR, biochemical and histopathological analyes. Taken together, this study indicates that hFoxa2 efficiently promoted the incorporation of MSCs into liver grafts and suggests that hFoxa2 genes could be used for structural or functional recovery of damaged liver cells.
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TABLE OF CONTENTS
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LIST OF TABLES∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼. ´
LIST OF FIGURES ...∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼.. ～
LIST OF ABBREVIATIONS ...∼∼∼∼∼∼∼∼∼∼∼∼∼∼.. ˘
ABSTRACT ∼...∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼.. ˚
CHAPTER I. INTRODUCTION ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼ 1
CHAPTER II. LITERATURE REVIEW ....∼∼∼∼∼∼∼∼∼∼.∼ 5
1. Mesenchymal stem cell ∼∼∼∼∼∼...∼∼∼∼∼∼∼.∼∼∼∼ 6
1.1. Characteristics of mesenchymal stem cell ∼∼∼∼∼∼∼∼.. 8
1.1.1. Morphology ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼ 8
1.1.2. Differentiation capacity ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼ 8
1.1.2.1. Adipogenic differentiation ∼∼∼∼∼...∼∼∼∼∼∼∼ 12
1.1.2.1.1. Adipocyte ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼ 12
1.1.2.1.2. Adipocyte differentiation ∼∼∼∼∼∼∼∼∼∼...∼... 13
1.1.2.1.3. Regulatory factors of adipogenesis ∼∼...∼∼∼∼∼... 17
1.1.2.1.3.1. Peroxisome proliferator？Cactivated receptor-┚ (PPAR-┚) ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼..∼∼... 17
1.1.2.1.3.2. CCAAT- enhancer binding proteins (C/EBPs) ∼∼.... 18
1.1.2.1.3.3. Mitochondrial uncoupling protein1 (UCP-1) ...∼∼... 20
1.1.2.1.3.4. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1┒) ∼∼∼∼..∼∼∼........ 22
1.1.2.2. Osteogenic differentiation ∼∼∼∼∼..∼∼∼∼∼∼∼. 23
1.1.2.2.1. Osteocyte ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼...∼∼. 23
1.1.2.2.2. Osteocyte differentiation ∼∼∼∼∼∼..∼∼∼∼∼∼ 25
1.1.2.2.3. Regulatory factors of osteogenesis ∼∼∼∼∼∼∼∼... 28
1.1.2.2.3.1. Bone morphogenetic proteins (BMPs) ∼∼∼∼∼∼.. 28
1.1.2.2.3.2. Runt-related transcription factor 2 (Runx2) ∼∼∼... 29
1.1.2.2.3.3. Osterix (Osx) ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼... 31
1.1.2.3. Hepatogenic differentiation ∼∼∼∼∼∼..∼∼∼∼∼.. 33
1.1.2.3.1. Hepatocyte ∼∼∼∼∼...∼∼∼∼∼∼∼∼∼∼∼∼.. 33
1.1.2.3.2. Hepatocyte differentiation ∼∼∼∼∼∼∼∼∼..∼∼. 34
1.1.2.3.3. Regulatory factors of hepatogenesis ∼∼∼∼...∼∼∼. 37
1.1.2.3.3.1. Hepatocyte growth factor (HGF) ∼∼∼∼∼∼∼∼.. 37
1.1.2.3.3.2. Oncostatin M (OSM) ∼∼∼∼∼∼∼∼∼∼∼∼∼.. 39
1.1.2.3.3.3. Hepatocyte nuclear factor 4 (HNF4) ∼∼...∼∼∼∼. 41
2. Forkhead box protein a2 (Foxa2) ∼∼∼∼∼∼∼...∼∼∼∼. 43
3. Liver disease ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼... 49
CHAPTER III. MATERIALS AND METHODS ∼∼...∼∼∼∼ 54
1. Isolation and culture of rMSC ∼∼∼∼∼∼∼...∼∼∼∼∼.. 55
2. In vitro differentiation of rMSC ∼∼∼∼∼∼...∼∼∼∼∼... 55
3. Transfection of cells ∼∼∼∼∼∼∼∼∼.∼..∼∼∼∼∼∼... 56
4. RNA isolation and RT-PCR analysis ∼∼∼.∼..∼∼∼∼∼∼ 57
5. Western bolt analysis ∼∼∼∼∼∼∼∼∼.∼..∼∼∼∼∼∼. 57
6. Immunocytochemistry ∼∼∼∼∼∼∼∼∼...∼∼∼∼∼∼... 58
7. CCl4-induced liver disease model and rMSCs transplantation ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼. 59
8. Histological analysis of the liver ∼∼∼∼∼∼∼..∼∼∼∼∼ 59
9. Biochemical analyses of serum ∼∼∼∼∼∼..∼∼∼∼∼∼.. 60
10. Stastical analyses ∼∼∼∼∼∼∼∼∼∼∼∼..∼∼∼∼∼.. 60
CHAPTER IV. RESULTS ∼∼∼∼∼∼∼∼∼∼∼∼.∼∼∼.. 62
1. Characterization of isolated rMSCs ∼∼∼∼..∼∼∼∼∼∼. 63
2. Hepatogenic differentiation of rMSCs ∼∼∼∼..∼∼∼∼∼. 65
3. Transfection of human Foxa2 gene into rMSCs ∼∼..∼∼∼.. 67
4. Induction of the CCl4-injured rat liver fibrosis model ∼..∼... 70
5. Transplantation of hFoxa2/MSCs and histopathology∼∼..∼... 72
6. Expression of liver-specific enzymes after cell transplantation ∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼∼...∼∼∼∼.. 74
CHAPTER V. DISCUSSION ∼∼∼∼∼..∼∼∼∼∼∼∼∼∼. 76
CHAPTER VI. REFERENCES ∼∼∼∼∼∼∼...∼∼∼∼∼∼∼. 82
