간섬유화에서 나타나는 간세포의 상피-중간엽세포 전이현상에 대한 연구
The epithelial-mesenchymal transition of hepatocyte in liver cirrhosis
- 주제(키워드) Liver cirrhosis , Hepatic dysfunction , Hepatocyte , Epithelial-mesenchymal transition (EMT) , Transforming growth factor-b (TGF-b) , 간경화 , 간부전 , 간세포 , 상피-중간엽세포 전이현상
- 발행기관 아주대학교 대학원
- 지도교수 김욱환
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 본문언어 영어
초록/요약
Liver cirrhosis is characterized by hepatic dysfunction with extensive accumulation of fibrous tissue in the liver. Even though the number of hepatocytes seems to be enough to maintain liver function, hepatic failure is one of the main causes of death in the patients with liver cirrhosis. The role of adult hepatocytes as contributors to the hepatocytes dysfunction and liver cirrhosis is yet undetermined. We surmised that epithelial-mesenchymal transition (EMT) of hepatocytes might contribute to both hepatocyte dysfunction and liver fibrogenesis. To address this issue, hepatocytes were isolated from normal rat liver and from dimethylnitrosamine (DMN)-induced fibrotic rat livers. Incubation of primary rat hepatocytes with TGF-β1, the central cytokine in fibrogenesis, and hepatocytes isolated from fibrotic rat liver showed decrease of epithelial cell markers such as c-Met, cytokeratin18 and zonula occludens-1 (ZO-1) in contrast to the increase of mesenchymal cell markers such as vimentin and α-SMA. Regarding the hepatocyte function, hepatocytes treated with TGF-β1 revealed a significant decrease in their ammonia-metabolizing activity and albumin production comparing to normal hepatocytes. Also, hepatocytes isolated from fibrotic liver revealed decrease in hepatic function. In addition, expression of the type I collagen was also increased in TGF-β1 treated hepatocytes and hepatocytes isolated from fibrotic liver. Interestingly, immunohistochemistry with fibrotic rat liver revealed the expression of TGF-β2 from inside fibrotic bands along with the expression of TGF-β2 from hepatocytes near the bands. Treatment of hepatocytes with TGF-β1 increased TGF-β2 secretion into the culture medium. Furthermore, in human liver cirrhotic tissues, albumin-positive cells, cytosolic E-cadherin and vimentin-positive cells were observed near the fibrotic bridges. Taken together, EMT of hepatocytes via TGF-β1 contributes to both the hepatocyte dysfunction and liver fibrosis
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―ABSTRACT― = ⅰ
LIST OF FIGURES = ⅴ
LIST OF TABLES = ⅵ
Ⅰ. INTRODUCTION = 1
A. Liver cirrhosis = 1
B. EMT (Epithelial-mesencymal transition) = 6
C. TGF-b and EMT = 9
D. Purpose of study = 9
II. MATERIALS AND METHODS = 11
A. Hepatocyte isolation and culture = 11
B. DMN-induced liver cirrhosis model = 12
C. Patients and paraffin-embedded tissue samples = 12
D. Western blot analysis = 14
E. Immunocytochemistry = 14
F. Isolation of RNA and RT-PCR = 15
G. Measurement of cellular viability = 17
H. Immunohistochemistry = 17
I. Assay for TGF-b1 and TGF-b2 = 18
J. Assay for ammonia metabolism = 18
K. Statistics = 19
Ⅲ. RESULTS = 20
A. Hepatocytes in primary culture go through spontaneous EMT = 20
B. TGF-b1 level increases in DMN-induced rat liver fibrosis = 23
C. Effect of TGF-b1 in hepatocytes = 25
D. TGF-b1 induces EMT of hepatocytes in vitro = 28
E. Decrease of hepatocyte function was accompanied with EMT = 31
F. Production of type I collagen from fibrotic hepatocytes = 36
G. Enhanced expression of TGF-b2 by EMT-transformed hepatocytes = 38
H. EMT of hepatocytes in human liver cirrhotic tissues = 41
IV. DISCUSSION = 44
V. CONCLUSION = 48
