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Hepatitis B virus X gene differentially modulate the cell cycle and apoptotic proteins in normal liver and hepatoma cell lines

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Abstract
1. PART Ⅰ
HBx 는 B형간염바이러스(HBV) 감염에 필수적이며, HBV 증식, HBV 감염에 의한 간질환 및 간암(HCC)의 발달에 영향을 미친다. 그러나 HBx의 정확한 역할은 아직 논란이 되고 있다. 본 연구에서, 우리는 논란이 되고 있는 HBx의 역할에 대해 일반 간세포와 간암세포주에서 HBx에 의한 세포주기와 세포자멸사 조절에 초점을 맞추고 실험을 하였다.
우리는 본 연구를 위해 HBx가 지속적으로 발현하는 Huh7-X 와 Chang-X 세포주를 만들었으며, HBx 단백질은 각 세포에서 세포주기와 p27, TGF-β 발현을 다르게 조절 하는 것을 보여주었다. 더욱이, HBx는 Chang-X 세포에서 특이적으로 Bcl-2 의 발현을 증가시켰으며, Cleaved PARP 단백질의 형성을 감소 시켰다. 그리고 HBx는 Chang 과 Huh7 세포에서 serum starvation과 같은 세포죽음을 발생할 수 있는 조건에서 세포자멸사를 억제시켰다. 이러한 결과는, HBx가 간암세포와 일반 간세포에서 다르게 세포주기와 세포자멸사를 조절 하는 것을 나타냈으며, 이는 논란이 되고 있는 HBx의 세포내 역할에 대한 단서를 제공 하였다.

2. PART Ⅱ
Hepatocellular carcinoma (HCC)는 일반적으로 발생빈도가 높은 암 중의 하나이며, HCC 와 HCC와 관련된 질병은 B형 간염바이러스(HBV)가 원인이 된다. HBV 는 basal core promoter (BCP) 지역에 종종 변이가 발생 하며, 이 지역은 HBx의 open reading frame (ORF)과 겹쳐져 발생한다. 또한 HBx 는 간암 발생에 있어 중요한 역할을 한다. 본 연구에서는 Huh-7 간암세포에서의 HBx 변이(BCP1) 의 영향에 대하여 조사를 하였다. 우리는 clinical samples 로 부터 HBx의 변이를 클로닝 하였으며, 이들을 Huh-7 세포에 일시적 또는 안정적으로 transfection 시켜 실험을 하였다. HBx 변이체(BCP1) 는 HBx 에 비해 특이적으로 CDK inhibitor p27kip1 의 발현을 감소 시켰으며, 세포주기를 더욱 촉진 시켰다. 또한 BCP1 은 HBx 에 비해 cleaved PARP 의 발생을 억제시켰으며, multifunctional cytokine인 TGF-β는 발현을 더욱 감소 시켰다. 따라서 BCP1 변이체는 HBx에 비해 Huh-7 세포에서 더욱 세포주기를 촉진시키며 세포자멸사를 억제하였으며, 이는 아마 TGF-β 의 발현 감소를 필요로 할 것이라는 것을 나타내었다.
1. ABSTRACT
HBx is essential for early virus infection, HBV genome replication, HBV associated liver dieses and development of hepatocellular carcinoma(HCC). However, a exact role of HBx is still controversy. In this study, we focus and studied controversial role of HBx at the regulation of cell cycle and apoptosis in normal liver and hepatoma cell lines. We established a Huh7-X and Chang-X cell line that constitutively expresses HBx mRNA and protein. HBx proteins showed some difference in cell cycle regulation and expression of p27 and TGF-β between each cell lines. In addition, HBx proteins dramatically increased expression of Bcl-2 and reduced Cleaved PARP protein in Chang-X cells and inhibited the apoptosis in unfavorable condition such as serum starvation in Chang and Huh7 cells. Our findings demonstrate that HBx does a different role on to control of cell cycle progression and apoptosis in hepatoma cell and normal liver cell lines and provide some clues at the controversial issue of intracellular roles of HBx.

2. ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers. HCC and HCC–associated disease are caused by hepatitis B virus (HBV). HBV often occur mutation at basal core promoter (BCP), which is overlapped with open reading frame (ORF) of hepatitis B virus X (HBx) protein. The HBx protein plays very important role in development of liver cancer. In this study, we have investigated the influence that a mutation of HBx (BCP1) had on to cell cycle in Hepatocellular carcinoma cell line, Huh-7. We has cloned mutant of HBx from clinical samples, and then transiently and stably transfected into Huh-7. The mutant HBx (BCP1) protein especially reduced expression of CDK inhibitor p27kip1 and more promoted the cell cycle progression than HBx. BCP1 also inhibited the production of cleaved PARP as a marker for apoptosis than HBx in Huh7 cells. In addition, multifunctional cytokine TGF-β was more down-regulated by BCP1 mutant. Therefore, BCP1 mutation more promotes the cell cycle progression and inhibits the apoptosis than HBx in Huh-7 cells, which may require the down-regulation of TGF-β.
Author(s)
양창희
Issued Date
2011
Awarded Date
2012. 2
Type
Dissertation
URI
https://oak.jejunu.ac.kr/handle/2020.oak/20403
Alternative Author(s)
Yang, Chang Hee
Affiliation
제주대학교
Department
대학원 의학과
Advisor
조문제
Table Of Contents
CONTENTS ⅰ
LIST OF FIGURES ⅱ
I. PART I . Hepatitis B virus X gene differentially modulate the cell cycle and apoptotic proteins in normal liver and hepatoma cell lines 1
1.ABSTRACT 2
2.INTRODUCTION 3
3.MATERIALS AND METHODS 6
3.1. Cell culture and transfection 6
3.2. Plasmids 6
3.3 Western blots and antibodies 7
3.4 Cell cycle analysis by flow cytometry 8
3.5 Intracellular localization 8
3.6 RT-PCR analysis 9
4. RESULTS 10
4.1. HBx protein was localized in cytoplasm and around the nuclear membrane in Huh7 and Chang cell lines 10
4.2. HBx differently regulate the expression of cyclin-dependent kinase inhibitor p27 proteins and cell cycle progression in Huh7-X and Chang-X cells 13
4.3. TGF-β mRNA expression was down-regulated in Huh7-X cells and up-regulated in Chang-X cells 17
4.4. HBx inhibit production of cleaved PARP as a marker for apoptosis protein via up-regulation of Bcl-2 in Chang cells 21
5. DISCUSSION 26
Ⅱ. PART Ⅱ. Hepatitis B virus X gene differentially modulate the cell cycle and apoptotic proteins in normal liver and hepatoma cell lines 30
1. ABSTRACT 31
2. INTRODUCTION 32
3. MATERIALS AND METHODS 35
3.1. Cell culture and transfection 35
3.2. Plasmids 35
3.3. Western blots and antibodies 36
3.4. Cell cycle analysis by flow cytometry 37
3.5. Intracellular localization 37
3.6. RT-PCR analysis 38
4. RESULTS
4.1. Intracellular localization of HBx and BCP1 39
4.2. BCP1 mutant especially down-regulate 42
4.3. BCP1 mutant inhibits production of cleaved PARP 47
4.4. Huh7-BCP1 cells more down-regulated expression of TGF-β 49
5. DISCUSSION 52
Ⅲ. REFERENCES 56
Ⅳ. 요약문 64
Degree
Master
Publisher
제주대학교 대학원
Citation
[1]양창희, “Hepatitis B virus X gene differentially modulate the cell cycle and apoptotic proteins in normal liver and hepatoma cell lines,” 제주대학교 대학원, 2011.
Appears in Collections:
General Graduate School > Medicine
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