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Effect of HBX on oxidative stress and apoptosis in hepatocellular carcinoma.

Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326193
Date January 2007
ContributorsLeung, Chung Yan., Chinese University of Hong Kong Graduate School. Division of Surgery.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xv, 116 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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