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.IIIAcknowledgements --- p.VList of figures --- p.VIList of tables --- p.VIIIAbbreviations --- p.IXTable of Contents --- p.XIIChapter Chapter 1: --- IntroductionChapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1Chapter 1.3 --- HBV genome structure --- p.2Chapter 1.4 --- HBV pathogenesis --- p.2Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3Chapter 1.6 --- Oxidative stress and antioxidant --- p.5Chapter 1.6.1 --- Glutathione (GSH) --- p.5Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8Chapter 1.7 --- Apoptosis and necrosis --- p.9Chapter 1.7.1 --- Apoptotic pathways --- p.9Chapter 1.8 --- Role of HBx in apoptosis --- p.10Chapter 1.9 --- Transcriptional activity by HBx --- p.12Chapter 1.10 --- Chemotherapy drug resistance --- p.13Chapter 1.11 --- Objectives of study --- p.14Chapter Chapter 2: --- Methods and materialsChapter 2.1 --- Construction of plasmid --- p.23Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23Chapter 2.1.2 --- Agarose gel extraction --- p.25Chapter 2.1.3 --- Restriction enzyme digestion --- p.26Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29Chapter 2.2 --- Transfection --- p.30Chapter 2.2.1 --- Cell line --- p.30Chapter 2.2.2 --- Lipofectamine transfection --- p.31Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32Chapter 2.3.1 --- RNA extraction --- p.32Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36Chapter 2.4.1 --- Sample preparation --- p.36Chapter 2.4.2 --- Measurement of protein concentration --- p.36Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38Chapter 2.4.5 --- Immunoblotting of protein --- p.38Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39Chapter 2.5.1 --- Sample preparation --- p.39Chapter 2.5.2 --- Measurement of GSH concentration --- p.39Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40Chapter 2.6.1 --- Sample preparation --- p.40Chapter 2.6.2 --- Measurement of total SOD activity --- p.41Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42Chapter 2.7 --- Cell proliferation assay --- p.43Chapter 2.7.1 --- Drugs and concentration --- p.43Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44Chapter 2.8.1 --- Cell culture --- p.44Chapter 2.8.2 --- Cell fixation --- p.45Chapter 2.8.3 --- Cell staining --- p.45Chapter 2.8.4 --- Flow cytometry analysis --- p.46Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46Chapter 2.9.1 --- Antibodies --- p.46Chapter 2.9.2 --- Sample Preparation --- p.47Chapter 2.9.3 --- Measurement of protein concentration --- p.48Chapter 2.9.4 --- Western blotting --- p.49Chapter Chapter 3: --- Establishment of HBx transfected stable cell linesChapter 3.1 --- Introduction --- p.55Chapter 3.2 --- Results --- p.56Chapter 3.2.1 --- Plasmid construction --- p.56Chapter 3.2.2 --- Stable transfection of cell lines --- p.57Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58Chapter 3.3 --- Discussion --- p.58Chapter Chapter 4: --- Antioxidant level in HBx transfected cell linesChapter 4.1 --- Introduction --- p.68Chapter 4.2 --- Results --- p.70Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71Chapter 4.2.2.1 --- Total SOD activity --- p.71Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71Chapter 4.2.2.3 --- MnSOD --- p.72Chapter 4.3 --- Discussion --- p.72Chapter Chapter 5: --- Involvement of HBx in apoptotic pathwayChapter 5.1 --- Introduction --- p.81Chapter 5.2 --- Results --- p.82Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84Chapter 5.3 --- Discussion --- p.85Chapter Chapter 6: --- Concluding remarks and general discussionChapter 6.1 --- General discussion --- p.93Chapter 6.2 --- Future work --- p.97Chapter 6.3 --- Summary --- p.99References --- p.100Appendix 1 --- p.11