Mitochondrial DNA mutations in hepatocellular carcinoma (HCC) of Chinese patients.

Fu Zhenming.
Thesis submitted in: December 2003.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 138-162).
s in English and Chinese.
List of abbreviations --- p.i
(in English) --- p.ii
摘要（中文） --- p.iii
Acknowledgement --- p.iv
Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1
Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2
Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2
Chapter 1.1.2 --- Pathology and staging --- p.4
Chapter 1.1.3 --- Treatment --- p.6
Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7
Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10
Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10
Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10
Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11
Chapter 1.2.2 --- The mitochondrial genome --- p.14
Chapter 1.2.2.1 --- Strucure --- p.14
Chapter 1.2.2.2 --- Genes for structure proteins --- p.16
Chapter 1.2.2.3 --- Genes for translation --- p.17
Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17
Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19
Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25
Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30
Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32
Chapter 1.3 --- MtDNA diseases --- p.35
Chapter 1.4 --- MtDNA mutation and HCC --- p.35
Chapter 1.5 --- Aims of the study --- p.39
Chapter Chapter 2. --- Materials and Methods --- p.41
Chapter 2.1 --- Materials --- p.42
Chapter 2.1.1 --- Chemicals --- p.42
Chapter 2.1.2 --- Primers --- p.42
Chapter 2.1.3 --- Enzymes --- p.45
Chapter 2.1.4 --- Cell line --- p.45
Chapter 2.1.5 --- Collection of specimens --- p.46
Chapter 2.2 --- Methodology --- p.47
Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47
Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47
Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49
Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50
Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50
Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51
Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51
Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51
Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51
Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52
Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52
Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53
Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53
Chapter 2.2.2.3.4 --- Purification of extension products --- p.54
Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55
Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55
Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55
Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55
Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56
Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57
Chapter 2.2.3.2.5 --- Electrophoresis --- p.57
Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58
Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59
Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60
Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60
Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60
Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61
Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61
Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62
Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62
Chapter 2.2.5 --- Statistics --- p.63
Chapter 2.2.5.1 --- The chi-square test --- p.63
Chapter 2.2.5.2 --- The Friedman test --- p.63
Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63
Chapter Chapter 3. --- Results --- p.64
Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65
Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65
Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76
Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76
Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77
Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77
Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82
Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82
Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82
Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82
Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82
Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87
Chapter Chapter 4. --- Discussion --- p.98
Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99
Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107
Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119
Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125
Chapter 4.5 --- Proposals for prospective studies --- p.126
Chapter 4.5.1 --- Function of C7 in D310 --- p.128
Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130
Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131
Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132
Chapter 4.6 --- Summary --- p.134
References --- p.137
http://library.cuhk.edu.hk/record=b5892082
Use 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/)