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Abstract I
中文摘要 II
Acknowledgment III
Table of Contents IV
List of Tables VI
List of Figures VII
List of Appendices VIII
List of Abbreviations and Symbols IX
1. Introduction 1
1.1 Hepatocellular carcinoma (HCC) 1
1.1.1 Epidemiology of HCC 1
1.1.2 Etiology of HCC 2
1.2 Hepatitis B virus (HBV) 2
1.2.1 Epidemiology of HBV 2
1.2.2 Virology of HBV 3
1.3 Pathogenesis of HBV-associated HCC 4
1.3.1 Integration of HBV DNA into host genome 5
1.3.2 HBV X protein (HBx) 5
1.3.2.1 HBx in cell cycle regulation 6
1.3.2.2 HBx in apoptosis 6
1.3.2.3 HBx in DNA repair 7
1.3.2.4 HBx in host gene transactivation 7
1.3.3 The pre-S2 mutant large hepatitis B surface antigen (Δ2-LHBS) 7
1.3.3.1 Δ2-LHBS in ER stress 8
1.3.3.2 Δ2-LHBS in cell cycle regulation 9
1.3.3.3 Δ2-LHBS in DNA damage 9
1.4 Genomic instability 9
1.4.1 Basis of genomic instability 10
1.4.2 Genomic instability in hepatocarcinogenesis 10
1.4.3 Genomic instability induced by HBV 10
2 Materials and Methods 13
2.1 Chemicals and reagents 13
2.2 Transgenic mice 13
2.3 Genomic DNA extraction 13
2.4 Array-based comparative genomic hybridization (aCGH) 14
2.5 Quantification of degree of aberrations 15
2.6 cDNA microarray 15
2.7 Pathways analysis of microarray data 15
2.8 RNA extraction 16
2.9 Reverse transcription-Polymerase chain reaction (RT-PCR) 16
3 Results 18
3.1 aCGH to identify genomic instability induced by ∆2-LHBS, HBx and ∆2-LHBS/HBx 18
3.1.1 Chromosomal aberrations in ∆2-LHBS, HBx and ∆2-LHBS/HBx double transgenic mice 18
3.1.2 ∆2-LHBS induced higher genomic instability than HBx 19
3.1.3 Carcinogenesis-related genes within the aberration regions 20
3.2 cDNA microarray to identify age-dependent gene expression profiles in transgenic mice 20
3.2.1 Genomic stability-related cellular pathways were greatly affected by transgenes 21
3.2.2 Age-dependent gene expression profile verified by RT-PCR 21
4 Discussions 23
4.1 HBV and host genomic instability 23
4.2 Role of Wee1 and Cdkn1a in the carcinogenesis 25
5 References 27
6 Appendices 84
List of Tables
Table 1 Chemicals and reagents 38
Table 2 Primers and PCR conditions 39
Table 3 Number of mice used in aCGH study 40
Table 4 Number of mice used in RT-PCR study 41
Table 5 Summaries of genomic aberrations in the ∆2-LHBS transgenic mice 42
Table 6 Summaries of genomic aberrations in HBx transgenic mice 43
Table 7 Summaries of genomic aberrations in the ∆2-LHBS/HBx double transgenic mice 45
Table 8 Common aberration regions in different transgenic strains 50
Table 9 The common genomic aberrations regions that identified in the ∆2-LHBS, HBx and ∆2-LHBS/HBx double transgenic mice 51
Table 10 Gain of oncogenes in the aberration regions of transgenic mice 52
Table 11 Loss of tumor suppressor genes in aberration regions of transgenic mice 53
Table 12 Gain of other oncogenesis-related genes in aberration regions of transgenic mice 54
Table 13 Loss of other oncogenesis-related genes in aberration regions of transgenic mice 55
Table 14 The rankings of cellular pathways that were most affected by ∆2-LHBS 56
Table 15 The rankings of cellular pathways that were most affected by HBx 57
Table 16 The rankings of cellular pathways that were most affected by ∆2-LHBS/HBx 58
Table 17 The fold changes of genomic stability-related genes 59
List of Figures
Figure 1 HBV LHBS genes and hepatocarcinogenesis in our transgenic mice model. 60
Figure 2 Chromosomal aberrations in the Δ2-LHBS transgenic mice. 62
Figure 3 Chromosomal aberrations in HBx transgenic mice. 65
Figure 4 Chromosomal aberrations in the Δ2-LHBS/HBx double transgenic mice. 69
Figure 5 CNV levels in Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 70
Figure 6 PCR of genomic DNA from transgenic mice liver tissues 71
Figure 7 Genomic stability-related genes expression changes in 3-month-old Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 73
Figure 8 Genomic stability-related genes expression changes in 6-month-old Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 75
Figure 9 Genomic stability-related genes expression changes in 12-month-old Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 77
Figure 10 Genomic stability-related genes expression changes in 18-month-old Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 79
Figure 11 Age-dependent Genomic stability-related genes expression changes analyzed by RT-PCR in Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 81
Figure 12 Age-dependent Genomic stability-related genes expression changes analyzed by gene expression microarray in Δ2-LHBS, HBx and Δ2-LHBS/HBx double transgenic mice. 83
List of Appendices
Appendix 1 HBV genome and replication cycle 84
Appendix 2 GGH and staining pattern of HBsAg 85
Appendix 3 Construction maps of the Δ2-LHBS and HBx transgenic mice 86
Appendix 4 Examples of cellular pathways that the selected genomic stability-related genes are involved in 90
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