Chan Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-153). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract 摘要 --- p.IV / List of Abbreviations --- p.VI / List of Figures --- p.VIII / List of Tables --- p.X / Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2 --- Molecular Information of Apolipoprotein E --- p.2 / Chapter 1.3 --- Protein Structure of Apolipoprotein E --- p.4 / Chapter 1.4 --- Polymorphism of Apolipoprotein E --- p.5 / Chapter 1.4.1 --- Genetic Polymorphism --- p.5 / Chapter 1.4.2. --- Posttranslational Glycosylation --- p.8 / Chapter 1.5 --- Tissue Distribution of Apolipoprotein E --- p.8 / Chapter 1.6 --- Functions of Apolipoprotein E --- p.10 / Chapter 1.6.1 --- Role of Apolipoprotein E in Lipid Metabolism --- p.10 / Chapter 1.6.1.1 --- Chylomicron Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.1.2 --- VLDL Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.1.3 --- HDL Metabolism and Apolipoprotein E --- p.11 / Chapter 1.6.2 --- Lipid-independent Role of Apolipoprotein E --- p.12 / Chapter 1.6.2.1 --- Stimulation on Neurite Extension --- p.13 / Chapter 1.6.2.2 --- Inhibition of Agonist-induced Platelet Aggregation --- p.13 / Chapter 1.6.2.3 --- Immunoregulation --- p.13 / Chapter 1.6.2.4 --- Regulation of Androgen Synthesis by Ovary --- p.14 / Chapter 1.7 --- Association Between Various Disorders and Apolipoprotein E --- p.15 / Chapter 1.7.1 --- Type III Hyperlipoproteinemia and Apolipoprotein E --- p.15 / Chapter 1.7.2 --- Alzheimer's Disease and Apolipoprotein E --- p.15 / Chapter 1.7.3 --- Atherosclerosis and Apolipoprotein E --- p.16 / Chapter 1.8 --- Cell Proliferation and Cell Cycle Progression --- p.18 / Chapter 1.8.1 --- Cyclin-dependent Kinases and Cyclins --- p.19 / Chapter 1.8.2 --- Cyclin-dependent Kinases and Their Inhibitors --- p.21 / Chapter 1.8.3 --- Restriction Point in Cell Cycle --- p.22 / Chapter 1.9 --- Cell Proliferation and Mitogen-activated Protein Kinase (MAPK) --- p.22 / Chapter 1.9.1 --- Organization of MAPK Signaling Pathway --- p.22 / Chapter 1.9.2 --- Regulation of Cell Proliferation via Activator Protein-1 (AP-1) --- p.24 / Chapter 1.9.3 --- Regulation of Cell Proliferation via c-Myc --- p.25 / Chapter 1.10 --- Cell Proliferation and Apolipoprotein E --- p.26 / Chapter 1.10.1 --- Effects of Exogenous Apolipoprotein E on Cell Proliferation --- p.26 / Chapter 1.10.2 --- Effects of Endogenous Apolipoprotein E on Cell Proliferation --- p.27 / Chapter 1.11 --- Aims and Scopes of Study --- p.27 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.29 / Chapter 2.1.1.1 --- Human ApoE3 cDNA --- p.29 / Chapter 2.1.1.2 --- Mammalian Expression Vector --- p.29 / Chapter 2.1.1.3 --- Reagents for Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.1.4 --- Reagents for Ligation Reaction --- p.31 / Chapter 2.1.1.5 --- Bacterial Culture Medium --- p.31 / Chapter 2.1.1.6 --- Reagents for Polymerase Chain Reaction (PCR) --- p.32 / Chapter 2.1.1.7 --- Site-directed Mutagenesis --- p.33 / Chapter 2.1.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.34 / Chapter 2.1.2.1 --- F111 Rat Embryonic Fibroblasts --- p.34 / Chapter 2.1.2.2 --- Reagents for Cell Culture --- p.34 / Chapter 2.1.2.3 --- Cell Culture Wares --- p.36 / Chapter 2.1.2.4 --- Reagents for Transfection --- p.36 / Chapter 2.1.2.5 --- Reagents for Protein Assay --- p.37 / Chapter 2.1.2.6 --- Reagents and Buffers for Western Blot Analysis --- p.37 / Chapter 2.1.2.7 --- Reagents for Enzyme-linked Immunosorbent Assay (ELISA) --- p.42 / Chapter 2.1.3 --- Study on Growth Properties Mediated by ApoE Isoforms --- p.44 / Chapter 2.1.3.1 --- Reagents for Growth Curve Construction --- p.44 / Chapter 2.1.3.2 --- Reagents for 3H-thymidine Incorporation Assay --- p.45 / Chapter 2.1.3.3 --- Reagents for Flow Cytometry --- p.45 / Chapter 2.1.4 --- Study on Signaling Mechanisms Mediated by ApoE Isoforms --- p.46 / Chapter 2.1.4.1 --- RNA Extraction --- p.46 / Chapter 2.1.4.2 --- Reagents for Gene Expression Profiles using Commercial Gene Array --- p.47 / Chapter 2.1.4.3 --- Reagents for Protein Quantification and Western Blot Analysis --- p.48 / Chapter 2.1.4.4 --- Reagents for Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.49 / Chapter 2.1.4.5 --- Reagents for Real Time PCR --- p.50 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.52 / Chapter 2.2.1.1 --- Subcloning --- p.52 / Chapter 2.2.1.2 --- Site-directed Mutagenesis --- p.53 / Chapter 2.2.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.55 / Chapter 2.2.2.1 --- Transfection --- p.55 / Chapter 2.2.2.2 --- Generation of Polyclonal Stable Cell Lines --- p.56 / Chapter 2.2.2.3 --- Protein Extraction --- p.57 / Chapter 2.2.2.4 --- Concentration of Media --- p.57 / Chapter 2.2.2.5 --- Quantification of Proteins --- p.58 / Chapter 2.2.2.6 --- Detection of ApoE Protein by Western Blot Analysis --- p.58 / Chapter 2.2.2.7 --- Enhanced Chemiluminescent (ECL´ёØ) Assay --- p.60 / Chapter 2.2.2.8 --- Quantification of ApoE Protein Level by ELISA --- p.61 / Chapter 2.2.2.9 --- Cell Morphology Study --- p.63 / Chapter 2.2.3 --- Characterization of Growth Properties of ApoE Isoform- expressing Cells --- p.63 / Chapter 2.2.3.1 --- Cell Proliferation Rates --- p.63 / Chapter 2.2.3.2 --- Measurement of DNA Synthesis --- p.63 / Chapter 2.2.3.3 --- Flow Cytometry Analysis of Cell Cycle Profile --- p.64 / Chapter 2.2.4 --- Study on Signaling Mechanisms mediated by ApoE Isoforms --- p.65 / Chapter 2.2.4.1 --- Isolation of Total RNA --- p.65 / Chapter 2.2.4.2 --- Study of Gene Expression Profile by Commercial Gene Array --- p.67 / Chapter 2.2.4.3 --- Determination of Activation Status of MAPK and Transcription Factors by Western Blot --- p.68 / Chapter 2.2.4.4 --- Quantification of Gene Expression Level by Real Time Polymerase Chain Reaction (PCR) --- p.70 / Chapter 2.2.5 --- Statistical Analysis --- p.75 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Construction of ApoE Isoform-encoding Plasmids --- p.76 / Chapter 3.2 --- Establishment of ApoE Isoform-expressing Cell Lines --- p.78 / Chapter 3.2.1 --- Cell Morphology Study --- p.81 / Chapter 3.3 --- Characterization of Growth Properties of ApoE Isoform- expressing Cells --- p.83 / Chapter 3.3.1 --- Cell Proliferation Rates --- p.83 / Chapter 3.3.2 --- DNA Synthesis --- p.86 / Chapter 3.3.3 --- Flow Cytometry Analysis of Cell Cycle Progression --- p.88 / Chapter 3.4 --- Study on Signaling Mechanisms Mediated by ApoE Isoforms --- p.92 / Chapter 3.4.1 --- Study of Gene Expression Profile by Commercial Gene Array --- p.92 / Chapter 3.4.2 --- Study of Activation Status of MAPKs and MEK1/2 --- p.95 / Chapter 3.4.3 --- Study of the Effects of ApoE Isoforms on Transcription Factors --- p.101 / Chapter 3.4.4 --- Real Time PCR Reaction Verification of Primers --- p.105 / Chapter 3.4.5 --- Quantification of Gene Expression Level by Real Time PCR --- p.107 / Chapter Chapter 4: --- Discussions / Chapter 4.1 --- Establishment of Stable Cell Lines Expressing ApoE Isoforms --- p.110 / Chapter 4.1.1 --- Rat Embryonic Fibroblasts as Cell Model --- p.110 / Chapter 4.1.2 --- PCR Amplification of ApoE cDNA --- p.111 / Chapter 4.1.3 --- Expression Level of ApoE Isoform Transgenesin F111Rat Fibroblasts --- p.112 / Chapter 4.2 --- Growth Properties Mediated by ApoE Isoforms --- p.115 / Chapter 4.2.1 --- Effects of ApoE Isoforms on Cellular Morphology --- p.115 / Chapter 4.2.2 --- Serum as the Stimulator of Cell Proliferation --- p.116 / Chapter 4.2.3 --- Isoform-specific Effects of Endogenous ApoE on Cell Proliferation --- p.118 / Chapter 4.2.4 --- Comparison of Effects Mediated by Endogenous and Exogenous ApoE --- p.119 / Chapter 4.3 --- Signaling Mechanisms Mediated by ApoE Isoforms --- p.121 / Chapter 4.3.1 --- Effects of ApoE Isoforms on MAPK Activities --- p.121 / Chapter 4.3.1.1 --- Effects of ApoE Isoforms on ERK1/2 MAPK --- p.122 / Chapter 4.3.1.1.1 --- Upstream Effectors of ERK1/2 --- p.123 / Chapter 4.3.1.1.2 --- Downstream Effectors of ERK1/2 --- p.124 / Chapter 4.3.1.2 --- Effects of ApoE Isoforms on ERK5 MAPK --- p.125 / Chapter 4.3.1.2.1 --- Upstream Effectors of ERK5 --- p.126 / Chapter 4.3.1.2.2 --- Downstream Effectors of ERK5 --- p.127 / Chapter 4.3.2 --- Effects of ApoE Isoforms on Cell Cycle Regulatory Molecules --- p.130 / Chapter 4.3.2.1 --- Effects of ApoE Isoforms on Cyclin Expression --- p.130 / Chapter 4.3.2.2 --- Effects of ApoE Isoforms on CKIs Expression --- p.132 / Chapter 4.3.3 --- Summary of the Signaling Mechanisms Underlying ApoE-mediated Responses --- p.135 / Chapter 4.3.4 --- Other Pathways Involved in ApoE-mediated Responses --- p.137 / Chapter 4.3.4.1 --- Synthesis of Nitric Oxide --- p.137 / Chapter 4.3.4.2 --- Synthesis of Perlecan --- p.138 / Chapter 4.4 --- Conclusions and Future Prospects --- p.139 / Chapter 4.4.1 --- Conclusions --- p.140 / Chapter 4.4.2 --- Future Prospects --- p.141 / Chapter Chapter 5: --- References --- p.142
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324552 |
| Date | January 2004 |
| Contributors | Chan, Wing Man., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 153 leaves : ill. ; 30 cm. |
| Rights | 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/) |
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