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Bioassay-guided isolation, characterization and mechanistic study of the bioactive components from Sophora flavescens for the anti-proliferative effect on human hepatoma cells.

by Tsang Kit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 179-188). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ABSTRACT IN CHINESE (摘要) --- p.iii / ACKNOWLEDGEMENTS --- p.v / CONTENTS --- p.vi / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.2 / Chapter 1.1.2 --- Therapies for Hepatocellular Carcinoma --- p.4 / Chapter 1.2 --- Multidrug Resistance of Tumor Cells --- p.8 / Chapter 1.3 --- Therapeutic Potential of Traditional Chinese Medicine on Human Hepatoma --- p.10 / Chapter 1.4 --- Sophora flavescens Ait --- p.13 / Chapter 1.5 --- Biological Activities of Sophorae Radix --- p.15 / Chapter 1.5.1 --- Antitumor Activities --- p.16 / Chapter 1.5.2 --- "Antibacterial, Antimalarial and Antiviral Activities" --- p.17 / Chapter 1.6 --- Objectives and Significance of Study --- p.19 / Chapter 1.6.1 --- Bioassay-guided Isolation of Active Compounds from Sophora flavescens --- p.19 / Chapter 1.6.2 --- Action Mechanisms of the Bioactive Compounds Isolated from Sophora flavescens --- p.20 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.21 / Chapter 2.1 --- Cell Culture --- p.22 / Chapter 2.1.1 --- Cell Lines --- p.22 / Chapter 2.1.2 --- Cell Culture Media --- p.24 / Chapter 2.2 --- Isolation of Bioactive Compounds from Sophora flavescens --- p.25 / Chapter 2.3 --- MTT assay --- p.27 / Chapter 2.4 --- Cell Cycle Analysis --- p.28 / Chapter 2.5 --- Detection of Phosphatidylserine Externalization with Annexin V-FITC and PI --- p.29 / Chapter 2.6 --- DNA Fragmentation Assay --- p.30 / Chapter 2.7 --- Western Blot Analysis --- p.32 / Chapter 2.7.1 --- Extraction of Total Cellular Protein --- p.32 / Chapter 2.7.2 --- Extraction of Cytosolic Protein --- p.32 / Chapter 2.7.3 --- Determination of Protein Concentration --- p.33 / Chapter 2.7.4 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.35 / Chapter 2.7.5 --- Electroblotting of Protein --- p.36 / Chapter 2.7.6 --- Probing of Proteins with Antibodies --- p.37 / Chapter 2.7.7 --- Enhanced Chemiluminescence (ECL) Assay --- p.39 / Chapter 2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent dye --- p.39 / Chapter 2.9 --- cDNA Microarray Analysis --- p.40 / Chapter 2.9.1 --- Isolation of Total RNA --- p.40 / Chapter 2.9.2 --- Microarray Hybridization and Analysis --- p.41 / Chapter 2.9.3 --- Validation of Candidate Genes --- p.44 / Chapter 2.9.3.1 --- Determination of RNA Concentration --- p.44 / Chapter 2.9.3.2 --- First-Strand cDNA Synthesis --- p.44 / Chapter 2.9.3.3 --- Reverse-Transcription Polymerase Chain Reaction (RT-PCR) of Candidate Genes --- p.45 / Chapter 2.10 --- Two-Dimensional Polyacrylamide Gel Electrophoretic Analysis (2D-PAGE) --- p.47 / Chapter 2.10.1 --- Extraction of Total Cellular Protein for 2-D Gel Electrophoresis --- p.47 / Chapter 2.10.2 --- Determination of Protein Concentration --- p.47 / Chapter 2.10.3 --- First-Dimension Isoelectric Focusing (IEF) --- p.49 / Chapter 2.10.4 --- Second-Dimension SDS-PAGE --- p.49 / Chapter 2.10.5 --- Visualization of 2-D Gel by Silver Staining --- p.50 / Chapter 2.10.6 --- Identification of Differentially Expressed Proteins with Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) --- p.51 / Chapter 2.11 --- Statistical Analysis --- p.53 / Chapter CHAPTER THREE: --- BIOASSAY-GUIDED ISOLATION AND CHARACTERISATION OF BIOACTIVE COMPOUNDS FROM SOPHORA FLAVESCENS --- p.54 / Chapter 3.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.55 / Chapter 3.2 --- Structure Identification of the Bioactive Compounds Isolated from Sophora flavescens --- p.64 / Chapter 3.3 --- In Vitro Anti-tumor Effect of the Bioactive Compounds Isolated from Sophora flavescens --- p.71 / Chapter CHAPTER FOUR: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G IN THE INDUCTION OF APOPTOSIS IN HEPATOCELLULAR CARCINOMA CELLS --- p.76 / Chapter 4.1 --- In Vitro Anti-tumor Effect of Sophoraflavanone G --- p.77 / Chapter 4.2 --- Cell Cycle Analysis of Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.81 / Chapter 4.3 --- Induction of Apoptosis in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.88 / Chapter 4.3.1 --- Induction of Phosphatidylserine Externalization in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.89 / Chapter 4.3.2 --- Induction of DNA Fragmentation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.94 / Chapter 4.3.3 --- Induction of Caspase-3 activation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.97 / Chapter 4.4 --- Underlying Mechanisms of Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.102 / Chapter 4.4.1 --- Involvement of Death Receptor Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.103 / Chapter 4.4.2 --- Involvement of Bid protein in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.105 / Chapter 4.4.3 --- Involvement of Mitochondrial Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.108 / Chapter 4.4.4 --- Induction of Mitochondrial Membrane Depolarization in Human Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.112 / Chapter 4.4.5 --- Involvement of Caspase-independent Pathway in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.116 / Chapter CHAPTER FIVE: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HUMAN HEPATOCELLULAR CARCINOMA CELLS BY USING cDNA MICROARRAY ANALYSIS --- p.119 / Chapter 5.1 --- Identification of Differentially Expressed Genes in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by cDNA Microarray Analyasis --- p.120 / Chapter CHAPTER SIX: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HEPATOCELLULAR CARCINOMA CELLS BY USING TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS --- p.136 / Chapter 6.1 --- Identification of Differentially Expressed Proteins in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by Two-Dimensional Polyacrylamide Gel Electrophoresis --- p.137 / Chapter CHAPTER SEVEN: --- DISCUSSION --- p.150 / Chapter 7.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.151 / Chapter 7.2 --- Induction of Apoptosis in Human Hepatocellular Carcinoma cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.154 / Chapter 7.3 --- Differential Gene Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells --- p.161 / Chapter 7.4 --- Differential Protein Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.164 / Chapter 7.5 --- Toxicity of Sophoraflavanone G against Normal Liver Cells --- p.170 / Chapter CHAPTER EIGHT: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.173 / Chapter 8.1 --- Conclusion --- p.174 / Chapter 8.2 --- Future Prospects --- p.176 / REFERENCES --- p.179

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325663
Date January 2006
ContributorsTsang, Kit Man., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xx, 188 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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