Li Tin Wai Olive. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 191-217). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.III / 中文摘要 --- p.IX / PUBLICATIONS --- p.XIV / TABLE OF CONTENTS --- p.XV / LIST OF ABBREVIATIONS --- p.XXI / LIST OF FIGURES AND TABLES --- p.XXIII / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1. --- Cardiovascular disease --- p.7 / Chapter 2.1.1. --- Introduction --- p.7 / Chapter 2.1.2. --- Atherosclerosis --- p.7 / Chapter 2.1.3. --- Cellular and molecular deregulation in early atherosclerosis --- p.10 / Chapter 2.1.3.1. --- Introduction --- p.10 / Chapter 2.1.3.2. --- Endothelial dysfunction --- p.11 / Chapter 2.1.3.3. --- Nitric oxide --- p.12 / Chapter 2.1.3.4. --- Adhesion molecules and the early events of atherogenesis --- p.13 / Chapter 2.1.3.4.1. --- Introduction --- p.13 / Chapter 2.1.3.4.2. --- Intracellular adhesion molecule-1 --- p.15 / Chapter 2.1.3.4.3. --- Nuclear factor kappa B --- p.18 / Chapter 2.1.3.5. --- Summary --- p.19 / Chapter 2.2. --- Nitric oxide in molecular vascular biology --- p.20 / Chapter 2.2.1. --- Introduction --- p.20 / Chapter 2.2.2. --- Nitric oxide synthase --- p.21 / Chapter 2.2.2.1. --- Introduction --- p.21 / Chapter 2.2.2.2. --- Endothelial nitric oxide synthase --- p.24 / Chapter 2.2.2.3. --- Inducible nitric oxide synthase --- p.25 / Chapter 2.2.2.4. --- Nitric oxide concentration dependent effector pathways --- p.26 / Chapter 2.2.3. --- Nitric oxide and its regulation in vascular events --- p.28 / Chapter 2.2.3.1. --- Introduction --- p.28 / Chapter 2.2.3.2. --- Regulation of vascular tone --- p.30 / Chapter 2.2.3.3. --- "Regulation of platelet adhesion, activation and aggregation" --- p.32 / Chapter 2.2.3.4. --- Regulation of endothelial adhesiveness and leukocyte adhesion - Anti-adhesive effect of nitric oxide --- p.32 / Chapter 2.2.3.5. --- "Regulation of vascular smooth muscle growth, migration and proliferation" --- p.33 / Chapter 2.2.3.6. --- Antioxidative effect of nitric oxide --- p.34 / Chapter 2.2.3.7. --- Regulation of endothelial apoptosis --- p.35 / Chapter 2.2.3.8. --- Nitric oxide and its relationship with other risk factors --- p.36 / Chapter 2.3. --- "Menopause, cardiovascular diseases and Traditional Chinese Medicine" --- p.37 / Chapter 2.3.1. --- Traditional Chinese Medicine and menopause --- p.37 / Chapter 2.3.2. --- Dang Gui Buxue Tang --- p.38 / Chapter 2.3.3. --- Danggui --- p.39 / Chapter 2.3.3.1. --- Botanic origins --- p.39 / Chapter 2.3.3.2. --- Usage --- p.39 / Chapter 2.3.4. --- Huangqi --- p.40 / Chapter 2.3.4.1. --- Botanic origins --- p.40 / Chapter 2.3.4.2. --- Usage --- p.40 / Chapter 2.3.5. --- Modern scientific research --- p.41 / Chapter 2.3.5.1. --- General cardioprotective role --- p.41 / Chapter 2.3.5.2. --- Vascular tone modulation --- p.42 / Chapter 2.3.5.3. --- Haemostasis --- p.42 / Chapter 2.3.5.4. --- Endothelial cell --- p.43 / Chapter 2.3.5.4.1. --- Nitric oxide pathway --- p.43 / Chapter 2.3.5.4.1.1. --- Direct alteration of nitric oxide secretion --- p.43 / Chapter 2.3.5.4.1.2. --- Alteration of Nitric oxide synthase expression or activity --- p.43 / Chapter 2.3.5.4.2. --- Alteration of adhesion molecule expression --- p.44 / Chapter 2.3.5.4.3. --- Alteration of adhesion molecule expression as an effect of nitric oxide secretion --- p.45 / Chapter 2.3.5.5. --- Antioxidant effect --- p.45 / Chapter 2.3.5.6. --- Estrogenicity of DBT --- p.46 / Chapter 2.4. --- Research plan --- p.47 / Chapter 2.4.1. --- Formulation of research hypotheses --- p.47 / Chapter 2.4.1.1. --- Hypotheses --- p.50 / Chapter 2.4.2. --- Plan of study --- p.50 / Chapter 2.4.2.1. --- Dang Gui Buxue Tang extraction and standardization of content --- p.50 / Chapter 2.4.2.2. --- Cell model development --- p.52 / Chapter 2.4.2.3. --- Experimental studies --- p.54 / Chapter 2.4.2.4. --- eNOS activity determination - the nitric oxide metabolite assay --- p.56 / Chapter 2.4.2.5. --- Endotoxin contamination in DBT --- p.57 / Chapter 2.4.3. --- Sample size and statistical analysis --- p.59 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.62 / Chapter 3.1. --- Dang Gui Buxue Tang extraction and content standardization --- p.62 / Chapter 3.1.1. --- Plant materials --- p.62 / Chapter 3.1.2. --- DBT authentication --- p.62 / Chapter 3.1.3. --- DBT processing prior to extraction --- p.63 / Chapter 3.1.4. --- DBT extraction --- p.63 / Chapter 3.1.5. --- Quantitative standardization of DBT markers by High Pressure Liquid Chromatography --- p.66 / Chapter 3.1.5.1. --- DBT markers: standard preparation --- p.66 / Chapter 3.1.5.2. --- Sample preparation --- p.67 / Chapter 3.1.5.3. --- Quantitative analysis of DBT constituents by HPLC --- p.67 / Chapter 3.1.6. --- DBT polysaccharide standardization --- p.68 / Chapter 3.1.6.1. --- Glucose standard preparation --- p.68 / Chapter 3.1.6.2. --- Sample preparation --- p.68 / Chapter 3.1.6.3. --- Quantitative determination of polysaccharide by Phenol-Sulfuric acid colorimetric assay --- p.68 / Chapter 3.1.7. --- DBT endotoxin contamination determination --- p.69 / Chapter 3.1.7.1. --- "Positive, negative and inhibition controls" --- p.69 / Chapter 3.1.7.2. --- Qualitative determination of sample endotoxin --- p.70 / Chapter 3.2. --- Cell culture --- p.70 / Chapter 3.2.1. --- Characterization of cultured cells --- p.72 / Chapter 3.2.2. --- Passage --- p.73 / Chapter 3.3. --- DBT treatment --- p.73 / Chapter 3.3.1. --- Solvent system of DBT treatment --- p.73 / Chapter 3.3.2. --- Dosage and duration of DBT treatment --- p.74 / Chapter 3.3.3. --- Positive and negative controls --- p.74 / Chapter 3.4. --- MTT-based cytotoxicity assay --- p.75 / Chapter 3.5. --- Reverse transcriptase- polymerase chain reaction --- p.76 / Chapter 3.5.1. --- Sample preparation --- p.76 / Chapter 3.5.1.1. --- Total RNA isolation --- p.76 / Chapter 3.5.1.2. --- DNase treatment --- p.77 / Chapter 3.5.1.3. --- RNAethanol precipitation --- p.78 / Chapter 3.5.1.4. --- Complementary DNA synthesis --- p.78 / Chapter 3.5.2. --- Polymerase chain reaction --- p.79 / Chapter 3.5.2.1. --- Polymerase chain reaction conditions --- p.79 / Chapter 3.5.2.2. --- Primers --- p.79 / Chapter 3.5.3. --- Visualization of the PCR products --- p.81 / Chapter 3.5.3.1. --- Gel electrophoresis --- p.81 / Chapter 3.5.3.2. --- Gel Doc software --- p.82 / Chapter 3.5.3.3. --- Densitometry --- p.82 / Chapter 3.5.4. --- Real time RT-PCR --- p.82 / Chapter 3.6. --- Quantitative Immunocytochemical studies --- p.84 / Chapter 3.6.1. --- Coverslip preparation --- p.84 / Chapter 3.6.2. --- Sample preparation --- p.84 / Chapter 3.6.3. --- Immunocytochemical staining preparation --- p.85 / Chapter 3.6.3.1. --- "Immunocytochemical staining for vWF, α-actin, iNOS, ICAM-1, NF-kB using DAKO catalyzed signal amplification (CSA) system (anti-mouse)" --- p.86 / Chapter 3.6.3.2. --- Immunocytochemical staining for eNOS using Santa Cruz immunoCruz staining system (anti-goat) --- p.87 / Chapter 3.6.4. --- Counterstaining and mounting --- p.88 / Chapter 3.6.5. --- Result interpretation --- p.89 / Chapter 3.6.5.1. --- Microscopy and digital image capture --- p.89 / Chapter 3.6.5.2. --- Determination of Image (file) Energy --- p.89 / Chapter 3.7. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.90 / Chapter 3.7.1. --- Sample preparation --- p.90 / Chapter 3.7.2. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.91 / Chapter CHAPTER 4. --- RESULTS --- p.93 / Chapter 4.1. --- Dang Gui Buxue Tang extraction and standardization of content --- p.93 / Chapter 4.1.1. --- DBT extraction - general data --- p.93 / Chapter 4.1.2. --- DBT polysaccharide standardization --- p.97 / Chapter 4.1.3. --- DBT marker standardization --- p.101 / Chapter 4.1.4. --- DBT endotoxin contamination determination --- p.104 / Chapter 4.2. --- Cell model development --- p.108 / Chapter 4.2.1. --- Endothelial morphology --- p.108 / Chapter 4.2.2. --- Immunocytochemistiy --- p.108 / Chapter 4.2.3. --- MTT cytotoxicity assay --- p.110 / Chapter 4.3. --- Study 1 --- p.112 / Chapter 4.3.1. --- Immunocytochemistry (Hypothesis 1) --- p.112 / Chapter 4.3.2. --- RT-PCR (Hypothesis 1) --- p.117 / Chapter 4.3.3. --- Real time RT-PCR (Hypothesis 1) --- p.121 / Chapter 4.3.4. --- Immunocytochemistry (Hypothesis 2) --- p.125 / Chapter 4.3.5. --- RT-PCR (Hypothesis 2) --- p.128 / Chapter 4.3.6. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.131 / Chapter 4.4. --- Study 2 --- p.133 / Chapter 4.4.1. --- Immunocytochemistry --- p.133 / Chapter 4.5. --- Study 3 --- p.138 / Chapter 4.5.1. --- Immunocytochemistry --- p.138 / Chapter 4.5.2. --- RT-PCR --- p.141 / Chapter 4.5.3. --- Real time RT-PCR --- p.145 / Chapter 4.6. --- Endotoxin contamination in DBT --- p.149 / Chapter 4.6.1. --- Effects of endotoxin on eNOS --- p.149 / Chapter 4.6.2. --- Immunocytochemistry on immunostained endothelial cells --- p.151 / Chapter 4.6.3. --- Effects of endotoxin on iNOS --- p.153 / Chapter 4.6.4. --- Effect of endotoxin on NF-kB --- p.157 / Chapter 4.6.5. --- Effects of endotoxin on ICAM-1 --- p.160 / Chapter CHAPTER 5. --- DISCUSSION --- p.165 / Chapter 5.1. --- DBT extraction and standardization of content --- p.165 / Chapter 5.1.1. --- Optimal DBT extraction conditions --- p.165 / Chapter 5.1.2. --- Evidence to support formulae usage --- p.166 / Chapter 5.1.3. --- Limitation of the methodology used --- p.166 / Chapter 5.2. --- Cell model development --- p.167 / Chapter 5.2.1. --- Choice of DBT concentration range in the study --- p.167 / Chapter 5.2.1.1. --- Choice of concentration range in consideration of endotoxin contamination --- p.167 / Chapter 5.2.1.2. --- Choice of concentration range in consideration of DBT's cytotoxicity effects --- p.168 / Chapter 5.2.1.3. --- Choice of concentration range in consideration of prevous studies --- p.168 / Chapter 5.3. --- Study 1 --- p.169 / Chapter 5.3.1. --- Action of DBT on eNOS expression --- p.169 / Chapter 5.3.2. --- Action of DBT on iNOS expression --- p.170 / Chapter 5.3.3. --- Action of DBT on Nitric oxide metabolite assay --- p.171 / Chapter 5.3.3.1. --- Result interpretation with rejected hypothesis 2 --- p.171 / Chapter 5.3.3.2. --- Assay limitations and improvements --- p.171 / Chapter 5.4. --- Study 2 --- p.172 / Chapter 5.4.1. --- Action of DBT on NF-kB expression --- p.172 / Chapter 5.4.2. --- Assay limitations and improvements --- p.173 / Chapter 5.5. --- Study 3 --- p.174 / Chapter 5.5.1. --- Action of DBT on ICAM-1 expression --- p.174 / Chapter 5.6. --- Endotoxin contamination in DBT --- p.175 / Chapter 5.6.1. --- Action of endotoxin contamination in DBT on various markers --- p.175 / Chapter 5.6:2. --- Experimental limitation --- p.176 / Chapter 5.6.3. --- Endotoxin removal --- p.177 / Chapter 5.6.3.1. --- Introduction --- p.177 / Chapter 5.6.3.2. --- Endotoxin removal methodologies suitable for herbal use --- p.179 / Chapter 5.7. --- Action of DBT on angiogenesis stimulation --- p.181 / Chapter 5.7.1. --- Evidence for DBT's proangiogenic effects from various studies --- p.181 / Chapter 5.7.2. --- Influence of endotoxin contamination on angiogenesis stimulation --- p.182 / Chapter 5.7.3. --- Assay limitations and future developments --- p.183 / Chapter CHAPTER 6. --- GENERAL DISCUSSION AND SUMMARY --- p.186 / Chapter CHAPTER 7. --- REFERENCES --- p.191
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324585 |
| Date | January 2004 |
| Contributors | Li, Tin Wai Olive., Chinese University of Hong Kong Graduate School. Division of Chinese Medicine. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxvi, 217 leaves : ill. (some col.) ; 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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