Lee Mei-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 123-133). / Abstracts in English and Chinese. / ACKOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / ABSTRACT in Chinese (摘要） --- p.iv / LIST OF FIGURES AND TABLES --- p.vi / LIST OF ABBREVIATIONS --- p.viii / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Discovery of polyphyllin D in pharmaceutical study --- p.1 / Chapter 1.2 --- Structure of Polyphyllin D --- p.4 / Chapter 1.3 --- Origin of Polyphyllin D --- p.4 / Chapter 1.4 --- Polyphyllin D in antitumor activities analysis --- p.6 / Chapter 1.5 --- Characteristic of Polyphyllin D --- p.9 / Chapter 1.6 --- General properties of Saponin --- p.9 / Chapter 1.6.1 --- Membrane-disrupting property --- p.10 / Chapter 1.6.2 --- Hemolytic Property --- p.10 / Chapter 1.7 --- Antitumor properties of saponins --- p.11 / Chapter 1.7.1 --- Induction of apoptosis by saponins --- p.11 / Chapter 1.7.2 --- Induction of cell cycle arrest by saponins --- p.12 / Chapter 1.7.3 --- Saponins with structure similar to polyphyllin D are found with antitumor activity --- p.13 / Chapter 1.8 --- Human breast cancer --- p.14 / Chapter 1.8.1 --- Incidence of Breast Cancer --- p.14 / Chapter 1.8.2 --- Classification of Breast Cancer --- p.16 / Chapter 1.8.3 --- Role of estrogen and estrogen receptor in breast cancer --- p.16 / Chapter 1.8.4 --- Today treatment in Breast Cancer --- p.18 / Chapter 1.8.4.1 --- Hormonal Therapy --- p.18 / Chapter 1.8.4.1.1 --- Tamoxifen --- p.19 / Chapter 1.8.4.2 --- Chemotherapy --- p.20 / Chapter 1.8.4.2.1 --- Doxorubicin (Adriamycin) --- p.20 / Chapter 1.9 --- The Hypothesis and the aim my project --- p.22 / Chapter CHAPTER 2: --- Materials and methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.1 --- MCF-7 --- p.24 / Chapter 2.1.1.2 --- MDA-MB-231 --- p.25 / Chapter 2.1.1.3 --- HS68 --- p.25 / Chapter 2.1.1.4 --- WRL-68 --- p.25 / Chapter 2.1.2 --- Culture Medium --- p.26 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.26 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-free Medium) --- p.27 / Chapter 2.1.2.3 --- Dulbecco's modified Eagle's medium --- p.27 / Chapter 2.2 --- Reagent and Buffers --- p.28 / Chapter 2.2.1 --- Regents for DNA fragmentation Assay --- p.28 / Chapter 2.2.2 --- Regents for Western Blot Analysis --- p.29 / Chapter 2.2.3 --- Reagent for Two Dimensional Electrophoresis Analysis --- p.31 / Chapter 2.2.3.1 --- Protein Preparation --- p.31 / Chapter 2.2.3.2 --- First dimension Electrophoresis --- p.31 / Chapter 2.2.3.3 --- Second dimension Electrophoresis --- p.32 / Chapter 2.2.3.4 --- Silver staining --- p.32 / Chapter 2.2.4 --- Reagent for ln-gel digestion --- p.33 / Chapter 2.2.4.1 --- Destaining --- p.33 / Chapter 2.2.4.2 --- Digestion --- p.33 / Chapter 2.2.4.3 --- Desalting of the peptide mixture for MS analysis --- p.33 / Chapter 2.2.5 --- Reagent for flow cytomertry analysis --- p.34 / Chapter 2.2.6 --- Reagent for immunofluorescent staining --- p.34 / Chapter 2.2.7 --- Reagent for Primary mouse speenocytes and macrophages preparation --- p.34 / Chapter 2.2.8 --- Reagent for Cell Cytotoxicity Assay --- p.35 / Chapter 2.2.9 --- Reagent for in vivo study --- p.35 / Chapter 2.2.10 --- Reagent for Estrogen Receptor Competitive Assay --- p.35 / Chapter 2.3 --- Chemicals --- p.36 / Chapter 2.4 --- Methods --- p.38 / Chapter 2.4.1 --- Polyphyllin D preparation --- p.38 / Chapter 2.4.1.1 --- in vitro application --- p.38 / Chapter 2.4.1.2 --- in vivo application --- p.39 / Chapter 2.4.2 --- Treatment of polyphyllin in vitro --- p.39 / Chapter 2.4.3 --- MTT assay --- p.39 / Chapter 2.4.4 --- Trypan Blue Exclusion Assay --- p.40 / Chapter 2.4.5 --- Analysis of Cell-Cycle Phase Distribution by Flow cytometry with PI staining --- p.41 / Chapter 2.4.6 --- Estrogen Receptor competitive Assay --- p.41 / Chapter 2.4.7 --- Nucleosome Detection Assay --- p.42 / Chapter 2.4.8 --- Quantification of Apoptosis by Flow Cytometry with Annexin V ´ؤ PI Staining --- p.42 / Chapter 2.4.9 --- Assessment of the Change in Mitochondrial Membrane Potential (Δφ m) --- p.43 / Chapter 2.4.10 --- Western Blotting Analysis --- p.44 / Chapter 2.4.10.1 --- Protein Extraction --- p.45 / Chapter 2.4.10.2 --- Protein concentration Determination --- p.47 / Chapter 2.4.10.3 --- SDS acrylamide gel electrohphoresis --- p.47 / Chapter 2.4.10.4 --- Electroblotting of Protein --- p.47 / Chapter 2.4.10.5 --- Probing of Proteins with Antibodies --- p.48 / Chapter 2.4.10.6 --- Enhanced Chemiluminescence (ECL) Assay --- p.48 / Chapter 2.4.11 --- Two Dimensional Electrophoretic Analysis --- p.49 / Chapter 2.4.11.1 --- Protein preparation --- p.50 / Chapter 2.4.11.2 --- First dimensional electrophoresis --- p.51 / Chapter 2.4.11.2.1 --- Rehydration of IPG strips --- p.51 / Chapter 2.4.11.2.2 --- IEF with IPGphor --- p.51 / Chapter 2.4.11.2.3 --- Running IPG strips --- p.52 / Chapter 2.4.11.2.4 --- Eauilibration of the IPG strip --- p.52 / Chapter 2.4.11.3 --- Second dimensional electrophoresis Equilibration of the IPG strip --- p.52 / Chapter 2.4.11.4 --- Visualization of the 2D gel by Silver staining --- p.53 / Chapter 2.4.11.5 --- Computer analysis of the 2d gel image --- p.54 / Chapter 2.4.12 --- Protein identification with Matrix assisted laser desorption- ionization Time-of-flight mass spectrometry (MALDI-TOF) --- p.54 / Chapter 2.4.12.1 --- In gel tryptic digestion --- p.54 / Chapter 2.4.12.2 --- Desalting of the peptide mixtures --- p.55 / Chapter 2.4.12.3 --- Database Searching --- p.55 / Chapter 2.5 --- Statisitc Analysis --- p.55 / Chapter CHAPTER 3: --- Cytotoxic effects of polyphyllin D / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Cytotoxic activities on human breast cancer cell lines --- p.58 / Chapter 3.2.1 --- In MCF-7 cells --- p.53 / Chapter 3.2.2 --- In MDA-MB-231 cells --- p.58 / Chapter 3.3 --- Cytotoxic activities on human normal cell lines --- p.63 / Chapter 3.3.1 --- "Human normal liver cell line, WRL-68" --- p.63 / Chapter 3.3.2 --- "Human skin fibroblast cell line, HS-68" --- p.63 / Chapter 3.4 --- Cytotoxic activities on primary culture --- p.65 / Chapter 3.4.1 --- Primary mouse spleenocytes --- p.65 / Chapter 3.5 --- Conclusion --- p.59 / Chapter CHAPTER 4: --- Induction of apoptosis / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Induction of apoptosis on MCF-7 cells --- p.71 / Chapter 4.2.1 --- Nucleosome formation by ELISA assay --- p.71 / Chapter 4.2.2 --- Phosphatidylserine Extemalization by flow cytometrial study --- p.73 / Chapter 4.3 --- Induction of apoptosis on MDA-MB-231 cells --- p.75 / Chapter 4.3.1 --- Apoptotic peak by flow cytometrial study --- p.75 / Chapter 4.3.2 --- Phosphatidylserine Extemalization by flow cytometrial study --- p.78 / Chapter 4.4 --- Conclusion --- p.80 / Chapter CHAPTER 5 --- : Induction of apoptosis via mitochondrial pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Mitochondrial Membrane Depolarization by flow cytometrial study --- p.82 / Chapter 5.2.1 --- In MCF-7 cells --- p.84 / Chapter 5.2.2 --- In MDA-MB-231 cells --- p.84 / Chapter 5.3 --- Alternation of mitochondrial protein expression by western blot analysis on MCF-7 and MDA-MB-231 cells --- p.87 / Chapter 5.4 --- Activation of caspase9 --- p.90 / Chapter 5.5 --- Conclusion --- p.92 / Chapter CHAPTER 6 --- : Antitumor activities independent of estrogen receptor / Chapter 6.1 --- Introduction --- p.93 / Chapter 6.2 --- Completion assay on Estrogen Receptor binding --- p.95 / Chapter 6.3 --- Expression level of Estrogen receptor --- p.97 / Chapter 6.4 --- Conclusion --- p.99 / Chapter CHAPTER 7 --- : Proteomic detection of intracellular changes / Chapter 7.1 --- Introduction --- p.100 / Chapter 7.2 --- Differential Protein expression profile of MCF-7 cells --- p.101 / Chapter 7.3 --- Elevated HLA-A antigen expression --- p.104 / Chapter 7.3.1 --- Mass fingerprinting with MALDI-TOF-MS --- p.104 / Chapter 7.3.2 --- Flow cytometrial analysis with immunofluoresent staining --- p.107 / Chapter 7.4 --- Conclusion --- p.109 / Chapter CHAPTER 8 --- : Discussion / Chapter 8.1 --- Polyphyllin D is Invaluable for further Investigation --- p.110 / Chapter 8.2 --- Induction of common mitochondrial pathway --- p.111 / Chapter 8.3 --- Induction of Fas receptor apoptotic pathway is unknown --- p.112 / Chapter 8.4 --- Polyphyllin D is Caspase-3 independent --- p.113 / Chapter 8.5 --- MDA-MB-231 is more sensitive to polyphyllin D --- p.113 / Chapter 8.6 --- Antitumor effects regardless of induction of apoptosis --- p.114 / Chapter 8.7 --- Advances in antitumor effect by MHC-1 antigen up-regulation --- p.114 / Chapter 8.8 --- Implication of polyphyllin D in up-regulating MHC-1 antigen --- p.115 / Chapter 8.9 --- Future aspect --- p.117 / Chapter 8.10 --- Conclusion --- p.121 / Chapter CHAPTER 9 : --- References --- p.122
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324400 |
| Date | January 2003 |
| Contributors | Lee, Mei-sze., 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, ix, 133 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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