Luk Wing Yan Vivien. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-239). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiv / List of Table --- p.xxv / Abbreviations --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ganoderma spp --- p.1 / Chapter 1.2 --- Bioactive components of Ganoderma spp --- p.3 / Chapter 1.2.1 --- Lingzhi polysaccharide --- p.3 / Chapter 1.2.2 --- Terpenes --- p.4 / Chapter 1.3 --- Ganoderma spp. as Chinese traditional medicine --- p.5 / Chapter 1.4 --- Artificial hybridisation of Ganoderma luciudm and G. tsugae --- p.6 / Chapter 1.4.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. lucidum --- p.8 / Chapter 1.5 --- Breast Cancer --- p.8 / Chapter 1.5.1 --- Anti-tumor effects of natural substances against breast cancer cell MCF-7 --- p.9 / Chapter 1.5.2 --- Anti-tumor effects of natural substances against breast cancer cell MDA-MB-231 --- p.11 / Chapter 1.5.3 --- Anti-proliferation of cancer --- p.12 / Chapter 1.5.3.1 --- Cell cycle arrest --- p.12 / Chapter 1.5.3.2 --- Cell death --- p.13 / Chapter 1.5.4 --- Anti-proliferation assays --- p.17 / Chapter 1.5.4.1 --- MTT assay --- p.17 / Chapter 1.5.4.2 --- Trypan blue cell viability assay --- p.18 / Chapter 1.5.4.3 --- BrdU assay --- p.18 / Chapter 1.6 --- Endocrine system and hormones --- p.19 / Chapter 1.6.1 --- Estrogen --- p.23 / Chapter 1.6.2 --- Estrogen receptors --- p.24 / Chapter 1.6.3 --- Estrogen action --- p.29 / Chapter 1.6.4 --- Estrogenicity assays --- p.32 / Chapter 1.6.4.1 --- Recombinant yeast assay --- p.33 / Chapter 1.6.4.2 --- E-screen assay --- p.35 / Chapter 1.6.4.3 --- Estrogen receptor competitor binding assay --- p.36 / Chapter 1.6.4.4 --- Endogenous estrogen-regulated gene expression assay --- p.39 / Chapter 1.6.4.4.1 --- Transforming growth factor --- p.39 / Chapter 1.6.4.4.2 --- Monoamine oxidase A --- p.40 / Chapter 1.6.4.4.3 --- pS2 --- p.40 / Chapter 1.6.4.5 --- Uterotrophic assay --- p.41 / Chapter 1.6.4.6 --- Comparison of in vitro and in vivo assay --- p.42 / Chapter 1.7 --- Aim of study --- p.45 / Chapter 1.7.1 --- Objectives --- p.45 / Chapter Chapter 2 --- Materials and Methods --- p.47 / Chapter 2.1 --- Fungal culture --- p.47 / Chapter 2.2 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.1 --- Protoplast isolation of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.2 --- Protoplast fusion of Ganoderma tsugae and G. lucidum --- p.48 / Chapter 2.3 --- Screening and selection of hybrid ´Ø --- p.49 / Chapter 2.3.1 --- Temperature screening --- p.49 / Chapter 2.3.2 --- DNA fingerprint by Arbitarily-primed polymerase chain reaction --- p.49 / Chapter 2.3.2.1 --- Extraction of genomic DNA --- p.49 / Chapter 2.3.2.2 --- Arbitrarily-primed polymerase chain reaction --- p.50 / Chapter 2.3.2.3 --- Gel electrophoresis --- p.51 / Chapter 2.4 --- Confirmation test --- p.51 / Chapter 2.4.1 --- Somatic incompatibility test --- p.51 / Chapter 2.4.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.52 / Chapter 2.4.2.1 --- Specific Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.4.2.2 --- Purification of PCR products --- p.52 / Chapter 2.4.2.3 --- Cycle-sequencing --- p.53 / Chapter 2.3.2.4 --- Sequencing --- p.54 / Chapter 2.3.2.5 --- Sequence analysis --- p.54 / Chapter 2.5 --- Characterization of the selected hybrid --- p.56 / Chapter 2.5.1 --- Scanning electron microscopy (SEM) --- p.56 / Chapter 2.5.1.1 --- Preparation of specimens for scanning electron microscopy --- p.56 / Chapter 2.5.1.2 --- "Cytological studies of pileus, stipe and spores of G. lucidum, G. tsugae and hybrid" --- p.57 / Chapter 2.5.2 --- Temperature effect --- p.57 / Chapter 2.5.3 --- Submerged fermentation --- p.57 / Chapter 2.5.4 --- Fruiting test --- p.58 / Chapter 2.6 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.58 / Chapter 2.6.1 --- Sample preparation --- p.58 / Chapter 2 6.2 --- Lingzhi polysaccharide --- p.59 / Chapter 2.6.3 --- Terpenes --- p.59 / Chapter 2.7 --- Effect of extracts against breast cancer cell lines --- p.60 / Chapter 2.7.1 --- Cell culture --- p.60 / Chapter 2.7.2 --- Lingzhi Extract preparation --- p.61 / Chapter 2.7.3 --- Optimization of cell density --- p.61 / Chapter 2.7.3.1 --- MTT assay --- p.61 / Chapter 2 7.3.2 --- Trypan blue cell viability assay --- p.62 / Chapter 2.7.3.3 --- BrdU assay --- p.62 / Chapter 2.7.3.4 --- Growth curve of MCF-7 --- p.63 / Chapter 2.7.3.5 --- Growth curve of MDA-MB-231 --- p.64 / Chapter 2.7.4 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.69 / Chapter 2.7.4.1 --- MTT assay --- p.69 / Chapter 2 7.4.2 --- Trypan blue cell viability assay --- p.69 / Chapter 2.7.4.3 --- BrdU assay --- p.70 / Chapter 2.7.5 --- Study of cultured medium effect of biomass and pileus extracts on MCF-7 cells --- p.71 / Chapter 2.7.5.1 --- Cultured medium effect ofbiomass and pileus extracts --- p.71 / Chapter 2.7.6 --- mRNA expression assay (RT-PCR) --- p.71 / Chapter 2.7.6.1 --- Effect of extract on gene expression --- p.71 / Chapter 2.7.6.2 --- Time effect of extract on gene expression --- p.72 / Chapter 2.7.6.3 --- Isolation of RNA --- p.72 / Chapter 2.7.6.4 --- Quantification and qualification of DNA and RNA by spectrophotometry --- p.73 / Chapter 2.7.6.5 --- First strand cDNA synthesis --- p.73 / Chapter 2.7.6.6 --- Amplification of cDNA --- p.74 / Chapter 2.7.7 --- Effect of biomass and pileus lingzhi polysacchandes and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.7.1 --- Effect of reconstitution of lingzhi polysacchande and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.8 --- Effect of biomass and pileus extracts on MDA-MB-231 cells --- p.76 / Chapter 2.8 --- Estrogenicigy assay --- p.76 / Chapter 2 8.1 --- E-screen test --- p.76 / Chapter 2.8.2 --- Estrogen receptor competitor binding assay --- p.77 / Chapter 2.8.3 --- pS2 mRNA expression assay --- p.78 / Chapter 2.9 --- DNA microarray analysis --- p.79 / Chapter 2.9.1 --- mRNA purification --- p.79 / Chapter 2.9.2 --- RT and LPR (Linear Polymerase Reaction) labeling --- p.80 / Chapter 2 9.3 --- pre-hybridization --- p.81 / Chapter 2.9.4 --- Hybridization --- p.82 / Chapter 2.9.5 --- Detection --- p.82 / Chapter 2.9.6 --- Image acquisition and analysis --- p.83 / Chapter Chapter 3 --- Result --- p.84 / Chapter 3.1 --- Artificial hybndization of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.1.1 --- protoplast isomation and fusion of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.2 --- Screening and selection of hybrid --- p.84 / Chapter 3.2.1 --- Temperature screening --- p.84 / Chapter 3.2.2 --- DNA fingerprint by Arbitrarily-primed polymerase chain reaction --- p.86 / Chapter 3.3 --- Confirmation tests --- p.88 / Chapter 3.3.1 --- Somatic incompatibility test --- p.88 / Chapter 3.3.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.90 / Chapter 3.4 --- Characterization of selected hybrid --- p.100 / Chapter 3.4.1 --- Scanning electron micscropy --- p.100 / Chapter 3.4.2 --- Temperature effect --- p.103 / Chapter 3.4.3 --- Submerged fermentation --- p.105 / Chapter 3.4.4 --- Fruiting test --- p.107 / Chapter 3.5 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.109 / Chapter 3.5.1 --- Lingzhi polysaccharide --- p.109 / Chapter 3.5.2 --- Terpenes --- p.109 / Chapter 3.6 --- Effect of extracts against breast cancer cell lines --- p.119 / Chapter 3.6.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.119 / Chapter 3.6.2 --- Study of medium effect of biomass and pileus extracts on MCF-7 cells --- p.139 / Chapter 3.6.3 --- mRNA expression assay (RT-PCR) --- p.143 / Chapter 3.6.4 --- Effect of biomass and pileus lingzhi polysaccharides and terpenes on MCF-7 cells --- p.150 / Chapter 3.6.5 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.159 / Chapter 3.7 --- Estrogenicity assay --- p.166 / Chapter 3.7.1 --- E-screen assay on biomass and pileus extracts --- p.166 / Chapter 3.7.2 --- E-screen assay on biomass and pileus terpenes and lingzhi polysaccharide --- p.166 / Chapter 3.7.3 --- Estrogen receptor competitor binding assay --- p.169 / Chapter 3.7.4 --- pS2 mRNA expression assay --- p.175 / Chapter 3.8 --- DNA microarray analysis --- p.177 / Chapter Chapter 4 --- Discussion --- p.184 / Chapter 4.1 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.184 / Chapter 4.1.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. luciudm --- p.184 / Chapter 4.1.2 --- Screening and selection of hybrid --- p.184 / Chapter 4.1.3 --- Characterization of the selected hybrid --- p.185 / Chapter 4.1.4 --- "Nature of hybrid, mutant and variant" --- p.189 / Chapter 4.2 --- Effect of extracts against breast cancer cell lines --- p.190 / Chapter 4.2.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.190 / Chapter 4.2.2 --- Study of effect of cultured medium of biomass and pileus extracts on MCF-7 cells --- p.193 / Chapter 4.2.3 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.194 / Chapter 4.2.4 --- mRNA expression assay (RT-PCR) --- p.195 / Chapter 4.3 --- Estrogenicity --- p.198 / Chapter 4.3.1 --- E-screen assay --- p.198 / Chapter 4.3.2 --- Estrogen receptor competitor binding assay --- p.199 / Chapter 4.3.3 --- pS2 mRNA expression assay --- p.200 / Chapter 4.3.4 --- Ganoderma spp. As hormonal therapy --- p.201 / Chapter 4.4 --- DNA microarray analysis --- p.201 / Chapter 4.5 --- Further investigation --- p.204 / Chapter Chapter 5 --- Conclusion --- p.205 / Chapter Chapter 6 --- Reference --- p.207

Qin, Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 162-218). / Abstract also in Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Table of Content --- p.vi / List of Figures --- p.x / List of Tables --- p.xv / Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ganoderma spp --- p.1 / Chapter 1.1.1 --- Introduction of Ganoderma spp --- p.1 / Chapter 1.1.2 --- Bioactivities of Ganoderma spp --- p.3 / Chapter 1.1.3 --- Endocrine system and breast cancer --- p.11 / Chapter 1.1.3.1 --- Estrogen --- p.11 / Chapter 1.1.3.2 --- Estrogen receptors --- p.12 / Chapter 1.1.3.3 --- Estrogen responsive genes --- p.15 / Chapter 1.1.3.3.1 --- pS2 --- p.15 / Chapter 1.1.3.3.2 --- Progesterone receptor --- p.18 / Chapter 1.1.3.4 --- Androgen --- p.21 / Chapter 1.1.3.5 --- Androgen receptor --- p.23 / Chapter 1.1.3.6 --- Androgen responsive gene --- p.24 / Chapter 1.1.3.6.1 --- Transmembrane prostate androgen-induced RNA --- p.24 / Chapter 1.1.3.6.2 --- Uridine diphosphate glucose dehydrogenase --- p.26 / Chapter 1.1.3.7 --- Breast cancer --- p.26 / Chapter 1.2 --- "Endocrine Disruption of p,p '-DDE" --- p.28 / Chapter 1.2.1 --- Introduction of p´ةp '-DDE --- p.28 / Chapter 1.2.2 --- "p,p '-DDE in environments" --- p.29 / Chapter 1.2.3 --- "p,p '-DDE in human body" --- p.32 / Chapter 1.2.4 --- "p,p '-DDE and reproductive system" --- p.33 / Chapter 1.2.5 --- Endocrine disruptor --- p.35 / Chapter 1.2.6 --- "Action mechanism of p,p '-DDE on endocrine system" --- p.37 / Chapter 1.2.7 --- Apoptosis --- p.39 / Chapter 1.3 --- Food therapy against endocrine disruption --- p.41 / Chapter 1.3.1 --- Food therapy and functional food --- p.41 / Chapter 1.3.2 --- Ganoderma as a Functional food --- p.47 / Chapter 1.3.3 --- Cancer prevention by dietary agents --- p.47 / Chapter 1.3.4 --- Hormone therapy --- p.48 / Chapter 1.3.5 --- Hormone-related properties of Ganoderma spp --- p.50 / Chapter 1.4 --- The aim of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Ganoderma samples --- p.52 / Chapter 2.2 --- Artificial cultivation of Ganoderma spp --- p.54 / Chapter 2.3 --- Molecular identification of Ganoderma spp --- p.55 / Chapter 2.3.1 --- Extraction of genomic DNA --- p.55 / Chapter 2.3.2 --- Gene-specific polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.3 --- Gel electrophoresis --- p.56 / Chapter 2.3.4 --- Purification of PCR amplified product for sequencing --- p.57 / Chapter 2.3.5 --- Cycle-sequencing --- p.57 / Chapter 2.3.6 --- Sequencing --- p.58 / Chapter 2.3.7 --- Sequence analysis --- p.58 / Chapter 2.4 --- Chemical analyses of Ganoderma spp --- p.59 / Chapter 2.4.1 --- Polysaccharide preparations --- p.59 / Chapter 2.4.2 --- Terpene profile --- p.60 / Chapter 2.4.3 --- Fatty acid profile --- p.60 / Chapter 2.5 --- Anti-oxidation activities --- p.61 / Chapter 2.5.1 --- Superoxide radical scavenging assay --- p.61 / Chapter 2.5.2 --- DPPH radical scavenging assay --- p.62 / Chapter 2.6 --- Anti-proliferation effect on human breast cancer cells --- p.62 / Chapter 2.7 --- Hormone-like effects --- p.63 / Chapter 2.7.1 --- E-screen test --- p.63 / Chapter 2.7.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.64 / Chapter 2.7.3 --- "Recombinant yeast cell based ER-, AR- and PGR-responsible promoter assays" --- p.65 / Chapter 2.7.3.1 --- Recombinant yeasts --- p.65 / Chapter 2.7.3.2 --- Growth medium for recombinant yeasts --- p.66 / Chapter 2.7.3.3 --- "ER, AR and PGR assays" --- p.67 / Chapter 2.7.3.4 --- β-Galactosidase assay --- p.67 / Chapter 2.7.4 --- Real time PCR --- p.68 / Chapter 2.8 --- Flow cytometry --- p.71 / Chapter 2.9 --- Comet assay --- p.71 / Chapter 2.10 --- DNA microarray --- p.73 / Chapter 2.10.1 --- Total RNA isolation --- p.73 / Chapter 2.10.2 --- cDNA synthesis --- p.73 / Chapter 2.10.3 --- Preparation of labelled cDNA --- p.74 / Chapter 2.10.4 --- cDNA purification --- p.74 / Chapter 2.10.5 --- Oligo GEArray hybridization --- p.75 / Chapter 2.10.6 --- Chemiluminescent detection --- p.76 / Chapter 2.10.7 --- Data analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- Analysis of Ganderma spp --- p.78 / Chapter 3.1.1 --- Mycelia and fruiting bodies --- p.78 / Chapter 3.1.2 --- Identification of Ganoderma spp --- p.79 / Chapter 3.1.3 --- Chemical properties of samples --- p.80 / Chapter 3.1.4 --- Anti-oxidation activities --- p.90 / Chapter 3.1.5 --- Anti-proliferation effect on human breast cancer cells --- p.90 / Chapter 3.1.6 --- Hormone-like bioactivities --- p.93 / Chapter 3.1.6.1 --- E-screen test --- p.93 / Chapter 3.1.6.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.94 / Chapter 3.1.6.3 --- "Recombinant yeast cell-based ER-, AR- and PGR-responsible promoter assays" --- p.95 / Chapter 3.1.6.4 --- ER- and AR-pathway gene expression by real time PCR --- p.97 / Chapter 3.2 --- "Action mechanism of p,p' -DDE" --- p.99 / Chapter 3.2.1 --- E-screen --- p.99 / Chapter 3.2.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.101 / Chapter 3.2.3 --- Recombinant yeast cell based ER- and AR-responsible promoter assays --- p.103 / Chapter 3.2.4 --- ER- and AR-pathway gene expression by real time PCR --- p.106 / Chapter 3.3 --- Ganoderma tsugae mycelia extract against p.p' -DDE --- p.109 / Chapter 3.3.1 --- E-screen test --- p.109 / Chapter 3.3.2 --- ER- and AR-pathway gene expression by real time PCR --- p.110 / Chapter 3.3.3 --- Analysis of cell cycle --- p.112 / Chapter 3.3.4 --- Analysis of DNA damage --- p.114 / Chapter 3.3.5 --- Analysis of sub-G1 peak --- p.117 / Chapter 3.3.6 --- DNA damage and apoptosis relative gene expression by real time PCR --- p.120 / Chapter 3.3.7 --- DNA microarray --- p.121 / Chapter Chapter 4 --- Discussion --- p.131 / Chapter 4.1 --- Analysis of Ganoderma spp --- p.131 / Chapter 4.2 --- Effects of p.p´ة-DDE --- p.144 / Chapter 4.3 --- Protective effects of G. tsugae against p.p' -DDE --- p.151 / Chapter 4.4 --- Further investigation --- p.159 / Chapter 4.5 --- Conclusion --- p.160 / References --- p.162