本論文報導了幾個系列的新型鋅酞菁配合物以及氟硼二吡咯染料的合成與表徵。 這些精心設計的化合物可作為高效的和選擇性的光敏劑應用於靶向性光動力療法和細菌的光動力失活。 / 第一章概述了光動力療法，包括歷史發展、光物理和生物機制及其臨床應用現狀。 重點介紹了用於靶向性光動力療法的第三代光敏劑，其中包括基於靶向性配體、納米載體的光敏劑以及可激活的光敏劑。 另外，本章還簡單介紹了用於抗菌光動力療法的光敏劑。 / 第二章報導了一種新型的由細胞核定位的短肽共軛修飾的鋅酞菁配合物的合成與表徵。 此短肽分子的氨基酸序列為：Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val。 我們研究了該化合物的光物理性質、聚集行為以及離體光動力活性，同時與其非肽共軛修飾的化合物進行了詳細的比較。 利用HT29人結腸腺癌細胞，研究發現此多肽共軛修飾的酞菁展示了較高的細胞吸收、更高的細胞內活性氧的產生效率和光毒性。 同時活體實驗證明此化合物增加了酞菁在裸鼠腫瘤的停留時間。 這些結果在本章中均進行了詳細的報導。 / 第三章敘述了另一種多肽共軛修飾的鋅酞菁化合物的製備和光物理性質。 這個多肽包含了一個環狀的氨基酸序列，即 Arg-Gly-Asp-D-Phe-Lys，此多肽被認為能以腫瘤相關的血管新生時的高表達的跨膜受體（如 α[subscript v]β₃ 整合素）為靶向。 利用 α[subscript v]β₃ 整合素高表達的 U87-MG 人惡性膠質瘤細胞，我們研究了這個化合物的細胞吸收、細胞內活性氧的產生、離體光動力活性以及亞細胞定位。 同時，用 α[subscript v]β₃ 整合素低表達的 MCF-7 人乳腺癌細胞作為對照。 / 通常，腫瘤細胞外的pH值比正常細胞組織的低，因此，我們合成一個由酸敏感的縮醛鍵連接的酞菁二聚體。 此二聚體會發生自身淬滅且對pH有響應。 通過電子吸收和熒光光譜， 我們詳細地研究了這個化合物在不同酸性條件下的光物理性質和斷開動力學。 由於酞菁環具有強的二聚化趨勢，這個二聚體能自身淬滅，因而呈現“失活狀態。 通過降低檸檬酸緩衝液的pH值，這個化合物的乙縮醛鍵能優先斷開，並且斷開的速率隨pH值的降低而增加。 兩個酞菁環的分開增強了熒光強度和單態氧的產生。 這個二聚光敏劑還能在 HT29 細胞內被激活，從而產生較強的細胞內熒光。 相比之下，由乙二醇鏈連接的類似物基本上沒有熒光發射。 同時，這個可斷開的二聚物對HT29細胞光毒性也比不可斷開的類似物高（半致死量：IC₅₀ = 0.35 vs. 0.59 μM）。 第四章對這些結果進行了詳細的報導。 / 在第五章中，我們報導了兩種以腫瘤靶向配體葉酸共軛修飾的二（苯乙烯基）－氟硼二吡咯衍生物的合成、光譜表徵以及光物理性質。 在這兩個化合物中，葉酸和二（苯乙烯基）－氟硼二吡咯是通過不同長度的乙二醇鏈連接的。 我們研究了這兩個化合物的鏈長對KB人鼻咽癌細胞和MCF-7細胞的吸收和離體光動力活性的影響。 前者能高表達葉酸受體，而後者作為低葉酸受體表達的一個對照。 與MCF-7細胞相比，兩個化合物都展示了對KB細胞較高的吸收和光毒性（半致死量：IC₅₀ = 0.062 vs. 2.56 μM 和0.177 vs. 0.995 μM）。 此外，具有較長鏈的化合物優先定位在溶酶體中，而較短鏈的那個化合物則較多停留在細胞的內質網。 / 第六章重點開發了一系列多胺以及不同長度的聚賴氨酸（包括2、4、8個賴氨酸）共軛修飾的鋅酞菁配合物，并用於抗菌光動力療法。 我們報導了它們的合成、光物理性質以及對甲氧西林青霉素敏感的格蘭陽性金黄色釀膿葡萄球菌和格蘭陰性綠膿桿菌的光動力抗菌活性。 其中，三-N-甲基化的酞菁顯示了特別高的效果，在濃度為16 nM時，能降低大於5 log10 的金黄色釀膿葡萄球菌。 / 第七章闡述了前面幾章的實驗部份。 論文的最後附上所有新化合物的核磁共振氫譜和碳譜。 / This thesis describes the synthesis and characterization of several series of novel zinc(II) phthalocyanines and boron dipyrromethenes (BODIPYs), which are carefully designed as efficient and selective photosensitizers for targeted photodynamic therapy (PDT) and photodynamic inactivation of bacteria. / Chapter 1 presents an overview of PDT, including its historical development, photophysial and biological mechanisms, and current research directions. Emphasis is placed on the third-generation photosensitizers for targeted PDT, including targeting ligand-based photosensitizers, nanoparticle-based photosensitizers, and activatable photosensitizers. A brief review of photosensitizers that can be used for antimicrobial PDT is also given. / Chapter 2 reports the synthesis and characterization of a novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, namely Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val. The photophysical properties, aggregation behavior, and in vitro photodynamic activity of this compound have been investigated and compared with its non-peptide-conjugated analogue. It has been found that the peptide-conjugated phthalocyanine shows an enhanced cellular uptake, higher efficiency in generating intracellular reactive oxygen species (ROS), higher photocytotoxicity against HT29 human colorectal adenocarcinoma cells, and enhanced tumor-retention property in tumor-bearing nude mice. The results are reported in detail in this chapter. / Chapter 3 describes the preparation and photophysical properties of another analogue conjugated with a peptide containing the cyclic Arg-Gly-Asp-D-Phe-Lys sequence, which is known to target the upregulated transmembrane protein receptors such as α[subscript v]β₃ integrin during angiogenesis. The cellular uptake, intracellular ROS generation, in vitro photodynamic activity, and subcellular localization of this conjugate have been investigated against U87-MG human glioblastoma cells, which have a high expression of α[subscript v]β₃ integrin. MCF-7 human breast adenocarcinoma cells, which have a low expression of α[subscript v]β₃ integrin, have been used as a negative control. / On the base that the extracellular pH in tumors is generally lower than that in normal tissues, we have developed a pH-responsive self-quenched phthalocyanine dimer connected with an acid-sensitive ketal linker. The basic photophysical properties of this compound and its cleavage kinetics upon exposure to different acidic conditions have been extensively studied by electronic absorption and fluorescence spectroscopy. Owing to the strong dimerization tendency of the phthalocyanine ring, this dimer is self-quenched and in the "OFF" state. By lowering the pH (< 6.5) in citrate buffer solutions, the linker is preferentially cleaved, and the rate of cleavage increases as the pH decreases. The separation of the phthalocyanine moieties leads to enhancement in fluorescence intensity and singlet oxygen production. This dimeric photosensitizer can also be activated inside HT29 cells causing strong intracellular fluorescence. By contrast, the fluorescence is hardly observed for the non-cleavable ethylene glycol-linked analogue. The photocytotoxicity of the cleavable dimer is also higher than that of the non-cleavable counterpart (IC₅₀ = 0.35 vs. 0.59 μM). The results are reported in detail in Chapter 4. / In Chapter 5, we describe the synthesis, characterization, and photophysical properties of two distyryl BODIPY derivatives conjugated with a folic acid as a tumor-targeting ligand via an ethylene glycol spacer with different chain length. The effects of the chain length on the cellular uptake and in vitro photodynamic activities of these compounds have been examined against KB human nasopharyngeal epidermal carcinoma cells and MCF-7 cells. The former are known to have a high expression of folate receptors, while the latter have been used as a negative control. Both compounds show enhanced cellular uptake and higher photocytotoxicity toward KB cells when compared with MCF-7 cells (IC₅₀ = 0.062 vs. 2.56 μM and 0.177 vs. 0.995 μM). The conjugate with a longer spacer shows preferential localization in the lysosomes, while the analogue with a shorter linker accumulates in the endoplasmic reticulum of the cells. / Chapter 6 focuses on the development of a series of zinc(II) phthalocyanines substituted with a polyamine moiety or a polylysine chain containing 2, 4, or 8 lysine units for antimicrobial PDT. Their synthesis, photophysical properties, and photodynamic antimicrobial activities against Gram (+) methicillin-sensitive Staphylococcus aureus and Gram (-) Pseudomonas aeruginosa are reported. The tri-N-methylated phthalocyanine is particularly potent showing a more than 5 log₁₀ reduction of the Staphylococcus aureus at 16 nM. / Chapter 7 gives the experimental details for the work described in the preceding chapters. ¹H and ¹³C{¹H} NMR of all the new compounds are given in the Appendix. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ke, Meirong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 159-176). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Abstract (in Chinese) --- p.v / Acknowledgment --- p.viii / Table of Contents --- p.xi / List of Figures --- p.xvi / List of Schemes --- p.xxiv / List of Tables --- p.xxv / Abbreviations --- p.xxvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction of Photodynamic Therapy --- p.1 / Chapter 1.2 --- Mechanisms of Photodynamic Therapy --- p.2 / Chapter 1.2.1 --- Photophysical Mechanisms --- p.2 / Chapter 1.2.2 --- Biological Mechanisms --- p.4 / Chapter 1.3 --- Clinical Status of Photodynamic Therapy --- p.5 / Chapter 1.4 --- Overview of Photosensitizers --- p.7 / Chapter 1.5 --- Targeted Photodynamic Therapy --- p.13 / Chapter 1.5.1 --- Targeting Ligand-Based Photosensitizers --- p.13 / Chapter 1.5.1.1 --- Synthetic Peptides --- p.13 / Chapter 1.5.1.2 --- Proteins --- p.16 / Chapter 1.5.1.3 --- Aptamers --- p.18 / Chapter 1.5.1.4 --- Folic acid --- p.19 / Chapter 1.5.1.5 --- Other Biological Ligands --- p.20 / Chapter 1.5.2 --- Nanoparticle-Based Photosensitizers --- p.20 / Chapter 1.5.2.1 --- Biodegradable Nanoparticle-Based Photosensitizers --- p.21 / Chapter 1.5.2.2 --- Non-Biodegradable Nanoparticle-Based Photosensitizers --- p.23 / Chapter 1.5.3 --- Activatable Photosensitizers --- p.25 / Chapter 1.5.3.1 --- Environment-Activated Photosensitizers --- p.26 / Chapter 1.5.3.2 --- Enzyme-Activated Photosensitizers --- p.27 / Chapter 1.5.3.3 --- Nucleic Acid-Activated Photosensitizers --- p.29 / Chapter 1.6 --- Antimicrobial Photodynamic Therapy --- p.32 / Chapter 1.6.1 --- General Introduction --- p.32 / Chapter 1.6.2 --- Photosensitizers for Antimicrobial Photodynamic Therapy --- p.34 / Chapter Chapter 2 --- A Phthalocyanine-Peptide Conjugate with High in vitro Photodynamic Activity and Enhanced in vivo Tumor-Retention Property --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Results and Discussion --- p.37 / Chapter 2.2.1 --- Molecular Design and Synthesis --- p.37 / Chapter 2.2.2 --- Electronic Absorption and Photophysical Properties --- p.42 / Chapter 2.2.3 --- In Vitro Photodynamic Activities --- p.44 / Chapter 2.2.4 --- In Vivo Studies --- p.50 / Chapter 2.3 --- Conclusion --- p.52 / Chapter Chapter 3 --- Synthesis, Characterization, and Photodynamic Activity of a cylic RGD-Conjugated Phthalocyanine --- p.53 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.2 --- Results and Discussion --- p.54 / Chapter 3.2.1 --- Molecular Design and Synthesis --- p.54 / Chapter 3.2.2 --- Electronic Absorption and Photophysical Properties --- p.57 / Chapter 3.2.3 --- In Vitro Photodynamic Activities --- p.59 / Chapter 3.3 --- Conclusion --- p.67 / Chapter Chapter 4 --- A pH-Responsive Fluorescent Probe and Photosensitizer Based on the Dimerization Property of Phthalocyanines --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Results and Discussion --- p.70 / Chapter 4.2.1 --- Synthesis and Characterization --- p.70 / Chapter 4.2.2 --- Electronic Absorption and Photophysical Properties --- p.74 / Chapter 4.2.3 --- In Vitro Studies --- p.80 / Chapter 4.3 --- Conclusion --- p.84 / Chapter Chapter 5 --- Synthesis, Characterization, and Photodynamic Activities of BODIPY-Folate Conjugates --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Results and Discussion --- p.87 / Chapter 5.2.1 --- Synthesis and Characterization --- p.87 / Chapter 5.2.2 --- Electronic Absorption and Photophysical Properties --- p.92 / Chapter 5.2.3 --- In Vitro Studies --- p.94 / Chapter 5.3 --- Conclusion --- p.100 / Chapter Chapter 6 --- Synthesis, Characterization, and Antimicrobial Photodynamic Activities of Cationic Phthalocyanines --- p.102 / Chapter 6.1 --- Introduction --- p.102 / Chapter 6.2 --- Results and Discussion --- p.103 / Chapter 6.2.1 --- Synthesis and Characterization --- p.103 / Chapter 6.2.2 --- Electronic Absorption and Photophysical Properties --- p.108 / Chapter 6.2.3 --- In Vitro Photodynamic Antimicrobial Activities --- p.112 / Chapter 6.3 --- Conclusion --- p.114 / Chapter Chapter 7 --- Experimental Section --- p.115 / Chapter 7.1 --- General --- p.115 / Chapter 7.2 --- Synthesis --- p.119 / Chapter 7.2.1 --- Synthesis for Chapter 2 --- p.119 / Chapter 7.2.2 --- Synthesis for Chapter 3 --- p.125 / Chapter 7.2.3 --- Synthesis for Chapter 4 --- p.128 / Chapter 7.2.4 --- Synthesis for Chapter 5 --- p.132 / Chapter 7.2.5 --- Synthesis for Chapter 6 --- p.138 / Chapter 7.3 --- pH-Response Properties of 4.6 and 4.7 in Citrate Buffer Solutions --- p.143 / Chapter 7.4 --- In Vitro Studies --- p.144 / Chapter 7.4.1 --- Cell Lines and Culture Conditions --- p.144 / Chapter 7.4.2 --- Photocytotoxicity Assay --- p.145 / Chapter 7.4.3 --- Photodynamic Antimicrobial Inactivatoin Studies --- p.147 / Chapter 7.4.4 --- Intracellular ROS Measurements --- p.148 / Chapter 7.4.5 --- Cellular Uptake (Determined by Extraction Method) --- p.149 / Chapter 7.4.6 --- Cellular Uptake (Determined by Confocal Microscopy) --- p.150 / Chapter 7.4.7 --- Cellular Uptake (Determined by Flow Cytometry) --- p.152 / Chapter 7.4.8 --- Fluorescence Microscopic Studies --- p.153 / Chapter 7.4.9 --- Subcellular Localization Studies --- p.153 / Chapter 7.4.10 --- pH-Dependent Intracellular Fluorescence Studies --- p.155 / Chapter 7.5 --- In Vivo Imaging and Ex Vivo Organ Biodistribution --- p.156 / Chapter Chapter 8 --- Conclusion and Outlook --- p.157 / References --- p.159 / Chapter Appendix --- NMR Spectra of New Compounds --- p.177

by Li Jian Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Hung Shieh-Jung Fanny. / Thesis submitted in: August 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-109). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Content --- p.vi / List of Abbreviations --- p.xiii / List of Figure --- p.xv / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease (AD) --- p.1 / Chapter 1.1.1 --- Clinical features --- p.2 / Chapter 1.2 --- Histopathological studies of AD --- p.2 / Chapter 1.2.1 --- Neuritic plaques --- p.2 / Chapter 1.2.2 --- Neurofibrillary tangles (NFT) --- p.4 / Chapter 1.2.3 --- Tau --- p.5 / Chapter 1.3 --- Kinases and Alzheimer's Disease --- p.7 / Chapter 1.4 --- Free radical damage --- p.8 / Chapter 1.5 --- Available treatment for AD --- p.7 / Chapter 1.6 --- A Chinese medicinal material 一 Danshen （（Salviae miltiorrhizcie) --- p.11 / Chapter 1.6.1 --- Chemical constituents --- p.11 / Chapter 1.6.1.1 --- Lipophilic Compounds of Danshen --- p.12 / Chapter 1.6.1.2 --- Water-soluble Compounds of Danshen --- p.17 / Chapter 1.6.2 --- Pharmacological usage --- p.20 / Chapter 1.6.2.1 --- Action on Coronary system --- p.20 / Chapter 1.6.2.2 --- Bacteriostatic action --- p.21 / Chapter 1.6.2.3 --- Actions on the immune system --- p.21 / Chapter 1.6.3 --- Biological activity on brain --- p.22 / Chapter 1.7 --- Objectives and scope of the project --- p.23 / Chapter Chapter 2 --- General Materials and Method --- p.24 / Chapter 2.1 --- Recombinant DNA techniques --- p.24 / Chapter 2.1.1 --- Preparation of E. coli strain DH-5a competent cells --- p.24 / Chapter 2.1.2 --- Transformation of plasmid DNA into competent cells --- p.25 / Chapter 2.1.3 --- Preparation of plasmid DNA using QIAGEN Plasmid Maxipreps kit --- p.25 / Chapter 2.1.4 --- Phenol/ choroform extraction of DNA --- p.26 / Chapter 2.1.5 --- Spectrophotometric quantitation of the amount and purity of DNA --- p.27 / Chapter 2.2 --- Drugs preparation --- p.27 / Chapter 2.2.1 --- Preparation of aqueous extracts of Traditional Chinese Medicine (TCM) --- p.27 / Chapter 2.2.2 --- Preparation of ethanol extracts of Traditional Chinese Medicine (TCM) --- p.27 / Chapter 2.3 --- "3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay " --- p.28 / Chapter 2.4 --- Analysis of proteins from culture cells --- p.28 / Chapter 2.4.1 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.4.2 --- Quantitation of protein by Bradford method --- p.29 / Chapter 2.4.3 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.4.4 --- Western blot analysis --- p.31 / Chapter 2.5 --- Reagents and buffers --- p.32 / Chapter 2.5.1 --- Reagents for competent cell preparation --- p.32 / Chapter 2.5.2 --- Reagents provided by QIAGEN Plasmid Maxipreps kit --- p.33 / Chapter 2.5.3 --- Reagents for SDS-PAGE --- p.34 / Chapter 2.5.4 --- Reagents and buffers for Western Blotting --- p.35 / Chapter 2.5.5 --- Cell lines --- p.36 / Chapter 2.5.6 --- Antibodies --- p.37 / Chapter 2.5.7 --- Plasmids --- p.37 / Chapter 2.5.8 --- Other Chemicals --- p.38 / Chapter Chapter 3 --- The effect of Danshen on GSK-3 induced hyperposphorylation of tau in Cos7 cells / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.1.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.39 / Chapter 3.1.2 --- Structure of GSK-3 --- p.39 / Chapter 3.1.3 --- The importance of GSK-3 in AD --- p.39 / Chapter 3.2 --- Materials and Methods --- p.41 / Chapter 3.2.1 --- Transfection of Gsk-3 and tau into Cos7 monkey kidney cells --- p.43 / Chapter 3.2.2 --- Extraction of total proteins from culture cells --- p.44 / Chapter 3.2.3 --- Quantitation of protein by Bradford method --- p.44 / Chapter 3.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.5 --- Western blot analysis --- p.44 / Chapter 3.3 --- Results --- p.45 / Chapter 3.3.1 --- Toxicity test on Cos7 cells --- p.45 / Chapter 3.3.2 --- The effect of ethanol extract of Danshen on GSK-3 β induced tau phosphorylation --- p.45 / Chapter 3.3.3 --- The effect of aqueous extract of Danshen on GSK-3 β induced tau phosphorylation --- p.48 / Chapter 3.3.4 --- The effect of Protocatechualdehyde on GSK-3β induced tau phosphorylation --- p.48 / Chapter 3.3.5 --- The effect of Salvianolic acid B on GSK-3β induced tau phosphorylation --- p.49 / Chapter 3.4 --- Discussion --- p.60 / Chapter Chapter 4 --- Cdk5 induced hyperposphorylation of tau in CHO cells / Chapter 4.1 --- Introduction --- p.63 / Chapter 4.1.1 --- Cyclin dependent kinase 5 (Cdk5) --- p.63 / Chapter 4.1.2 --- Structure of Cdk5 --- p.63 / Chapter 4.1.3 --- Neurological functions of Cdk5 --- p.64 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Transfection of p35 and tau into CHO cells --- p.66 / Chapter 4.2.2 --- Extraction of total proteins from culture cells --- p.67 / Chapter 4.2.3 --- Quantitation of protein by Bradford method --- p.67 / Chapter 4.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.67 / Chapter 4.2.5 --- Western blot analysis --- p.67 / Chapter 4.3 --- Results --- p.68 / Chapter 4.3.1 --- Toxicity test on CHO cells --- p.68 / Chapter 4.3.2 --- Tau transfection in Cdk5/p35 and TauON3R transiently transfected in CHO cells --- p.68 / Chapter 4.3.3 --- Effect of roscovitine treatment on the transiently tau and p35 transfection in CHO cells --- p.74 / Chapter 4.3.4 --- "Effects of aqueous active components of Danshen, PCAH and SAB on the transiently tau and p35 transfection in CHO cells " --- p.74 / Chapter 4.4 --- Discussion --- p.79 / Chapter Chapter 5 --- Antioxidant effect of Danshen and its active components on lipid peroxidation / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.1.1 --- Red-blood-cell hemolysis model --- p.82 / Chapter 5.2 --- Materials and methods --- p.84 / Chapter 5.2.1 --- Red-blood-cell hemolysis model --- p.84 / Chapter 5.2.2 --- Materials --- p.85 / Chapter 5.2.2.1 --- Animals --- p.85 / Chapter 5.2.2.2 --- Chemicals --- p.85 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Aqueous and ethanol extracts of Danshen --- p.86 / Chapter 5.3.2 --- Active components ´ؤ Protocatechualdehyde and Salvianolic acid B --- p.87 / Chapter 5.4 --- Discussion --- p.91 / Chapter Chapter 6 --- General discussion and Outlook / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Proposed study in the future --- p.95 / Chapter 6.2.1 --- In vitro kinase assay using gamma32 P ATP and substrate with or without TCM --- p.95 / Chapter 6.2.2 --- Use of neuroblastoma cells (SHSY-5Y) to study the effect of Danshen and its active components on tau phosphorylation --- p.95 / Chapter 6.2.3 --- Thiobarbituric acid-reacting substances (TBARS) assay --- p.96 / Chapter 6.2.4 --- In vitro phosphatase kinase assay --- p.96

Chan Tai-po. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 94-101). / Abstracts in English and Chinese. / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 2 --- HYPOTHESIS --- p.3 / Chapter 3 --- OBJECTIVE --- p.4 / Chapter 4 --- CLINICAL IMPLICATIONS --- p.5 / Chapter 5 --- METHODOLOGY --- p.6 / Chapter 5.1 --- Materials --- p.8 / Chapter 5.2 --- Study method --- p.13 / Chapter 5.3 --- Venues of the research --- p.22 / Chapter 5.4 --- Data acquisition --- p.23 / Chapter 5.5 --- Data management and analysis --- p.24 / Chapter 5.6 --- Ethical considerations --- p.25 / Chapter 5.7 --- Participations of persons in the research --- p.28 / Chapter 6 --- RESULTS --- p.34 / Chapter 6.1 --- Problems and fate of rats in the model development group --- p.34 / Chapter 6.2 --- Morbidity and mortality after LEM administration --- p.38 / Chapter 6.3 --- Results of radiological findings --- p.39 / Chapter 6.4 --- Results of liver function tests --- p.48 / Chapter 6.5 --- Results of liver morphology --- p.52 / Chapter 6.6 --- Histological results --- p.53 / Chapter 7 --- DISCUSSION --- p.69 / Chapter 7.1 --- Problems encountered in the development group --- p.69 / Chapter 7.2 --- The pilot study group --- p.71 / Chapter 7.3 --- The need for the present study --- p.74 / Chapter 7.4 --- LEM in cirrhotic rat compared with the normal liver rat --- p.75 / Chapter 7.5 --- Liver function markers in cirrhotic liver --- p.76 / Chapter 7.6 --- Discussion on the assumptions of the research --- p.80 / Chapter 7.7 --- Assessment on measurement error --- p.82 / Chapter 7.8 --- Errors in the pilot study --- p.83 / Chapter 8 --- CONCLUSIONS --- p.84 / Chapter 9 --- Future experiments that may be performed using this model --- p.85 / Chapter 10 --- APPENDICES --- p.86 / Chapter 10.1 --- Appendix 1: Copy on the letter of ethics approval from the Animal Research Ethics Committee of the Chinese University of Hong Kong --- p.86 / Chapter 10.2 --- Appendix 2: Copy on the licences issued by the Department of Health of Hong Kong --- p.88 / Chapter 11 --- REFERENCES --- p.94

Lai Kin Ming Connie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-141). / Abstracts in English and Chinese. / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction on growth cycle of mushroom --- p.1 / Chapter 1.2 --- Literature review of mushroom biological activities --- p.3 / Chapter 1.2.1 --- Various bioactivities of mushroom --- p.3 / Chapter 1.2.2 --- Components responsible for various bioactivities of mushrooms --- p.3 / Chapter 1.3 --- Mushroom polysaccharides and polysaccharide-protein complexes --- p.5 / Chapter 1.3.1 --- Polysaccharides important for antitumor effects --- p.5 / Chapter 1.3.2 --- Polysaccharide-protein complexes important for antitumor effects --- p.7 / Chapter 1.4 --- Structure-function relationship of antitumor activities of polysaccharides --- p.8 / Chapter 1.4.1 --- Effect of molecular mass --- p.8 / Chapter 1.4.2 --- Effect of linkages --- p.9 / Chapter 1.4.3 --- Effect of degree of branching --- p.9 / Chapter 1.4.4 --- Effect of conformation --- p.10 / Chapter 1.5 --- Immunomodulatory effects of mushroom polysaccharides and polysaccharide-protein complexes --- p.11 / Chapter 1.5.1 --- Immunomodulatory effects of polysaccharides --- p.11 / Chapter 1.5.1.1 --- Bioactive polysaccharides in Lentinus edodes --- p.11 / Chapter 1.5.1.2 --- Bioactive polysaccharides in Ganoderma lucidum --- p.12 / Chapter 1.5.2 --- Immunomodulatory effects of polysaccharide-protein complexes --- p.12 / Chapter 1.5.2.1 --- Bioactive polysaccharide-protein complexes in Trametes versicolor --- p.13 / Chapter 1.5.3 --- Immunotherapeutic effects of mushroom polysaccharides --- p.14 / Chapter 1.6 --- Cell cycle and apoptosis --- p.14 / Chapter 1.6.1 --- Introduction of cell cycle --- p.14 / Chapter 1.6.2 --- Cell cycle regulation --- p.15 / Chapter 1.6.3 --- Antitumor effects through apoptotic gene regulation --- p.17 / Chapter 1.7 --- Mushroom sclerotium with antitumor activity --- p.20 / Chapter 1.7.1 --- Literature review on Pleurotus tuber-regium --- p.20 / Chapter 1.7.2 --- Literature review on Poria cocos --- p.22 / Chapter 1.7.3 --- Literature review on Polyporus rhinocerus --- p.23 / Chapter 1.8 --- Objectives --- p.23 / Chapter Chapter 2. --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Mushroom sclerotia --- p.25 / Chapter 2.1.2 --- Animal Model --- p.25 / Chapter 2.1.3 --- Cell lines --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Extraction Scheme for mushroom sclerotia --- p.28 / Chapter 2.2.1.1 --- Hot water extraction only --- p.28 / Chapter 2.2.1.2 --- Sequential extraction scheme --- p.28 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.31 / Chapter 2.2.2.1 --- Acid Depolymerisation --- p.31 / Chapter 2.2.2.2 --- Neutral Sugar Derivatization --- p.31 / Chapter 2.2.2.3 --- Gas Chromatography (GC) --- p.32 / Chapter 2.2.3 --- High Pressure Liquid Chromatography (HPLC) --- p.33 / Chapter 2.2.3.1 --- Size exclusion chromatography --- p.33 / Chapter 2.2.3.2 --- Anion exchange chromatography --- p.34 / Chapter 2.2.4 --- Linkage analysis by methylation --- p.34 / Chapter 2.2.4.1 --- Preparation of partially methylated polysaccharides --- p.34 / Chapter 2.2.4.2 --- Preparation of partially methylated alditol acetates (PMAAs) --- p.37 / Chapter 2.2.4.3 --- Gas chromatography-Mass spectrometry (GC-MS) analysis --- p.37 / Chapter 2.2.5 --- Determination of total sugar by phenol-sulphuric acid Method --- p.38 / Chapter 2.2.6 --- Determination of acidic sugars by measurement of uronic acid content --- p.39 / Chapter 2.2.7 --- Determination of protein content by Lowry-Folin method --- p.40 / Chapter 2.2.8 --- Chemical modification by carboxymethylation --- p.41 / Chapter 2.2.9 --- In vitro antitumor assay --- p.41 / Chapter 2.2.9.1 --- Trypan blue exclusion assay --- p.42 / Chapter 2.2.9.2 --- MTT Assay --- p.42 / Chapter 2.2.10 --- Cell cycle analysis by Flow Cytometry --- p.43 / Chapter 2.2.11 --- In vivo antitumor and immunomodulatory assay --- p.44 / Chapter 2.2.11.1 --- Measurement on tumor growth --- p.44 / Chapter 2.2.11.2 --- Blood sampling for immunostimulatory effects --- p.45 / Chapter 2.2.12 --- Mouse Cytokine Array --- p.45 / Chapter 2.2.13 --- Quantification of Mouse IL-13 by ELISA --- p.46 / Chapter 2.2.14 --- Enumeration of peritoneal cells --- p.47 / Chapter 2.2.15 --- Enumeration of splenocytes --- p.49 / Chapter 2.2.16 --- Statistical methods --- p.50 / Chapter Chapter 3. --- Results and Discussion --- p.51 / Chapter 3.1 --- Yield of crude mushroom sclerotial extracts --- p.51 / Chapter 3.2 --- Chemical composition of crude mushroom sclerotial extracts --- p.57 / Chapter 3.2.1 --- Total carbohydrate content --- p.57 / Chapter 3.2.2 --- Uronic acid content --- p.58 / Chapter 3.2.3 --- Soluble protein content --- p.58 / Chapter 3.3 --- Monosaccharide profiles of mushroom sclerotial extracts by GC --- p.60 / Chapter 3.4 --- Chromatographic analyses of mushroom sclerotial extracts --- p.65 / Chapter 3.4.1 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.65 / Chapter 3.4.2 --- Charge distribution by ion exchange chromatography (IEC) --- p.73 / Chapter 3.5 --- Antitumor effects of mushroom sclerotial extracts from hot water extraction alone --- p.73 / Chapter 3.5.1 --- In vitro antiproliferative study by HL-60 --- p.73 / Chapter 3.5.2 --- In vitro antiproliferative study by MCF-7 --- p.74 / Chapter 3.5.3 --- In vivo antitumor study by BALB/c mice --- p.75 / Chapter 3.6 --- Antitumor effects of extracts from sequential extraction scheme --- p.76 / Chapter 3.6.1 --- In vitro antiproliferative study by HL-60 --- p.76 / Chapter 3.6.2 --- In vitro antiproliferative study by MCF-7 --- p.78 / Chapter 3.6.3 --- In vivo antitumor study by BALB/c mice --- p.80 / Chapter 3.7 --- Comparison of in vitro and in vivo activities of mushroom sclerotial extracts --- p.82 / Chapter 3.8 --- Dose-response relationship of hot water extract from PR on cancer cell lines --- p.85 / Chapter 3.8.1 --- In vitro dose-response antiproliferation of PR-W and PR-HWE on HL-60 --- p.85 / Chapter 3.8.2 --- In vitro dose-response antiproliferation of PR-W on K562 and S180 --- p.88 / Chapter 3.8.3 --- In vivo dose-response relationship of PR-W on S180 --- p.91 / Chapter 3.9 --- Flow cytometric analysis of PR-W on cancer cell lines --- p.92 / Chapter 3.9.1 --- Antiproliferative effect of PR-W on HL-60 --- p.92 / Chapter 3.9.2 --- Antiproliferative effect of PR-W on K562 --- p.95 / Chapter 3.9.3 --- Proposed mechanisms of cell cycle arrest by PR-W --- p.98 / Chapter 3.10 --- Host-mediated antitumor mechanism of PR-W --- p.100 / Chapter 3.10.1 --- Mouse cytokine array --- p.100 / Chapter 3.10.2 --- Quantification of IL-13 by ELISA --- p.105 / Chapter 3.10.3 --- Immunostimulatory effects of PR-W on mice --- p.109 / Chapter 3.11 --- Correlation between antitumor activity and structure of mushroom sclerotial extract from hot water extraction alone --- p.114 / Chapter Chapter 4. --- Conclusions and Future works --- p.118 / List of References --- p.121 / Related Publications --- p.142

神經母細胞瘤是一種交感神經系統的腫瘤。它是最常見的兒童顱外實體瘤。神經母細胞瘤約佔兒童腫瘤的8-10%，佔15%的兒童腫瘤死亡率。目前神經母細胞瘤的治療方法包括外科手術， 化學藥物治療， 放射治療， 幹細胞移植， 誘導分化治療和免疫治療。 然而，這些治療方法通常會導致許多無法避免的嚴重的副作用。因此，開發能高效抑制神經母細胞瘤但對正常細胞無明顯副作用的新型藥物顯得至關重要。最近，用來源於天然產物或中藥的化合物治療癌症引起了科學家的廣泛關注。靛玉紅-3’-肟（Indirubin-3’-oxime, I3M）和色胺酮（tryptanthrin）分別是從板藍根中分離得到的靛藍生物鹼和吲哚喹唑啉類生物鹼。據研究報導，這兩種生物鹼具有多種生物學和藥理學作用，包括抗菌，抗炎症和抗腫瘤作用。它們對體外的多種人腫瘤細胞具有抗腫瘤作用。然而，它們對人神經母細胞瘤的調節作用和作用機理仍不太清楚。在我的博士研究課題中，我們對板藍根生物鹼包括靛玉紅-3’-肟和色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了研究和探討。 / 首先，我們研究了靛玉紅-3’-肟對人神經母細胞瘤的抗腫瘤活性和作用機制。實驗結果表明，靛玉紅-3’-肟能夠抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，並且其抑制效果呈時間和濃度依賴性。然而，靛玉紅-3’-肟對正常細胞無顯著的細胞毒性作用。對其生長抑制作用機制的研究結果表明靛玉紅-3’-肟能夠特異性地減少LA-N-1細胞系中線粒體的調節子ERR和 PGC-1的表達，從而導致線粒體生成減少，線粒體膜電位降低以及線粒體活性氧（ROS）增加。靛玉紅-3’-肟還增加週期蛋白依賴性蛋白激酶（CDK）抑制蛋白p27{U+1D37}{U+2071}{U+1D56}¹的蛋白水平並降低週期蛋白依賴性蛋白激酶2（CDK2）和細胞週期蛋白E（Cyclin E）的表達，從而導致細胞週期阻滯在G0/G1期。 另外，我們發現靛玉紅-3’-肟能減少SH-SY5Y細胞系的線粒體生成，增加細胞內活性氧的水準從而導致細胞週期停滯在G0/G1期和細胞凋亡。以上結果表明靛玉紅-3’-肟可能通過破壞線粒體的功能從而導致LA-N-1和SH-SY5Y細胞的細胞週期阻滯和誘導SH-SY5Y細胞的細胞凋亡來發揮其抗腫瘤的作用。 / 接著，我們對色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了探討。我們研究的結果表明，色胺酮可以時間和濃度依賴性地抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，而對正常的細胞無顯著的細胞毒性。對色胺酮抑制人神經母細胞瘤生長的機制研究表明，色胺酮能顯著地降低細胞週期蛋白（Cyclin D1和 Cyclin D3）和週期蛋白依賴性蛋白激酶（CDK4和CDK6）的蛋白水平從而導致細胞週期停滯在G0/G1期。色胺酮可以激活半胱氨酸天冬氨酸蛋白酶8,9和3/7（caspase 8, caspase 9 和 caspase 3/7）從而誘導LA-N-1細胞凋亡。色胺酮還可以誘導LA-N-1細胞分化，表現為神經細胞分化的細胞形態，乙醯膽鹼酯酶活性的增加和多種細胞分化的分子標記的表達上調。另外，色胺酮還能降低LA-N-1細胞中N-myc的表達。有趣的是，通過RNA干擾技術降低N-myc的表達能誘導LA-N-1細胞的分化。總的來說，以上結果顯示色胺酮通過誘導細胞週期阻滯，細胞凋亡和細胞分化從而發揮其抗腫瘤的作用。它可能被開發為治療有N-myc基因擴增的高危的人神經母細胞瘤的潛在藥物。 / 此外，我們還研究了靛玉紅-3’-肟和色胺酮是否具有抗血管生成的作用。體外實驗的研究結果表明，靛玉紅-3’-肟和色胺酮能夠濃度依賴性地抑制人微血管上皮細胞 （HMEC-1細胞）的增殖，遷徙和血管生成，但對HMEC-1細胞卻沒有顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮能顯著地抑制小鼠體內的基質膠栓（Matrigel plug）的血管生成。對它們抑制血管生成的機制的研究表明，靛玉紅-3’-肟能下調血管生成素1（Ang-1）和基質金屬蛋白酶2（MMP2）的表達，上調血管生成素2（Ang-2）的表達。靛玉紅-3’-肟能結合到血管內皮生長因數受體2（VEGFR2） 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和下游的MEK/ERK和PI3K/AKT/GSK信號轉導通路。色胺酮同樣可以抑制多種血管生成因子（Ang-1,PDGFB 和MMP2）的表達。此外，它可以結合到血管內皮生長因數受體2 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和血管內皮生長因數受體2介導的ERK1/2信號通路。以上的體外和體內實驗研究結果表明靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。它們可能被開發為治療血管生成相關疾病的潛在藥物。 / 總而言之，我們的研究結果表明靛玉紅-3’-肟和色胺酮通過誘導人神經母細胞瘤細胞的細胞週期阻滯，細胞凋亡或誘導神經細胞分化從而抑制人神經母細胞瘤細胞的生長。然而，它們對正常細胞無顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。未來的研究將進一步探討靛玉紅-3’-肟和色胺酮對人神經母細胞瘤細胞的分子作用機理。另外，通過人神經母細胞瘤細胞的裸鼠移植瘤動物模型可進一步去了解這些板藍根生物鹼在體內的抗腫瘤效果。 / Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid cancer in childhood. It accounts for 8% to 10% of all childhood cancers and for approximately 15% of cancer deaths in children. Current treatment modalities consist of surgery, chemotherapy, radiation therapy, stem cell transplantation, differentiation therapy and immunotherapy. However, these treatments often cause severe and inevitable side effects. It is important to develop novel drugs with higher efficacy on neuroblastoma cells and minimal side effects on normal cells. The use of new promising therapeutic compounds derived from natural products or Chinese herbs have attracted much attention of scientist as an alternative strategy in cancer treatment. Indirubin-3’-oxime (I3M) is an indigo alkaloid and tryptanthrin is an indoloquinazoline alkaloid which can be isolated from the dried roots of medicinal indigo plants known as Banlangen. These two alkaloids have been reported to possess various biological and pharmacological activities, such as anti-microbial, anti-inflammatory, and anti-tumor effects. They were found to exhibit potent anti-tumor activities on various types of human cancer cells in vitro. However, their modulatory effects on human neuroblastoma and the underlying mechanisms remain poorly understood. In my PhD project, the possible anti-tumor activities and action mechanisms of Banlangen alkaloids, including I3M and tryptanthrin, on human neuroblastoma cells were investigated. / Firstly, the anti-cancer effects of I3M on human neuroblastoma cells and the underlying mechanisms were investigated. I3M was found to inhibit the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies showed that I3M specifically decreased the expression of mitochondrial regulators ERRγ and PGC-1βand resulted in decreased mitochondrial mass and altered mitochondrial function characterized by reduction in mitochondrial membrane potential and elevation of reactive oxygen species (ROS) level in LA-N-1 cells. I3M also increased the level of cyclin-dependent kinase (CDK) inhibitor p27{U+1D37}{U+2071}{U+1D56}¹ and reduced the levels of CDK2 and cyclin E in LA-N-1 cells, leading to cell cycle arrest at the G0/G1 phase. In addition, I3M was also found to reduce the mitochondrial mass and increase the ROS level leading to cell cycle arrest at G0/G1 phase and apoptosis in SH-SY5Y cells. These results, when taken together, suggest that I3M might exert its anti-tumor activity by causing mitochondrial dysfunction which led to cell cycle arrest in LA-N-1 cells and resulted in cycle arrest and apoptosis in SH-SY5Y cells. / The anti-tumor effects and action mechanisms of tryptanthrin on the human neuroblastoma cells were also examined. Our results showed that tryptanthrin inhibited the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies indicated that tryptanthrin significantly reduced the protein levels of cyclin D1, cyclin D3, CDK4 and CDK6 leading to cell cycle arrest at G0/G1 phase. In addition, tryptanthrin activated caspase 8, caspase 9 and caspase 3/7 resulting in apoptosis of the human neuroblastoma LA-N-1 cells. Moreover, tryptanthrin induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Tryptanthrin treatment also led to the significant reduction of N-myc expression in LA-N-1 cells. Interestingly, down-regulating N-myc expression using siRNA induced neuronal differentiation of LA-N-1 cells. Collectively, these results indicate that tryptanthrin might exert its anti-tumor activity on the human neuroblastoma LA-N-1 cells by inducing cell cycle arrest, apoptosis and neuronal differentiation. It might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. / Moreover, the anti-angiogenic activities of I3M and tryptanthrin were studied. Our results showed that I3M and tryptanthrin inhibited the proliferation, migration, and tube formation of the human microvascular endothelial HMEC-1 cells in vitro in a concentration-dependent manner but exhibited no significant cytotoxicity on these cells. Moreover, I3M and tryptanthrin markedly suppressed the in vivo angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that I3M down-regulated the expression of Ang-1 and MMP2 and up-regulated the expression of Ang-2. It also bound to the ATP-binding site of VEGFR2 and inhibited the phosphorylation of VEGFR2 leading to suppression of the down-stream MEK/ERK and PI3K/AKT/GSK signaling pathways in HMEC-1 cells. Similarly, tryptanthrin also reduced the expression of several angiogenic factors (Ang-1, PDGFB and MMP2) in HMEC-1 cells. In addition, tryptanthrin also bound to the ATP-binding site of VEGFR2 and suppressed the phosphorylation of VEGFR2 and VEGFR2-mediated ERK1/2 signaling pathway in HMEC-1 cells. Collectively, our results demonstrated that I3M and tryptanthrin exhibited anti-angiogenic activity both in vitro and in vivo by specifically targeting the VEGFR2-mediated signaling pathways and might be exploited as potential therapeutic candidates for the treatment of angiogenesis-related diseases. / In conclusion, our findings indicate that I3M and tryptanthrin might exert their growth-inhibitory effect on the human neuroblastoma cells by causing cell cycle arrest, inducing apoptosis or inducing neuronal differentiation. However, they exhibited minimal cytotoxicity towards the normal cells. Moreover, I3M and tryptanthrin were found to possess anti-angiogenic activities by targeting the VEGFR2-mediated signaling pathways. In the future, investigations should be focused on further elucidation of the molecular action mechanisms of I3M and tryptanthrin on human neuroblastoma cells and to test the anti-tumor efficacy of I3M and tryptanthrin in animal models, using human neuroblastoma xenografts in nude mice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Xuemei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 206-229). / Abstract also in Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Publications --- p.vi / Abstract --- p.vii / 摘要 --- p.xii / Table of Contents --- p.xvi / Chapter One / General Introduction --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- Epidemiology of neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.6 / Chapter 1.1.3 --- Clinical symptoms and diagnosis of neuroblastoma --- p.10 / Chapter 1.1.4 --- Molecular pathogenesis of neuroblastoma --- p.13 / Chapter 1.1.4.1 --- Genetic alterations in neuroblastoma --- p.13 / Chapter 1.1.4.2 --- Disruption of cell division cycle, apoptotic and signaling pathways --- p.16 / Chapter 1.1.5 --- Treatment strategies --- p.19 / Chapter 1.1.5.1 --- Low-risk neuroblastoma treatment strategy --- p.19 / Chapter 1.1.5.2 --- Intermediate-risk neuroblastoma treatment strategy --- p.20 / Chapter 1.1.5.3 --- High-risk neuroblastoma treatment strategy --- p.21 / Chapter 1.1.5.4 --- Side effects of treatment --- p.23 / Chapter 1.2 --- Banlangen alkaloids --- p.23 / Chapter 1.2.1 --- Overview of Banlangen alkaloids --- p.23 / Chapter 1.2.2 --- Biological and pharmacological effects of Banlangen alkaloids --- p.28 / Chapter 1.2.2.1 --- Anti-inflammatory activity --- p.28 / Chapter 1.2.2.2 --- Anti-microbial activity --- p.29 / Chapter 1.2.2.3 --- Anti-tumor activity --- p.30 / Chapter 1.2.2.4 --- Other biological activities --- p.32 / Chapter 1.2.3 --- Bioavailability of Banlangen alkaloids --- p.33 / Chapter 1.2.4 --- Toxicity of Banlangen alkaloids --- p.34 / Chapter 1.3 --- Aims and scope of this project --- p.36 / Chapter Two / Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media --- p.41 / Chapter 2.1.4 --- Drugs and chemicals --- p.42 / Chapter 2.1.5 --- Reagents and buffers for cell culture --- p.44 / Chapter 2.1.6 --- General staining solutions --- p.47 / Chapter 2.1.7 --- Reagents and buffers for cell growth assays --- p.48 / Chapter 2.1.8 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.9 --- Reagents and buffers for acetylcholine esterase activity assay --- p.50 / Chapter 2.1.10 --- Reagents and buffers for immunocytochemistry --- p.51 / Chapter 2.1.11 --- Reagents and buffers for total RNA extraction --- p.53 / Chapter 2.1.12 --- Reagents and buffers for reverse transcription --- p.54 / Chapter 2.1.13 --- Reagents for quantitative real-time polymerase chain reaction (qRT-PCR) --- p.56 / Chapter 2.1.14 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.1.15 --- Assay kits --- p.65 / Chapter 2.2 --- Methods --- p.68 / Chapter 2.2.1 --- Culture of cells --- p.68 / Chapter 2.2.2 --- MTT assay --- p.69 / Chapter 2.2.3 --- Cell proliferation assay --- p.70 / Chapter 2.2.4 --- Trypan blue exclusion test --- p.70 / Chapter 2.2.5 --- Cytotoxicity assay --- p.71 / Chapter 2.2.6 --- Colony-forming assay --- p.72 / Chapter 2.2.7 --- Cell cycle analysis --- p.72 / Chapter 2.2.8 --- Assessment of apoptosis --- p.73 / Chapter 2.2.9 --- Caspase activity determination --- p.74 / Chapter 2.2.10 --- Mitochondrial mass assay --- p.75 / Chapter 2.2.11 --- Reactive oxygen species (ROS) assay --- p.75 / Chapter 2.2.12 --- Mitochondrial membrane potential determination --- p.76 / Chapter 2.2.13 --- Morphological detection of cell differentiation --- p.76 / Chapter 2.2.14 --- Acetylcholine esterase activity determination --- p.77 / Chapter 2.2.15 --- Immunocytochemistry --- p.77 / Chapter 2.2.16 --- RNA interference --- p.78 / Chapter 2.2.17 --- Wound healing assay --- p.79 / Chapter 2.2.18 --- Tube formation assay --- p.79 / Chapter 2.2.19 --- In vivo Matrigel plug assay --- p.80 / Chapter 2.2.20 --- Phospho-VEGFR2 Sandwich ELISA assay --- p.80 / Chapter 2.2.21 --- Isolation of total cellular RNA --- p.81 / Chapter 2.2.22 --- Reverse transcription (RT) --- p.82 / Chapter 2.2.23 --- Quantitative real-time PCR --- p.83 / Chapter 2.2.24 --- Total protein extraction --- p.84 / Chapter 2.2.25 --- Protein concentration determination --- p.84 / Chapter 2.2.26 --- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) --- p.85 / Chapter 2.2.27 --- Semi-dry Western blotting --- p.85 / Chapter 2.2.28 --- Enhanced chemiluminescence (ECL) assay --- p.87 / Chapter 2.2.29 --- Molecular docking --- p.87 / Chapter 2.2.30 --- Statistical analysis --- p.88 / Chapter Three / Modulatory effects and action mechanisms of indirubin-3'-oxime on human neuroblastoma cells --- p.89 / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Indirubin-3’-oxime inhibited the growth and colony formation of human neuroblastoma cells in vitro --- p.94 / Chapter 3.2.2 --- Indirubin-3’-oxime exhibited no significant cytotoxicity on normal cells --- p.101 / Chapter 3.2.3 --- Indirubin-3’-oxime induced G0/G1 cell cycle arrest in LA-N-1 cells --- p.103 / Chapter 3.2.4 --- Indirubin-3’-oxime caused mitochondrial dysfunction in LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Indirubin-3’-oxime selectively reduced ERR γ and PGC-1β protein and mRNA levels in LA-N-1 cells --- p.111 / Chapter 3.2.6 --- Indirubin-3’-oxime induced cell cycle arrest at G0/G1 phase and apoptosis of SH-SY5Y cells --- p.113 / Chapter 3.2.7 --- Indirubin-3’-oxime reduced mitochondrial mass and elevated mitochondrial ROS level in SH-SY5Y cells --- p.115 / Chapter 3.2.8 --- Indirubin-3’-oxime increased the caspase 8, caspase 9 and caspase 3/7 activities in SH-SY5Y cells --- p.117 / Chapter 3.3 --- Discussion --- p.119 / Chapter Four / Modulatory effects and action mechanisms of tryptanthrin on human neuroblastoma cells --- p.125 / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Results --- p.129 / Chapter 4.2.1 --- Tryptanthrin inhibited the cell growth and colony formation of human neuroblastoma cells --- p.129 / Chapter 4.2.2 --- Tryptanthrin exhibited no significant cytotoxicity on normal cells --- p.136 / Chapter 4.2.3 --- Tryptanthrin induced cell cycle arrest at G0/G1 phase --- p.138 / Chapter 4.2.4 --- Tryptanthrin induced apoptosis of LA-N-1 cells --- p.140 / Chapter 4.2.5 --- Tryptanthrin induced morphological neuronal differentiation in LA-N-1 cells --- p.143 / Chapter 4.2.6 --- Tryptanthrin induced the expression of neuronal differentiation markers --- p.146 / Chapter 4.2.7 --- Tryptanthrin down-regulated the expression of N-myc in LA-N-1 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Five / Anti-angiogenesis effects and action mechanisms of indirubin-3'-oxime and tryptanthrin --- p.158 / Chapter 5.1 --- Introduction --- p.159 / Chapter 5.2 --- Results --- p.163 / Chapter 5.2.1 --- Indirubin-3’-oxime and tryptanthrin inhibited the proliferation of endothelial cells --- p.163 / Chapter 5.2.3 --- Indirubin-3’-oxime and tryptanthrin reduced the tube formation of endothelial cells --- p.168 / Chapter 5.2.4 --- Indirubin-3’-oxime and tryptanthrin blocked angiogenesis in the in vivo Matrigel plug model --- p.171 / Chapter 5.2.5 --- Indirubin-3’-oxime and tryptanthrin reduced the angiogenic gene expression in endothelial cells --- p.174 / Chapter 5.2.6 --- Indirubin-3’-oxime and tryptanthrin attenuated VEGFR2-mediated signaling pathways in endothelial cells --- p.176 / Chapter 5.2.7 --- Indirubin-3’-oxime bound to the ATP-binding site of VEGFR2 kinase domain --- p.181 / Chapter 5.2.8 --- Tryptanthrin bound to the ATP-binding site of VEGFR2 kinase domain --- p.182 / Chapter 5.3 --- Discussion --- p.184 / Chapter Six / Conclusions and future perspectives --- p.191 / References --- p.206

研究背景：常规骨科临床在治疗大段骨缺损时需要移植骨和（或）支架材料，尤其复合有治疗性生物活性成分的复合材料尤为理想。本研究的策略在于发展开发一种具有生物活性和生物降解特性的的合并有植物小分子icaritin（外源性生长因子）或者骨形态发生蛋白2（BMP-2, 内源性生长因子）的复合骨支架用于骨再生。基于聚乳酸乙交酯共聚物和磷酸三钙，我们利用先进的快速成型技术编制了新型的符合有BMP-2 或者icaritin 的支架材料， 命名为PLGA/TCP （ 对照材料组） ，PLGA/TCP/BMP-2（BMP-2 编织复合治疗材料组）， PLGA/TCP/icaritin （低，中，高剂量icaritin 编织复合治疗材料组）。 / 研究目标：本研究的总体目标是通过系统的体外实验和兔骨缺损的体内实验，建立和评估一种优化的复合递送系统，用于骨再生的应用。体内效果的研究体现在终点关于合并有外源性生长因子icaritin 和内源性生长因子BMP-2 的复合材料之间的比较研究。 / 材料和方法：低温快速成型机器用于复合材料的编制。PLGA 和TCP 作为基本载体材料，icaritin 和BMP-2 作为具有生物活性的外源性和内源性生长因子，分别进行编织复合。最终编织复合的支架材料命名为P/T 对照组，P/T/BMP-2 和低，中，高剂量P/T/icaritin 治疗组。另外，我们通过液体完全浸泡并在真空橱内干燥24 小时的方法制备了BMP-2 和icaritin 浸泡复合支架材料，分别是P/T+BMP-2(阳性对照组)和中剂量P/T+icaritin(比较组)。体外成骨潜能是通过兔骨髓干细胞和支架材料共培养的方法检测细胞接种，增殖效率，碱性磷酸酶活性，钙沉积以及成骨基因定量mRNA 表达检测。兔尺骨双侧阶段性缺损并植入复合支架材料的模型用于探讨支架材料体内成骨和成血管功效，影像学和活体检测CT 技术用于评估骨再生；借助CT的血管造影术和组织学检测新生血管；动态核磁共振技术用于检测骨缺损局部血液灌注功能，以及宿主组织和支架材料之间的相互作用。 / 研究结果： 对编织的支架材料的体外特性和成骨潜能进行鉴定和评估。显微CT 定量结果显示此支架材料具有互联大孔隙，平均孔隙率75±3.27%，平均孔径458±25.6μm。和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，与材料共培养的兔骨髓干细胞（BMSCs）表现了较高的细胞接种效率，碱性磷酸酶活性和上调的胶原酶I，骨桥蛋白mRNA 表达，以及较多的钙结节沉积。同时，BMP-2 浸泡复合组表现了最佳的效果（n=6, p<0.01）。兔尺骨缺损模型体内试验结果显示，术后2，4，8周影像学和显微CT 显示，和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组材料（n=6, p<0.01）植入的骨缺损区域有更多新生成骨。BMP-2 浸泡复合组表现了最多的新骨形成（n=6,p<0.01）。组织学结果同样也验证了在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，存在较多的骨样组织和典型的板层骨。BMP-2 浸泡复合组也具有最多的新骨组织生成（n=6, p<0.01）。此外， 在icaritin 编织复合支架材料（n=6, p<0.05）尤其中剂量组（n=6, p<0.01）中，借助显微CT 的血管造影术检测发现，骨缺损区域出现较大的新生血管体积，动态核磁共振检查发现较好的局部血液灌注功能。在三种icaritin 剂量浓度的编织复合材料组之间比较，我们发现中浓度icaritin 复合比例的编织复合材料组显示了最佳的成骨潜能。 / 研究结论： 编织复合有外源性植物分子icaritin 的PLGA/TCP 支架材料在体内体外试验中均表现了预期的成骨分化潜能和骨再生能力，尤其是中剂量icaritin 编织复合材料。传统的应用前做体外复合的BMP-2 浸泡复合支架材料和更具吸引力和方便应用的植物分子icaritin 编织复合支架材料，都可以较好的增强骨修复，这很可能为新型生物复合材料潜在的临床有效性验证提供很好的基础。 / Background: Treatment of large bone defect in routine orthopaedic clinics requires bonegrafting and/or scaffold materials, especially desirable with composite material combined with therapeutic and bioactive agents for achieving better treatment outcome. The strategy of this study was to develop such a bioactive biodegradable composite bone scaffold incorporating a phytomolecule icaritin as an exogenous growth factor or bone morphogenetic protein-2 (BMP-2) as a known endogenous growth factor for bone regeneration. Based on polylactide-co-glycolide (PLGA) and Tricalcium Phosphate (TCP), we fabricated innovative BMP-2 or icaritin incorporated scaffold materials, namely PLGA/TCP (Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin with three different dosages of icaritin (Treatment groups) by an advanced prototyping technology. / Aims: The overall aim of the study was to establish and evaluate a local delivery system with slow release of bioactive agents for acceleration of bone regeneration in a bone defect model in rabbits. In vivo efficacy study served as end-point of this comparative study between composite scaffold incorporating exogenous growth factor icaritin and endogenous growth factor BMP-2. / Materials & Methods: Composite scaffolds were fabricated at -28ºC by a lowtemperature rapid-prototyping machine. PLGA and TCP were used as basic carrier materials, and icaritin or BMP-2 was incorporated as exogenous or endogenous bioactive growth factors, respectively. The incorporated scaffolds were named by PLGA/TCP (P/T, Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin (Treatment groups). In addition, we prepared BMP-2 and icaritin loading scaffolds, namely PLGA/TCP+BMP-2 as positive control group and PLGA/TCP+middle-icaritin as comparative group by entire immersion in the solution and dry in vacuum cabinet for 24 hours. In vitro osteogenic potentials of the designed bioactive composite scaffolds were tested in scaffold-co-cultured rabbit bone marrow stem cells (BMSCs) for measurement of cell seeding and proliferation efficiency, alkaline phosphatase (ALP) activity, calcium deposition, and quantitative mRNA expression of relative osteogenic genes. In vivo efficacy investigation was designed to evaluate osteogenesis and angiogenesis in a bilateral ulna bone segmental defect model implanted with composite scaffold in rabbits, with radiography and in vivo micro-CT for studying new bone regeneration and micro-CT-based angiography and histology for neovascularization, dynamic MRI for local blood perfusion function, as well as host tissue and scaffold material interactions. / Results: The in vitro characterization and osteogenic potential of the fabricated scaffolds were performed and confirmed, respectively. Micro-CT quantitation showed that the scaffolds had interconnected macropores with an average porosity of 75±3.27 % and pore size or diameter of 458±25.6 μm. Compared to P/T, P/T+icaritin and P/T/BMP-2 scaffolds, P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin (n=6, p<0.01) presented higher cell seeding efficiency, ALP activity and calcium nodules and up-regulated mRNA expressions of Collagen type I and Osteopontin of co-cultured BMSCs. P/T+BMP-2 showed the best osteogenic effects among all groups (n=6, p<0.01). In vivo measurement of x-ray and micro-CT in rabbit ulna bone defect model at week 2, 4 and 8 post-surgery showed more newly formed bone in the defects treated with P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin scaffold (n=6, p<0.01) compared with that of P/T, P/T+icaritin and P/T/BMP-2 groups. P/T+BMP-2 also showed the best bone formation among all groups (n=6, p<0.01). Histological results also demonstrated that there were more osteoid tissues and typical lamellar bone in surface and internal of the implants, as well as along the adjacent host bone in P/T/icaritin groups (n=5, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01). P/T+BMP-2 group showed the most newly formed bone (n=6, p<0.01). In addition, newly formed vessels in the defects were identified with micro-CT-based angiography and functionally supported by dynamic MRI for reflecting blood perfusion. The results showed more ingrowing new vessels in P/T/icaritin groups (n=6, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01), compared to P/T and P/T/BMP-2 groups. For comparing dose effects among three scaffolds incorporating different concentration of icaritin, we found that middle dose PLGA/TCP/icaritin composite scaffold showed the best osteogenic potential. / Conclusion: PLGA/TCP scaffolds incorporating exogenous phytomolecule icaritin demonstrated the desired osteogenic differentiation potential and bone regeneration capability as investigated in vitro and in vivo, where the middle dose of icaritin incorporating PLGA/TCP composite scaffold showed the best effects. These findings may form a good foundation for potential clinical validation of this innovative bioactive composite scaffold with either conventional endogenous BMP-2 for in vitro loading before application or more attractively and user-friendly incorporated with exogenous phytomolecule icaritin as a ready product for enhancing bone defect repair. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Shihui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 173-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.viii / Abstract --- p.x / 中文摘要 --- p.xiii / List of Abbreviations --- p.xvi / List of Tables --- p.xix / List of Figures --- p.xx / Journal Publications --- p.xxv / Journal Supplements --- p.xxv / Conference Abstracts --- p.xxvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Bone Defect in Orthopaedics --- p.2 / Chapter 1.2 --- Human Skeletons --- p.2 / Chapter 1.2.1 --- Bone Types and Function --- p.2 / Chapter 1.2.2 --- Bone Development --- p.4 / Chapter 1.2.3 --- Bone Physiology and Structure --- p.6 / Chapter 1.2.4 --- Bone Specific Markers --- p.7 / Chapter 1.2.5 --- Bone Cells --- p.9 / Chapter 1.2.6 --- Bone Marrow Stromal Cells --- p.12 / Chapter 1.3 --- Bone Regeneration and Remodeling --- p.13 / Chapter 1.3.1 --- Bone Defect Healing --- p.13 / Chapter 1.3.2 --- Non-union and Segmental Defect --- p.15 / Chapter 1.3.3 --- Bone Defect Treatment --- p.16 / Chapter 1.4 --- Angiogenesis in Bone Healing --- p.19 / Chapter 1.4.1 --- Blood Vessels Formation Process --- p.20 / Chapter 1.4.2 --- Growth Factor in Angiogenesis --- p.21 / Chapter 1.5 --- Biomaterials in Bone Tissue Engineering --- p.22 / Chapter 1.6 --- Scaffold-Based Therapy --- p.23 / Chapter 1.6.1 --- Bone Grafts --- p.23 / Chapter 1.6.1.1 --- Autografts --- p.23 / Chapter 1.6.1.2 --- Allografts --- p.25 / Chapter 1.6.2 --- Bone Graft Substitutes --- p.25 / Chapter 1.6.2.1 --- Bone Formation in Porous Scaffolds --- p.25 / Chapter 1.6.2.2 --- Degradable Polymers --- p.27 / Chapter 1.6.2.3 --- Non-Degradable Polymers --- p.29 / Chapter 1.6.2.4 --- Ceramics --- p.29 / Chapter 1.6.2.5 --- Bioactive Composite Materials --- p.30 / Chapter 1.7 --- Growth Factor-Based Therapy --- p.31 / Chapter 1.7.1 --- Endogenous Growth Factor--Bone Morphogenetic Proteins --- p.31 / Chapter 1.7.2 --- Exogenous phytomoleculeIcaritin--Icaritin --- p.31 / Chapter 1.7.3 --- Delivery of Growth Factor in Tissue Engineering --- p.34 / Chapter 1.8 --- Fabrication of Porous Composite Scaffolds --- p.37 / Chapter 1.8.1 --- Architectural Parameters of Bone Scaffolds --- p.37 / Chapter 1.8.2 --- Three-Dimensional Scaffold Fabrication --- p.37 / Chapter 1.9 --- Animal Models for Testing Bone Defects Healing --- p.39 / Chapter Chapter 2 --- Research Rationale and Study Objectives / Chapter 2.1 --- Research Rationale --- p.42 / Chapter 2.2 --- Study Objectives --- p.46 / Chapter Chapter 3 --- Bioactive Composite Scaffolds: Preparation, Morphology and Release Assay / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.50 / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Fabrication of PLGA/TCP Incorporating BMP-2 or Icaritin --- p.51 / Chapter 3.2.3 --- Morphological Analysis of Composite Scaffolds --- p.53 / Chapter 3.2.3.1 --- Analysis of Porosity and Macropores Diameter Using High-resolution Micro-CT --- p.53 / Chapter 3.2.3.2 --- Analysis of Surface Morphology and Elements Composition Using Scanning Electron Microscopy --- p.54 / Chapter 3.2.4 --- Icaritin Content Assay in PLGA/TCP Scaffolds Incorporating Icaritin --- p.54 / Chapter 3.2.5 --- Preparation of PLGA/TCP Scaffold Coating BMP-2 or Icaritin --- p.55 / Chapter 3.2.6 --- In vitro Release Assay --- p.55 / Chapter 3.2.6.1 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.55 / Chapter 3.2.6.2 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.56 / Chapter 3.2.7 --- Mechanical Properties of Composite Scaffolds --- p.56 / Chapter 3.2.8 --- Statistical Analysis --- p.57 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- Morphological Analysis of Composite Scaffolds --- p.57 / Chapter 3.3.1.1 --- Porosity and Macroscopic Diameter --- p.57 / Chapter 3.3.1.2 --- Surface Morphology and Elements Composition --- p.58 / Chapter 3.3.2 --- Icaritin Content in Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60 / Chapter 3.3.3 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60 / Chapter 3.3.4 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.61 / Chapter 3.3.5 --- Mechanical Properties of Composite Scaffolds --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.5 --- Summary --- p.71 / Chapter Chapter 4 --- Bioactive Composite Scaffolds: In vitro Degradation and Characterization Studies / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and Methods --- p.74 / Chapter 4.2.1 --- Preparation of Composite Scaffolds for in vitro Degradation Assay --- p.74 / Chapter 4.2.2 --- Characterizations --- p.75 / Chapter 4.2.2.1 --- Scaffold Volume Changes --- p.75 / Chapter 4.2.2.2 --- Scaffold Weight Changes --- p.75 / Chapter 4.2.2.3 --- pH Value Changes --- p.75 / Chapter 4.2.2.4 --- Calcium Ion Release from Scaffolds --- p.76 / Chapter 4.2.3 --- Mechanical Properties Changes --- p.76 / Chapter 4.2.4 --- Statistical Analysis --- p.77 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- Volume Decrease --- p.78 / Chapter 4.3.2 --- Weight Loss --- p.78 / Chapter 4.3.3 --- pH Value Reduction --- p.79 / Chapter 4.3.4 --- Calcium Ion Release --- p.79 / Chapter 4.3.5 --- Mechanical Properties --- p.80 / Chapter 4.4 --- Discussion --- p.81 / Chapter 4.5 --- Summary --- p.84 / Chapter Chapter 5 --- In vitro Evaluation of Bone Marrow Stem Cells (BMSCs) Growing on Bioactive Composite Scaffolds / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.90 / Chapter 5.2.1 --- Preparation of Composite Scaffolds for in vitro Evaluation --- p.90 / Chapter 5.2.2 --- BMSCs Seeding Rate and Proliferation on Composite Scaffolds --- p.90 / Chapter 5.2.3 --- Alkaline Phosphate (ALP) Activity Assay --- p.92 / Chapter 5.2.4 --- Osteogenic Gene Expression Assay Using Quantitative Real-time PCR --- p.92 / Chapter 5.2.5 --- Calcium Deposition Assay Using Alizarin Red Staining --- p.93 / Chapter 5.2.6 --- Statistical Analysis --- p.94 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Cells Seeding Efficiency and Proliferation --- p.94 / Chapter 5.3.2 --- ALP Activity --- p.97 / Chapter 5.3.3 --- Osteogenic Gene mRNA Expression --- p.97 / Chapter 5.3.4 --- Calcium Deposition --- p.98 / Chapter 5.4 --- Discussion --- p.99 / Chapter 5.5 --- Summary --- p.102 / Chapter Chapter 6 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds / Chapter 6.1 --- Introduction --- p.105 / Chapter 6.2 --- Materials and Methods --- p.106 / Chapter 6.2.1 --- Preparation of Composite Scaffolds for Implantation --- p.106 / Chapter 6.2.2 --- Establishment of Ulna Bone Segmental Defect in Rabbits --- p.107 / Chapter 6.2.3 --- Radiographic Evaluation of New Bone Area Fraction --- p.109 / Chapter 6.2.4 --- XtremeCT Evaluation of New Bone Formation and Bone Mineral Density (BMD) --- p.110 / Chapter 6.2.5 --- Histological Evaluation of New Bone Formation --- p.111 / Chapter 6.2.6 --- Evaluation of Rate of New Bone Formation and Mineral Apposition Rate (MAR) --- p.114 / Chapter 6.2.7 --- Evaluation of Neovascularization Using Micro-CT-based Microangiography --- p.116 / Chapter 6.2.8 --- Blood Perfusion Function Using Dynamic Magnetic Resonance Imaging (MRI) --- p.119 / Chapter 6.2.9 --- Statistical Analysis --- p.120 / Chapter 6.3 --- Results --- p.121 / Chapter 6.3.1 --- Radiographic Area Fraction of New Bone Formation --- p.123 / Chapter 6.3.2 --- XtremeCT New Bone Volume Fraction and BMD --- p.128 / Chapter 6.3.3 --- Histological New Bone Fraction --- p.133 / Chapter 6.3.4 --- Rate of New Bone Formation and MAR --- p.136 / Chapter 6.3.5 --- New Vessels Volume Evaluated Using Micro-CT-Based Microangiography --- p.140 / Chapter 6.3.6 --- Dynamic Blood Perfusion Function --- p.144 / Chapter 6.4 --- Discussion --- p.146 / Chapter 6.5 --- Summary --- p.151 / Chapter Chapter 7 --- Summaries, Conclusions, Limitations and Future Studies / Chapter 7.1 --- Introduction --- p.153 / Chapter 7.2 --- Bioactive Composite Scaffolds: Preparation, Morphology and in vitro Release Evaluation --- p.155 / Chapter 7.3 --- Bioactive Composite Scaffolds: in vitro Degradation and Characterization Studies --- p.159 / Chapter 7.4 --- In vitro Evaluation of the Response of Bone Marrow Stem Cells Growing on Bioactive Composite Scaffolds --- p.160 / Chapter 7.5 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds --- p.162 / Chapter 7.6 --- Evaluation of Dose-dependent Effects of Icaritin Mechanical Property, Degradation, and Osteogenic Potentials --- p.164 / Chapter 7.7 --- Conclusions --- p.170 / Chapter 7.8 --- Limitations and Future Studies --- p.171 / Chapter 7.9 --- References --- p.173 / Chapter 7.10 --- Appendix --- p.199 / Chapter 7.10.1 --- Animal Licence and Ethics --- p.199 / Chapter 7.10.2 --- Safety Approval --- p.201 / Chapter 7.10.3 --- Journal Supplements --- p.202 / Chapter 7.10.4 --- Conference Abstracts--Posters --- p.205 / Chapter 7.10.5 --- Conformation of Paper Submission --- p.208 / Chapter 7.10.6 --- Published Paper --- p.209

Athymic nude mice were employed as the in vivo model to study the detailed mechanism of how the three sclerotial polysaccharides act to inhibit the growth of human xenografted tumors in vivo. Using immunohistochemical staining, it was found that the presence of F4/80 + macrophages was related to the reduction of tumor size of the HL-60 xenograft. mRNA extracted from the spleens were reverse-transcribed to cDNA and detected by real-time PCR so that a variety of genes related to the toll-like receptors being up-regulated or down-regulated due to the injection of mushroom sclerotial polysaccharides were determined. Combining the results from dectin-1 regulation, it was concluded that both hot water-soluble sclerotial polysaccharides, PTRW and PRW, having a structure of polysaccharide-protein complexes were responsible for activating and thus binding to CR3 or toll-like receptors while PRSon with structure of pure beta-glucan was responsible for activating the expression of dectin-1 receptor, which led to the subsequent activation of host immune system in immunopotentiation and antitumor activities. / In the future, further investigation of the detailed structure of mushroom sclerotial polysaccharides is required to explain the immunomodulatory mechanism so that the effective dosage for immunomodulation as well as antitumor effects can be determined. Furthermore, phage display can be applied to find out any novel glucan receptors specific to the mushroom sclerotial polysaccharides. / In vitro antitumor study indicated that PTRW had a significant (p&lt;0.05) inhibitory effect (>40%) on the human monocytic leukemic cells (THP-1) in addition to HL-60 and K562 cells. In vitro immunomodulatory study showed that both PRW and PRSon had significant proliferative effects (p&lt;0.05) on human normal spleen monocyte/macrophage cell, MD. Moreover, PRSon was shown to have a significant increase (p&lt;0.05) in the growth of human natural killer cells, NK-92M1; however, PTRW showed a significant inhibition (p&lt;0.05) on this cell line. / Mushroom sclerotia have a rich source of polysaccharides when compared with fruit bodies. It was previously reported that the polysaccharides from novel mushroom sclerotia, namely, Pleurotus tuber-regium and Polyporus rhinocerus, had potent in vitro and in vivo antitumor effects. In this project, hot water-soluble sclerotial polysaccharides of Pleurotus tuber-regium (PTRW), hot water-soluble and sonication-assisted cold alkali-soluble sclerotial polysaccharides of Polyporus rhinocerus (PRW and PRSon, respectively) were chosen for investigation of their in vivo and in vitro immunomodulatory effects. / Polysaccharides have long been proposed to exert their antitumor and thus immunomodulating functions through glucan receptors and among the four being discovered, Dectin-1 has drawn most attention recently. In the in vivo study, PRSon showed an increase in the expression of Dectin-1 on mice spleen MNCs while PTRW showed an increase in the expression of the previously widely-reported complement receptor (CR3). There was also an increase of Dectin-1 expression on PEC in the mice injected with PRSon. In the in vitro study, the three mushroom sclerotial polysaccharides were incubated with NK-92M1, MD and THP-1 cells. There was a significant increase (p&lt;0.05) of Dectin-1 expression on NK-92MI cells incubated with PTRW. On the other hand, PTRW caused a significant decrease ( p&lt;0.05) of Dectin-1 expression while PRSon showed a significant increase (p&lt;0.05) on THP-1 cells. The cytokine profile of extra-cellular media indicated that the inhibition of THP-1 cells by PTRW should be related to the innate immunity. In the in vitro study, human primary immune cells, CD56+ NK cells were used to incubate with sclerotial polysaccharides and there was a significant stimulation (p&lt;0.05) of their growth when compared with the control. / The in vivo immunomodulatory study was carried out by injecting the abovementioned sclerotial polysaccharides intraperitoneal to 7-8 weeks old healthy male BALB/c mice. The spleens excised from groups injected with PTRW and PRW were found to have significant increase of weight ( p&lt;0.001). Flow cytometric analysis revealed that the NK cell population in spleen mononuclear cells (MNCs) of mice injected with PRW and PRSon was increased when compared with the control. In addition, ail three sclerotial polysaccharides showed a large increase of T helper cell population as well as CD4+/CD8+ ratio in spleen MNCs. On the other hand, the macrophage population in peritoneal exudates cells (PEC) was found to be increased in the groups of mice injected with PTRW and PRW. / Lai, Kin Ming Connie. / Adviser: Cheung Chi Keung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3412. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis. / Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA. / Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins. / by Guo, Yuming. / Adviser: Chung Hale Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 188-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Aneurysmal subarachnoid haemorrhage (SAH) accounts for only 3-5% of all strokes but has the highest morbidity and mortality rates among all types of stroke. Experimental studies have confirmed that magnesium inhibits excitatory amino acid (EAA) release, blocks N-methyl-D-aspartate (NMDA) receptors and prevents calcium from entering cells. These changes may minimise neuronal injury during episodes of cerebral vasospasm. / Based on the aforementioned biological evidence, a pilot study of magnesium sulphate (MgSO4) infusion for aneurysmal SAH was conducted. This pilot study indicated that MgSO4 infusion for aneurysmal SAH is safe and has the potential to improve clinical outcomes. The pilot study results are supported by the findings of other research groups. / In summary, our results do not support a clinical benefit of intravenous magnesium sulphate infusion in patients with acute aneurysmal SAN. / Studies were carried out to investigate the distribution of magnesium in the cerebrospinal fluid (CSF) and the brain after intravenous magnesium sulphate infusion. We found that magnesium sulphate infusion resulted in an 11% to 21% increase in CSF magnesium, which was sustained for at least nine days. We further investigated intracellular free magnesium using 31 P-MRS, in aneurysmal SAH patients receiving and not receiving intravenous magnesium sulphate infusion. Intravenous magnesium sulphate infusion was found to produce a significant increase (15.6%) in the level of intracellular free magnesium during the first week, which covered the time frame required for neuroprotection, to improve outcomes in patients with aneurysmal SAH. / Three hundred and twenty-seven patients with aneurysmal SAH were recruited and randomly assigned to receive magnesium sulphate infusion or a saline placebo for 10 to 14 days. In the primary outcome analysis, the proportions of patients with a favourable outcome at six months (Extended Glasgow Outcome Scale [GOSE] score of 5-8) were similar, 64% in the MgSO4 group and 63% in the saline group (OR 1.0, 0.7-1.6). In the secondary outcome analyses, there were also no significant differences between the two groups. Analysis of the plasma magnesium concentration levels did not suggest that higher levels of plasma magnesium concentration result in better clinical outcomes. / Wong Kwok Chu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 132-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.