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

Steroid-associated osteonecrosis (SAON) accounts for a major fraction in non-traumatic osteonecosis (ON) and generally has poor prognosis even after surgical interventions. This suggests that prevention is one of the important intervention strategies for SAON. So far, there is lacking of proven prevention modalities for SAON. / Study I was to establish an alternative SAON model. Based on the proposed pathogenesis of SAON that the intravascular thrombosis and extravascular marrow fat deposition are the two important contributors to the development of ON lesion, lipopolysaccharides (LPS) could induce vascular dysfunction and even thrombosis, and methylprednisolone (MPS) could induce the adipogenesis of marrow mesenchymal stem cells (MSCs). They were accordingly used in a combination for ON induction in animals. / Study II was to investigate the effect of Herbal Epimedium-derived formula for prevention of ON using the validated SAON animal model. Efficacy of the herbal Epimedium-derived formula was assessed for prevention of SAON using the animal model. Thirty adult male rabbits were used in this study. The ON incidence was set as the end-point for evaluation of the prevention efficay. For the potential intervention targets, the intravascular thrombosis and extravascular marrow fat formation were evaluated hematologically and histopathologically. The vascular structure and function were evaluated by advanced bioimaging modalities of micro-CT and MRI. / Study III was to investigate the bioactive compound(s) from the herbal Epimedium-derived herbal formula for prevention of SAON. Phytochemical analysis identified seven compounds in this efficacy-proven formula, with icariin as the major compound accounting for more than 80% in weight. Icariin was therefore tested for its prevention efficacy using the SAON animal model. / Study IV was to investigate the underlying mechanism(s) of bioactive compound Icariin in effective prevention of SAON using in vitro cell models. As activation of endothelial cells and adipogenesis of MSCs are suggested to be the two major events involving in vascular dysfunction and marrow fat formation in SAON animal model, Icariin were accordingly hypothesized to be able to prevent activation of endothelial cells and inhibit adipogenesis of MSCs. / Summary. After summarizing the major findings of these four logically interrelated studies, it was able to conclude that Icariin was the identified bioactive compound from the herbal Epimedium-derived formula, which was able to reduce the SAON incidence with inhibition of intravascular thrombosis and extravascular marrow fat formation in an established rabbit model. The underlying mechanisms might be related to its effects on protection of endothelial cells activation and inhibition of MSCs adipogenesis (This can be summarized in the following picture). This study provides a new bioactive agent Icariin for SAON prevention and potential future clinical application. (Abstract shortened by UMI.) / The following research questions were addressed in the present study: (1) Is there an alternative SAON animal model? (Study I); (2) Whether the herbal Epimedium-derived formula is able to prevent SAON in this animal model? (Study II); (3) What is the bioactive compound(s) in this herbal Epimedium-derived formula? (Study III); (4) How does this bioactive compound prevent SAON? (Study IV) / Sheng, Hui. / Adviser: Ling Qin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3421. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / 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.

激素性骨壞死是由於經常使用脈衝性激素處理非骨科性問題引起的一種常見的骨科疾病。在組織病理學上，激素性骨壞死指骨死亡，血管內血栓閉塞和血管外骨髓脂肪沉積會引起缺血導致骨修復不足。上游的分子細胞病理學機制研究表明間充質幹細胞細胞池活性下降，成骨細胞凋亡和骨小梁基質退變導致的不充分修復是激素性骨壞死發生的重要因素。 / 間充質幹細胞是骨髓的基質組成部分，具有分化成多種細胞的潛能。最近的研究表明，激素性骨壞死可能是骨細胞和/或間質幹細胞病變引起的一種疾病。研究發現，在接受類固醇治療而發生骨壞死的病人中，骨髓間充質幹細胞活性下降和分化潛能發生改變。在骨髓細胞中，激素能夠誘導脂肪發生。盛輝等發現來源於激素性骨壞死兔子中的間充質幹細胞成脂分化增強，謝新薈等進一步發現發生激素性骨壞死的兔子，骨缺損修復延遲，這可能是由激素導致的間充質幹細胞潛能發生改變引起的。綜合以上研究表明，間充質幹細胞在骨壞死發生和修復過程中起著重要作用。我們之前報導過淫羊藿黃酮（EFs）的腸代謝產物淫羊藿素Icaritin通過抑制血栓的形成和脂肪沉澱預防激素性骨壞死。最近，我們把Icaritin整合到聚乳酸聚乙醇酸共聚物/磷酸三鈣（PLGA/TCP）支架材料中，形成PLGA/TCP/Icaritin複合支架材料。我們發現PLGA/TCP/Icaritin複合材料可以促進激素性骨壞死骨缺損的修復，肌肉移植發現PLGA/TCP/Icaritin也能促進新生血管的發生。我們也發現單純PLGA/TCP複合材料也能夠促進激素性骨壞死骨缺損的修復，但是潛在的機制尚不清楚。 / 骨是一個高度血管化的組織，依賴於血管和骨細胞密切的時空連結維持骨骼的完整性。因此，血管生成在骨骼發育和骨折修復過程中發揮著舉足輕重的作用。血管為骨的發育和再生提供氧氣，為基質輸送刺激間充質細胞特異性成骨的重要信號，另一方面，骨為血管生成輸送生長因數和細胞。 / 本論文分為以下四個主要部分： / 第一部分: 研究Icaritin對人源間充質幹細胞分化的作用及其機制。流式細胞分選鑒定結果表明我們使用的人源間充質幹細胞能夠特異表達間充質幹細胞表面標誌物。MTT實驗結果顯示Icaritin不影響間充質幹細胞的增殖；分化實驗表明Icaritin在沒有成骨誘導試劑存在的情況下無法影響間充質幹細胞的分化。在成骨誘導試劑存在的情況下，Icaritin促進間充質幹細胞成骨分化，抑制其成脂分化；即時螢光實時定量聚合酶鏈式擴增（RT-PCR）結果顯示Icaritin在間充質幹細胞分化過程中上調成骨基因的表達，下調成脂基因表達。進一步研發發現在成骨分化過程中，Icaritin能夠促進BMP2和beta-catenin 蛋白的表達，而BMP2抑制劑Noggin能夠能夠逆轉Icaritin促進的成骨發生。這些發現表明Icaritin能夠促進而非誘導間充質幹細胞的成骨分化，Icaritin調解間充質幹細胞成骨分化具有BMP2信號通路依賴性。 / 第二部分: 評估激素性骨壞死兔源間充質幹細胞的分化潛能及Icaritin 對異常分化的間充質幹細胞分化潛能的影響。結果表明Icaritin促進正常兔源間充質幹細胞的成骨分化，抑制其成脂分化。激素性骨壞死兔源間充質幹細胞的成骨分化潛能降低，成脂分化升高；而Icaritin能夠劑量依賴性地部分恢復降低的成骨分化潛能，抑制升高的成脂分化活性。激素性骨壞死兔源間充質幹細胞的增殖活性也下降但是不能被Icaritin恢復。Icaritin對激素性骨壞死兔源間充質幹細胞中下降的VEGF的表達無影響。這些發現顯示間充質幹細胞的分化潛能在激素性骨壞死發生過程中遭到破壞，但是能夠被Icaritin部分恢復。 / 第三部分: 評估Icaritin對體外成血管的影響。我們對Icaritin對人臍帶靜脈內皮細胞（HUVECs）的增殖、遷移、管狀結構形成及成血管相關基因的表達的影響進行了檢測。結果表明Icaritin不影響HUVECs的增殖、遷移和管狀結構的形成；RT-PCR結果顯示Icaritin對HUVECs中的VEGF, HIF1a, FGF2 and TGF-beta表達也沒有影響。這些發現表明Icaritin在體外並不能直接作用于血管生成。結果謝新薈和陳詩慧等人的體內研究結果可以推測在骨缺損修復過程中，Icaritin通過促進成骨間接促進血管生成。 / 第四部分: 主要研究Icaritin及複合生物材料在體外體內對間充質幹細胞歸巢的影響。結果表明Iaritin能夠促進間充質幹細胞的遷移並上調血管細胞黏附分子1（VCAM1）的表達。複合材料PLGA/TCP和PLGA/TCP/Icaritin在體外培養的條件下能夠募集間充質幹細胞到材料周圍及進入材料。間充質幹細胞體外用修飾性超順磁性氧化鐵（SPIO@SiO₂-NH₂）納米顆粒標記後，其分化潛能依然保留，增殖和潛能能力稍微下降。兔激素性骨壞死造模完成後，股骨遠端髓芯減壓壞死骨缺損手術，PLGA/TCP和PLGA/TCP/Icaritin複合材料植入缺損孔道，同時把SPIO@SiO₂-NH₂標記的間充質幹細胞注射到距離缺損區20毫米的骨髓腔內。結果顯示只有標記的間充質幹細胞植入而沒有材料植入時，缺損區被脂肪細胞充滿，並沒有標記的間充質幹細胞出現，而在缺損區附近和遠離缺損區的部位有標記的間充質幹細胞出現。同時植入PLGA/TCP複合材料和標記的間充質幹細胞時，標記的間充質幹細胞出現在缺損區的材料中，在缺損區附近沒有標記的間充質幹細胞出現，而在遠離缺損區的部位，有標記的間充質幹細胞出現。同時植入PLGA/TCP/Icaritin和標記的間充質幹細胞時，得到跟植入PLGA/TCP複合材料和標記的間充質幹細胞相似的結果，但是在缺損區域，SPIO陽性的間充質幹細胞數目在PLGA/TCP和PLGA/TCP/Icaritin組別中並未發現有顯著性差異。以上發現表明Icaritin和PLGA/TCP複合材料能夠在體外和體內促進間充質幹細胞的歸巢。 / 綜上所述，複合支架材料PLGA/TCP/Icaritin通過調節間充質幹細胞的歸巢和分化促進激素性骨壞死骨缺損的修復。Icaritin通過BMP2和Wnt/beta-catenin通路調解間充質幹細胞的成骨分化。這是首次研究發現Icaritin及PLGA/TCP支架材料影響骨缺損修復過程中幹細胞歸巢，但是分子細胞生物學機制還需要進一步的研究。 / Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem as the pulsed steroids are frequently prescribed for the treatment of non-orthopaedic medical conditions. Histopathologically, SAON refers to death of bone. Intravascular thrombus occlusion and extravascular marrow lipid deposition cause ischemia, which leads to an inadequate repair of the bone. Recent study revealed upstream pathological mechanism at cellular and molecular level. The decrease in activity of mesenchymal stem cell (MSC) pool, apoptosis of osteocytes, and trabecular bone matrix degeneration may cause bone inadequate repair, a key pathological feature found in SAON. / MSCs are the stromal component of bone marrow (BM) and have the potential to differentiate into several cell types. Recent studies have suggested that SAON may be a disease of bone cells and/or MSCs. With corticosteroid therapy in patients, the MSCs activity decreased and differentiation potential changed. Steroids have been also shown to produce adipogenesis in bone-marrow cells. It has been found adipogenesis of MSCs from SAON rabbits elevated (Sheng et al., 2007a) and bone defect repair was delayed in rabbits with SAON (Xie et al., 2011), this may be caused by altered MSCs potentials. All these findings imply MSCs play a vital role in SAON development and bone defect repair. It had been reported that Icaritin, an intestinal metabolite of Epimedium-derived avonoids (EF) reduced SAON incidence with inhibition of both thrombosis and lipid deposition (Zhang et al., 2009a). More recently, we found integrating Icaritin into PLGA/TCP to form PLGA/TCP/Icaritin composite scaffold could promote SAON bone defect repair and more neovascularization formed in an intramuscular implantation model, and further found PLGA/TCP scaffold only also could promote SAON bone defect repair in rabbits (Wang et al., 2012a). But the underlying mechanism remains unclear. / Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate MSC specification to promote bone formation and repair. On the other hand, bone also supplies growth factors and cells for angiogenesis. The content of this thesis is divided into the following four major parts: / Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real- time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner. / Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin. / Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair. / Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO@SiO₂-NH₂, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO@SiO₂-NH₂ labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects. / In summary, the composite scaffold PLGA/TCP/Icaritin enhanced bone defect repair in rabbit with SAON by promoting homing and osteogenesis of MSCs. Icaritin promoted osteogenic differentiation of MSCs through BMP2 mediated signal pathway, such as Wnt/beta-catenin signal pathway. It is first time to report that PLGA/TCP scaffold promoted MSCs homing during bone defect repair, but underlying molecular and cellular mechanism need to be further studied. / 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. / Detailed summary in vernacular field only. / Yao, Dong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 137-158). / Abstract also in Chinese; some appendixes also in Chinese. / ACKNOWLEDGEMENTS --- p.i / TABLE OF CONTENTS --- p.iii / ABSTRACT (IN ENGLISH) --- p.x / ABSTRACT (IN CHINESE) --- p.xiv / FLOWCHART --- p.xviii / LIST OF PUBLICATIONS --- p.xix / LIST OF ABBREVIATIONS --- p.xxi / LIST OF FIGURES --- p.xxiv / Chapter CHAPTER 1: --- Introduction --- p.1 / Chapter 1 --- Osteonecrosis --- p.2 / Chapter 1.1. --- Etiology --- p.2 / Chapter 1.2. --- Anatomy of femoral head --- p.3 / Chapter 1.3. --- Pathogenesis --- p.4 / Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4 / Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4 / Chapter 1.3.3. --- Coagulation --- p.5 / Chapter 1.4. --- Development stages of osteonecrosis --- p.5 / Chapter 2. --- Steroids-associated osteonecrosis --- p.11 / Chapter 2.1. --- Epidemiology --- p.12 / Chapter 2.2. --- Histopathology --- p.12 / Chapter 2.3. --- Etiopathogenesis --- p.13 / Chapter 2.3.1. --- Steroid and fat metabolism --- p.14 / Chapter 2.3.2. --- Steroid and endothelial cells --- p.15 / Chapter 2.3.3. --- Steroid and coagulation --- p.16 / Chapter 2.3.4. --- Steroid and angiogenesis --- p.17 / Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18 / Chapter 2.5. --- Treatment strategies for SAON --- p.18 / Chapter 2.5.1. --- Prevention --- p.19 / Chapter 2.5.2. --- Nonoperative treatment --- p.19 / Chapter 2.5.3. --- Operative treatment --- p.19 / Chapter 2.5.3.1. --- Core decompression strategy --- p.20 / Chapter 2.5.3.2. --- Tissue engineering approach --- p.22 / Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22 / Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24 / Chapter 3.1.1. --- Anti-tumor activity --- p..25 / Chapter 3.1.2. --- Neuroprotective effects --- p.25 / Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25 / Chapter 3.1.4. --- Osteogenic differentiation --- p.26 / Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26 / Chapter 5. --- PLGA/TCP/Icaritin --- p.28 / Chapter 6. --- Hypothesis of this study --- p.28 / Chapter 7. --- Objective --- p.29 / Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30 / Chapter 1. --- Introduction --- p.31 / Chapter 2. --- Material and Methods --- p.33 / Chapter 2.1. --- Ethics --- p.33 / Chapter 2.2. --- Reagents and cell culture --- p.33 / Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33 / Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34 / Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34 / Chapter 2.6. --- ALP staining --- p.35 / Chapter 2.7. --- ALP activity assay --- p.35 / Chapter 2.8. --- Alizarin Red S staining --- p.35 / Chapter 2.9. --- Oil Red O staining --- p.35 / Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36 / Chapter 2.11. --- Reverse transcription --- p.36 / Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.13. --- Western blotting --- p.37 / Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39 / Chapter 2.15. --- Statistical analysis --- p.39 / Chapter 3. --- Results --- p.40 / Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40 / Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41 / Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42 / Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44 / Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45 / Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48 / Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50 / Chapter 4. --- Discussion --- p.52 / Chapter 5. --- Conclusion --- p.56 / Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57 / Chapter 1. --- Introduction --- p.58 / Chapter 2. --- Methods and materials --- p.59 / Chapter 2.1. --- SAON model establishment --- p.59 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60 / Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61 / Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62 / Chapter 2.5. --- ALP Staining --- p.62 / Chapter 2.6. --- ALP Activity Assay --- p.62 / Chapter 2.7. --- Alizarin Red S Staining --- p.62 / Chapter 2.8. --- Oil Red O Staining --- p.63 / Chapter 2.9. --- RNA Isolation --- p.63 / Chapter 2.10. --- Reverse transcription --- p.64 / Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64 / Chapter 2.12. --- Western blotting performance --- p.65 / Chapter 2.13. --- Statistical analysis --- p.65 / Chapter 3. --- Results --- p.66 / Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66 / Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68 / Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71 / Chapter 3.4. --- PPAR-γ and aP2 proteins expression increased in SAON rabbit while inhibited by Icaritin both in normal and SAON rabbit --- p.74 / Chapter 3.5. --- Proliferation ability of MSCs derived from SAON rabbit declined and Icaritin had no effect on proliferation both derived from normal and SAON rabbit --- p.75 / Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76 / Chapter 4. --- Discussion --- p.76 / Chapter 5. --- Conclusion --- p.81 / Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82 / Chapter 1. --- Introduction --- p.83 / Chapter 2. --- Material and Methods --- p.85 / Chapter 2.1. --- Cell culture --- p.85 / Chapter 2.2. --- Proliferation assay --- p.85 / Chapter 2.3. --- Scratch-wound healing assay --- p..86 / Chapter 2.4. --- Migration Assay --- p.86 / Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87 / Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87 / Chapter 2.7. --- Statistical Analysis --- p.88 / Chapter 3. --- Results --- p.88 / Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88 / Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92 / Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94 / Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95 / Chapter 4. --- Discussion --- p.96 / Chapter 5. --- Conclusion --- p.100 / Chapter CHAPTER 5: --- Effect of PLGA/TCP and PLGA/TCP/Icaritin composite scaffolds on stem cell homing during bone defect repair with SAON --- p.101 / Chapter 1. --- Introduction --- p.102 / Chapter 2. --- Material and Methods --- p.106 / Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106 / Chapter 2.3. --- Wound healing assay --- p.107 / Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107 / Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108 / Chapter 2.6. --- Prussian blue staining --- p.108 / Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108 / Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109 / Chapter 2.9. --- Real time PCR --- p.109 / Chapter 2.10. --- Animal model establishment --- p.109 / Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110 / Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112 / Chapter 2.13. --- Statistical analysis --- p.112 / Chapter 3. --- Results --- p.112 / Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112 / Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114 / Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118 / Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- Conclusion --- p.136 / Chapter CHAPTER 6: --- Summary of the study and future research --- p.137 / Chapter 1. --- Summary of the study --- p.138 / Chapter 2. --- Limitations and further studies --- p.139 / APPENDIXES --- p.142 / REFERENCES --- p.147

Liu, Xiaozhuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-169). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgments --- p.vi / List of Abbreviations --- p.vii / Table of Contents --- p.xiv / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia --- p.6 / Chapter 1.1.2.1 --- An Overview of Leukemia --- p.6 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.8 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.12 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.15 / Chapter 1.2 --- Sophoraflavanone G: A Bioactive Compound Isolated from Kushen --- p.18 / Chapter 1.2.1 --- An Overview of Kushen: A Traditional Chinese Medicine --- p.19 / Chapter 1.2.2 --- An Overview of Lavandulyl Flavanones --- p.22 / Chapter 1.2.3 --- Historical Development and Occurrence of Sophoraflavanone G --- p.24 / Chapter 1.2.4 --- Biological Activities of Sophoraflavanone G --- p.25 / Chapter 1.2.4.1 --- Anti-microbial and Insecticidal Activities --- p.25 / Chapter 1.2.4.2 --- Anti-tumor Activities --- p.26 / Chapter 1.2.4.3 --- Pharmacodynamics of Sophoraflavanone G --- p.27 / Chapter 1.3 --- Objectives and Scopes of the Present Study --- p.30 / Chapter Chapter Two: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Animals --- p.32 / Chapter 2.1.2 --- Cell lines --- p.32 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.34 / Chapter 2.1.4 --- Reagents and Buffers for Flow Cytometry --- p.37 / Chapter 2.1.5 --- Reagents for DNA Extraction --- p.39 / Chapter 2.1.6 --- Reagents for Measuring Caspase Activity --- p.40 / Chapter 2.1.7 --- "Reagents, Buffers and Materials for Western Blotting" --- p.43 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Extraction and Isolation of Sophoraflavanone G from Kushen --- p.48 / Chapter 2.2.2 --- Culture of Tumor Cell Lines --- p.49 / Chapter 2.2.3 --- "Isolation, Preparation and Culturing of Human Peripheral Blood Leukocytes and Murine Bone Marrow Cells" --- p.50 / Chapter 2.2.4 --- Assays for Anti-proliferation and Cytotoxicity --- p.51 / Chapter 2.2.5 --- Determination of Anti-leukemic Activity In Vivo (In Vivo Tumorigenicity Assay) --- p.52 / Chapter 2.2.6 --- Cell Cycle Analysis by Flow Cytometry --- p.53 / Chapter 2.2.7 --- Measurement of Apoptosis-induced Activities --- p.54 / Chapter 2.2.8 --- Protein Expression Study --- p.59 / Chapter 2.2.9 --- Assessment of Differentiation-associated Characteristics --- p.64 / Chapter 2.2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Studies on the Anti-proliferative Effect of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Results --- p.69 / Chapter 3.2.1 --- Structure Identification of Sophoraflavanone G Isolated from Sophora flavescens --- p.69 / Chapter 3.2.2 --- Anti-proliferative Activity of Sophoraflavanone G on Various Myeloid Leukemia Cell Lines --- p.72 / Chapter 3.2.3 --- Effect of Sophoraflavanone G on the Viability of the Human Promyelocytic Leukemia HL-60 Cells --- p.80 / Chapter 3.2.4 --- Cytotoxic Effect of Sophoraflavanone G on Primary Normal Cells In Vitro --- p.83 / Chapter 3.2.5 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Sophoraflavanone G on the Human Promyelocytic Leukemia HL-60 Cells --- p.85 / Chapter 3.2.6 --- Effect of Sophoraflavanone G on the In Vivo Tumorigenicity of the HL-60 Cells --- p.88 / Chapter 3.2.7 --- Effect of Sophoraflavanone G on the Cell Cycle Profile of the HL-60 cells In Vitro --- p.90 / Chapter 3.2.8 --- Effect of Sophoraflavanone G on the Expression of Cell Cycle-regulatory Proteins in the HL-60 Cells --- p.93 / Chapter 3.2.9 --- Anti-proliferative Effect of Sophoraflavanone G on Multidrug-resistant (MDR) Leukemia Cell Line HL-60/MX2 Cells --- p.95 / Chapter 3.3 --- Discussion --- p.101 / Chapter Chapter Four: --- Studies on the Apoptosis- and Differentiation-inducing Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.114 / Chapter 4.2.1 --- Induction of DNA Fragmentation in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.114 / Chapter 4.2.2 --- Induction of Phosphatidylserine Externalization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G as Detected by Annexin V-GFP and PI Double Staining Method --- p.116 / Chapter 4.2.3 --- Effects of Sophoraflavanone G on the Caspase Activities in the Human Promyelocytic Leukemia HL-60 Cells --- p.119 / Chapter 4.2.4 --- Induction of Mitochondrial Membrane Depolarization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.124 / Chapter 4.2.5 --- Involvement of Bcl-2 Family Members in Sophoraflavanone G-induced Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells --- p.128 / Chapter 4.2.6 --- Effects of Sophoraflavanone G on the Induction of Reactive Oxygen Species in the Human Promyelocytic Leukemia HL-60 Cells --- p.131 / Chapter 4.2.7 --- Effect of Sophoraflavanone G on the Intracellular Ca2+ Level in the Human Promyelocytic Leukemia HL-60 Cells --- p.134 / Chapter 4.2.8 --- Morphological Studies on the Sophoraflavanone G-treated Human Promyelocytic Leukemia HL-60 Cells --- p.136 / Chapter 4.2.9 --- Effect of Sophoraflavanone G on the NBT Reducing Activity of the Human Promyelocytic Leukemia HL-60 Cells --- p.138 / Chapter 4.3 --- Discussion --- p.140 / Chapter Chapter Five: --- Conclusions and Future Perspectives --- p.148 / References --- p.156

Xie, Xinhui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 163-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1. / Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC. / This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases. / This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals. / This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model. / Li, Ming. / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 199-232). / 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. / Abstract also in Chinese.

Absorption study showed that gossypetin was methoxylated and conjugated to form glucuronide during the first-pass metabolism after oral administration. Glucuronide conjugate was the major circulating form in the plasma. As determined by HPLC analysis, the total gossypetin concentration in the plasma was higher than the unchanged gossypetin indicating that most of gossypetin underwent first-pass metabolism. Moreover, urinary excretion was not a main elimination route. / Uses of foods and dietary supplements present a safe chemopreventive strategy. The application of phytochemicals for cancer prevention currently receives a great deal of attention. Flavonoids are known to be antiproliferative and may play an important role in the prevention of carcinogenesis. In addition to epidemiologic studies, basic science research to elucidate mechanisms and evaluate chemopreventive potential of phytochemicals is also necessary. In this study, gossypetin was found to have stronger antiproliferative activity when compared with quercetin, a well studied flavonoid, in human hepatocellular carcinoma (HepG2) cells and human breast carcinoma (MCF-7) cells. The results demonstrated that gossypetin induced growth inhibition in MCF-7 cell line by arresting cell cycle at G0/G1 phase. The inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression, cdk6 kinase activity and phosphorylation of retinoblastoma protein (pRb). Although the cdk inhibitor p21 could not be detected, its upstream protein, p53 tumor suppressor protein, was activated by gossypetin in the MCF-7 cell line. Also, the proliferation of MCF-7 cells was suppressed through down-regulating the Erk1/2 pathway. / Ngai Lei-ka. / "August 2005." / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6156. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 222-250). / 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.

Ampelopsis grossedentata is a wildly used herb in South China as sleep aid beverage for many years. Yet the active ingredients and mechanisms of this herb were unknown. In the present study, extract from Ampelopsis grossedentata which we named ECBRC-AG, and one of its active ingredient myricetin were proved having significant hypnotic/sedative effects in multiple animal models. ECBRC-AG shortened sleep latency, increase NREM sleep and decrease locomotor activity when treated before the onset of light period in rats. ECBRC-AG could decrease active awake and increase REM sleep in the late part of light period. ECBRC-AG also decreased the caffeine induced hyperactivity in rats. Among the three suspected active ingredients from ECBRC-AG, myricetin showed similar active profile with ECBRC-AG. Myricetin increased NREM and REM sleep, decreased sleep latency, decreased locomotor activity and also active awake. All the above evidences have implicated that myricetin is the most important active ingredient of ECBRC-AG ECBRC-AG and myricetin did not show any obvious side effects on rats. / Based on these findings, we propose that myricetin facilitates GABA function on PVN neurons through a T-type calcium channel and CaM-KII mechanism. The hypnotic/sedative effects of ECBRC-AG and myricetin are mediated by PVN. ECBRC-AG treatment decreased corticosterone levels in rats, which also indicated that PVN/HPA axis was the target of these herbal derivates. PVN has broad interactions with GABAergic, hypocretinergic, cytokine and NPY system and all these systems are proved to be deeply involved in sleep regulation. / In conclusion, the present study has identified that myricetin is the most important active ingredient of the herbal extract ECBRC-AG. We confirmed the hypnotic/sedative effects of ECBRC-AG and myricetin on rats, and also revealed the different action profiles of these herbal derivates compared with zolpidem. T-type calcium channels and the HPA axis were shown to be involved in the mechanisms of ECBRC-AG and myricetin, indicating that they may be the new targets for insomnia treatment with these herbal derivates. / Insomnia is the most common sleep disorder and affects about one third of the general population. Insomnia is always combined with physical and mental illness, as either a consequence or a contributing factor. Insomnia produces sleepiness, impairment in psychomotor performance, absenteeism, frequent accidents, memory impairment and a high risk of depression. Pharmacologic therapies are the most important interventions for insomnia. However, the currently available hypnotics are associated with residual effects and risks of abuse and dependence. More efficient and safe hypnotics are needed. / The DNA array and RT-PCR studies revealed that GABA, hypocretin, cytokine and NPY systems were involved in the mechanisms of ECBRC-AG and myricetin. In calcium imaging study, we found that myricetin induced a transient Ca 2+ influx in the primary culture of rat hypothalamus neurons. This Ca2+ influx could be blocked by T-type channel blocker mibefradil. RT-PCR study also showed that ECBRC-AG and myricetin treatment changed the mRNA expression level of T-type calcium channel al G subunit in rat hypothalamus. The present results are consistent with our previous study showing that myricetin enhanced GABA function in the neurons of rat hypothalamic paraventricular nucleus (PVN), and that blocking CaM-KII pathway eliminated this effect. / Zhang, Xiaohu. / "March 2008." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1516. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 156-174). / 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.

Yeung, Tak Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 181-197). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / List of Publications --- p.vii / Presentation --- p.vii / List of Abbreviations --- p.viii / Abbreviations in Figures --- p.xiii / Abbreviations in Symbols --- p.xiv / List of Cell Lines Used in this Study --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cellular Redox State and Cancer Biology --- p.1 / Chapter 1.2 --- Reactive Oxygen Species (ROS) --- p.1 / Chapter 1.3 --- Regulation of Cellular Redox State by Intrinsic and Extrinsic Antioxidant Systems --- p.5 / Chapter 1.3.1 --- Intrinsic Antioxidant System --- p.6 / Chapter 1.3.2 --- Extrinsic Antioxidant System --- p.8 / Chapter 1.4 --- Glutathione --- p.9 / Chapter 1.4.1 --- General Information of Glutathione --- p.9 / Chapter 1.4.2 --- Functions of Glutathione --- p.12 / Chapter 1.4.2.1 --- As an Antioxidant and Free Radical Scavenger --- p.12 / Chapter 1.4.2.2 --- As a Detoxifier --- p.13 / Chapter 1.4.2.3 --- As a Regulator of Cell Signaling --- p.14 / Chapter 1.4.3 --- Synthesis of Glutathione --- p.15 / Chapter 1.4.4 --- Catabolism of Glutathione --- p.15 / Chapter 1.4.5 --- Transport and Uptake of Glutathione --- p.16 / Chapter 1.4.6 --- Glutathione in Cancer Biology --- p.18 / Chapter 1.4.6.1 --- "Role of Glutathione in the Regulation of Carcinogenesis, Growth and Apoptosis of Cancer Cells" --- p.18 / Chapter 1.4.6.1.1 --- Role of Glutathione in Carcinogenesis --- p.18 / Chapter 1.4.6.1.2 --- Role of Glutathione in the Growth of Cancer Cells --- p.20 / Chapter 1.4.6.1.3 --- Role of Glutathione in Apoptosis of Cancer Cells --- p.21 / Chapter 1.4.6.2 --- Role of Glutathione in the Regulation of Metastasis --- p.23 / Chapter 1.4.6.3 --- Role of Glutathione in Cancer Resistance and Therapy --- p.24 / Chapter 1.4.6.3.1 --- Role of Glutathione in Cancer Resistance --- p.24 / Chapter 1.4.6.3.2 --- Role of Glutathione in Cancer Therapy --- p.24 / Chapter 1.5 --- Aims of the Present Study --- p.25 / Chapter Chapter 2 --- In Vitro Study of Bαicαlein and Baicalin on Glutathione Depletion --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Scutellaria bαicαlensis Georgi --- p.28 / Chapter 2.1.1.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.28 / Chapter 2.1.1.2 --- As an Antioxidant and Free Radical Scavenger --- p.29 / Chapter 2.1.1.3 --- Long History for Treatment of Cancers with the Obscure Mechanism --- p.30 / Chapter 2.1.1.4 --- Major Components --- p.31 / Chapter 2.1.2 --- Baicalein and Baicalin --- p.32 / Chapter 2.1.2.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.32 / Chapter 2.1.2.2 --- As an Antioxidant and Free Radical Scavenger --- p.33 / Chapter 2.1.3 --- Hypothesis: Baicalein and Baicalin Induce Cancer Cell Death Via Glutathione Depletion --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Chemicals --- p.36 / Chapter 2.2.2 --- Buffers and Solutions --- p.36 / Chapter 2.2.3 --- Animals --- p.37 / Chapter 2.2.4 --- Preparation of Rat Brain Microsomes --- p.37 / Chapter 2.2.5 --- Glutathione Depletion Assay In Vitro and Thiol Depletion Assay in Rat Brain Microsomes --- p.38 / Chapter 2.2.6 --- Statistical Analysis --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Glutathione --- p.42 / Chapter 2.3.2 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Rat Brain Microsomes --- p.42 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Effects of Baicalein and Baicalin on Proliferation of Different Human Cancer and Normal Cells --- p.45 / Chapter 3.1 --- Introduction-Importance of Developing A Novel Compound Inducing Cancer Cells to Cell Death with the Least Side Effects on Normal Cells --- p.45 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Instruments --- p.46 / Chapter 3.2.2 --- Chemicals and Cell Culture Reagents --- p.46 / Chapter 3.2.3 --- Buffers --- p.46 / Chapter 3.2.4 --- Cell Lines --- p.47 / Chapter 3.2.5 --- Cell Culture --- p.48 / Chapter 3.2.6 --- Determination of Cell Proliferation by MTT Assay --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.51 / Chapter 3.3.2 --- Effects of Baicalein on Different Normal Cell Lines --- p.56 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.58 / Chapter 3.4.2 --- Effects of Baicalein on Cell Proliferation on Different Human Normal Cell Lines --- p.60 / Chapter Chapter 4 --- Glutathione-Depleting Effects of Baicalein on Cell Proliferation of Different Cell Lines --- p.61 / Chapter 4.1 --- Introduction-Brain Tumors --- p.61 / Chapter 4.1.1 --- Types and Classifications of Brain Tumors --- p.61 / Chapter 4.1.2 --- "Incidence Time, Patient Survival Time and Rate for" --- p.65 / Chapter 4.1.3 --- Symptoms and Diagnostic Methods for Brain Tumors --- p.66 / Chapter 4.1.4 --- "Treatments, Side Effects and Difficulties of Treatments for Brain Tumors" --- p.67 / Chapter 4.1.5 --- Glutathione Levels in Brain Normal and Cancer Cells --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Instruments --- p.70 / Chapter 4.2.2 --- Chemicals --- p.70 / Chapter 4.2.3 --- Buffers --- p.70 / Chapter 4.2.4 --- Determination of Cell Proliferation by MTT Assay --- p.70 / Chapter 4.2.5 --- Determination of Intracellular Glutathione Depletion by Fluorescent Dye CMAC --- p.71 / Chapter 4.2.6 --- Determination of Cellular Reduced Glutathione Levels by DTNB-Coupled Glutathione Reductase Recycling Assay --- p.73 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of Baicalein on Intracellular GSH Levels and Cell Proliferation for Different Cell Lines --- p.75 / Chapter 4.3.2 --- Basal Intracellular Glutathione in Different Cell Lines --- p.81 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Intracellular Glutathione Depletion and Cell Death Induction Effects of Baicalein on Different Cell Lines --- p.84 / Chapter 4.4.2 --- Relationship between Basal Glutathione Levels and Drug Susceptibilities --- p.85 / Chapter Chapter 5 --- Effects of Baicalein on Apoptosis and Caspase Pathways --- p.88 / Chapter 5.1 --- Introduction-Modes of Cell Death --- p.88 / Chapter 5.1.1 --- Necrosis --- p.88 / Chapter 5.1.2 --- Apoptosis --- p.89 / Chapter 5.2 --- Materials and Methods --- p.92 / Chapter 5.2.1 --- Chemicals --- p.92 / Chapter 5.2.2 --- Buffers --- p.92 / Chapter 5.2.3 --- Determination of Change of Mitochondrial Membrane Potential by JC-1 --- p.93 / Chapter 5.2.4 --- Determination of Apoptosis by Annexin V-Propidium Iodide Staining --- p.94 / Chapter 5.2.5 --- Determination of Cell Cycle Arrest by Propidium Iodide Staining --- p.95 / Chapter 5.2.6 --- "Determination of Caspase-3, -8 and -9 Activities by Fluorescent-Labeled Peptides" --- p.96 / Chapter 5.2.7 --- Determination of DNA Fragmentation --- p.97 / Chapter 5.2.8 --- Terminal Deoxynucleotidyl Transferase Mediated dUTP End Labeling (TUNEL) Assay --- p.99 / Chapter 5.2.9 --- Flow Cytometry --- p.101 / Chapter 5.3 --- Results --- p.102 / Chapter 5.3.1 --- Effects of Baicalein on Mitochondrial Membrane Potential by JC-1 Staining --- p.102 / Chapter 5.3.2 --- Effects of Baicalein on Apoptosis and Necrosis by Annexin V-Propidium Iodide Staining --- p.104 / Chapter 5.3.3 --- Effects of Baicalein on Cell Cycle Arrest by Propidium Iodide Staining --- p.108 / Chapter 5.3.4 --- "Effects of Baicalein on Caspase-3, -8 and -9 Activities" --- p.110 / Chapter 5.3.5 --- Effeets of Baiealein on DNA Fragmentation --- p.115 / Chapter 5.3.6 --- Effects of Baicalein on TUNEL Assay --- p.117 / Chapter 5.4 --- Discussion --- p.120 / Chapter Chapter 6 --- Pro-Oxidant Role of Baicalein on Reactive Oxygen Species Generation --- p.122 / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.122 / Chapter 6.2.1 --- Chemicals --- p.122 / Chapter 6.2.2 --- Determination of Cellular Reactive Oxygen Species Generation by Fluorescent Dye cDCFDA --- p.123 / Chapter 6.2.3 --- Determination of Mitochondrial Reactive Oxygen Species Generation by Fluorescent Dye Rhl23 --- p.124 / Chapter 6.3 --- Results --- p.125 / Chapter 6.3.1 --- Effects of Baicalein on Cellular ROS Generation by Fluorescent Dye cDCFDA --- p.125 / Chapter 6.3.2 --- Effects of Baicalein on Mitochondrial ROS Generation by Fluorescent Dye Rhl23 --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- The Anticancer Mechanistic Study of Baicalein --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Materials and Methods --- p.134 / Chapter 7.2.1 --- Chemicals --- p.134 / Chapter 7.2.2 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.134 / Chapter 7.2.3 --- Reversibility of Baicalein-Induced Cellular ROS Generation --- p.136 / Chapter 7.2.4 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.137 / Chapter 7.2.5 --- "Reversibility of Baicalein-Induced Caspase-3, -8 and -9 Activation by Co-Treatment of Different Antioxidants" --- p.138 / Chapter 7.3 --- Results --- p.139 / Chapter 7.3.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.139 / Chapter 7.3.1.1 --- Pre-treatments --- p.139 / Chapter 7.3.1.2 --- Co-treatments --- p.141 / Chapter 7.3.1.3 --- Post-treatments --- p.144 / Chapter 7.3.2 --- Reversibility of Baicalein-Induced Cellular ROS Generation by Co-Treatment of Different Antioxidants --- p.147 / Chapter 7.3.3 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.152 / Chapter 7.3.4 --- Reversibility of Baicalein-Induced Caspase-3 Activation by Co-Treatment of Different Antioxidants --- p.156 / Chapter 7.3.5 --- Reversibility of Baicalein-Induced Caspase-8 and -9 Activation by Co-Treatment of Different Antioxidants --- p.160 / Chapter 7.4 --- Discussion --- p.164 / Chapter 7.4.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death --- p.164 / Chapter 7.4.2 --- "Reversibility of Baicalein-Induced ROS Generation," --- p.167 / Chapter 7.5 --- Concluding Remarks --- p.168 / Chapter Chapter 8 --- General Discussion --- p.169 / Chapter 8.1 --- Drug Delivery to Brain --- p.169 / Chapter 8.2 --- Protective Roles of Baicalein on Brain Cells --- p.170 / Chapter 8.2.1 --- Actions Against Oxidative Stress --- p.170 / Chapter 8.2.2 --- Actions Against Other Neurotoxic Damages --- p.171 / Chapter 8.2.3 --- Actions Against Neuronal Diseases --- p.172 / Chapter 8.3 --- Anticancer Roles of Baicalein on Astrocytoma --- p.173 / Chapter 8.4 --- Implications on the Dual Roles of Baicalein: Antioxidant and Pro-oxidant --- p.175 / Chapter 8.5 --- Future Perspectives --- p.175 / Chapter 8.5.1 --- Effects of Baicalein on Antioxidant System --- p.175 / Chapter 8.5.2 --- Effects of Baicalein on GSH Synthesis --- p.176 / Chapter 8.5.3 --- In Vivo Studies on Cytotoxic Effects of Baicalein --- p.177 / Chapter 8.5.4 --- In Vivo Studies on Anti-Tumor Effects and In Vitro Studies on Anti-Metastasis Effects of Baicalein --- p.178 / Reference List --- p.181

Li, Wing Yan Kate. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 130-154). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Overview of cancer --- p.1 / Chapter 1.2. --- Apoptosis and cancer --- p.4 / Chapter 1.3. --- Roles and regulation of caspase-dependent apoptosis --- p.7 / Chapter 1.3.1. --- Extrinsic death receptor pathway --- p.8 / Chapter i. --- TNFR1 and TNFa --- p.13 / Chapter ii. --- CD95/Fas and CD95 Ligand/FasL --- p.14 / Chapter iii. --- "TRAIL-R1(DR4), TRAIL-R2 (DR5) and TRAIL" --- p.14 / Chapter 1.3.2. --- Intrinsic mitochondrial pathway --- p.16 / Chapter i. --- Bcl-2 family of proteins --- p.17 / Chapter ii. --- Reactive Oxygen Species (ROS) --- p.19 / Chapter 1.4. --- Phytochemicals from Traditional Chinese Medicine (TCM) as a source of new therapeutics --- p.22 / Chapter 1.5. --- Biological effects of baicalein --- p.25 / Chapter 1.5.1 --- Roles of baicalein as a lipoxygenase inhibitor --- p.28 / Chapter 1.5.2 --- Dual roles of baicalein as an antioxidant and prooxidant --- p.28 / Chapter 1.5.3 --- "Roles of baicalein as an anti-carcinogenic, anti-proliferative and anti-metastatic agent" --- p.29 / Chapter 1.6. --- Aims of current study --- p.30 / Chapter Chapter 2 --- Effects of Baicalein on Growth and Survival of Human Cancer Cells / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Cell culture --- p.35 / Chapter 2.2.2 --- Measurement of growth and survival of various cell lines --- p.36 / Chapter 2.2.3 --- Statistical analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Baicalein retards the growth and survival of human melanoma A375 and colorectal carcinoma Caco-2 --- p.37 / Chapter 2.3.2 --- Baicalein reduces the growth and survival of melanoma A375 but not in normal skin fibroblast Hs68 cells --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Effects of Baicalein on Cell Cycle and the Apoptosis in Human Melanoma A375 Cells / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Determination of cell cycle changes and quantification of apoptosis --- p.51 / Chapter 3.2.2 --- Immunoblotting --- p.52 / Chapter 3.2.3 --- Inhibition of caspase-8 by caspase-8 inhibitor --- p.54 / Chapter 3.2.4 --- Fluorometric measurement of caspase-3 activity --- p.54 / Chapter 3.2.5 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Baicalein induces S-phase arrest in cell cycle and triggers apoptosis --- p.55 / Chapter 3.3.2 --- Baicalein induces proteolytic inactivation of PARP and activation of caspases --- p.59 / Chapter 3.3.3 --- Caspase-8 is the major initiator caspase eliciting the baicalein-induced apoptosis --- p.62 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Immunoblotting --- p.75 / Chapter 4.2.2 --- Determination of sub-lethal dose of exogenous TRAIL --- p.76 / Chapter 4.2.3 --- Determination of the combinatory effect of exogenous TRAIL and baicalein --- p.76 / Chapter 4.2.4 --- Statistical analysis --- p.77 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Baicalein upregulates the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) --- p.77 / Chapter 4.3.2 --- Baicalein sensitizes the melanoma cells to sub-lethal dose of exogenous TRAIL --- p.80 / Chapter 4.4 --- Discussion --- p.84 / Chapter Chapter 5 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells Cancer Cells / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Materials and Methods / Chapter 5.2.1 --- Analysis of mitochondrial membrane potential --- p.94 / Chapter 5.2.2 --- Fractionation of cell lysates into cytosolic and mitochondrial fractions for immunoblotting --- p.95 / Chapter 5.2.3 --- Immunoblotting --- p.95 / Chapter 5.2.4 --- Determination of cellular reactive oxygen species (ROS) production --- p.96 / Chapter 5.2.5 --- Verification of ROS generation via the addition of Trolox´ёØ --- p.96 / Chapter 5.2.6 --- Statistical analysis --- p.97 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Baicalein induces mitochondrial membrane depolarization --- p.97 / Chapter 5.3.2 --- Cytochrome c is released in the baicalein-induced mitochondrial membrane depolarization --- p.100 / Chapter 5.3.3 --- Baicalein does not elicit the intrinsic apoptotic pathway via modulation of some better-characterized Bcl-2 family proteins in A375 cells --- p.102 / Chapter 5.3.4 --- Baicalein induces ROS production --- p.105 / Chapter 5.3.5 --- Baicalein induces mitochondrial permeabilization via ROS-mediated mechanisms --- p.108 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General Discussion --- p.119 / References --- p.130