Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

有證據顯示基質細胞在腫瘤的發生發展中可以發揮重要的作用，基質細胞可以提供適宜腫瘤細胞增殖的腫瘤微環境。腫瘤相關成纖維細胞是一種特殊的與腫瘤生成高度相關的基質細胞。而通过我们的论证，小鼠胚胎成纖維細胞可以作為一種腫瘤相關成纖維細胞的細胞模型。 / 腫瘤誘導因子（TIF）是本實驗室在成瘤實驗中發現的一種新的倉鼠CXC 趨化因子。基于蛋白質序列的分析，TIF 属于Gro CXC 趨化因子家族。這個家族主要通過激活其受體CXCR2 來發揮作用。為了研究TIF 在腫瘤發生中的作用，我們在CHO-K1 細胞中建立了過表達TIF 的穩定細胞株。 / 我們發現共同注射的永生化MEF 與過表達TIF 的D12 細胞導致了腫瘤生長的抑制。為了研究這種現象，重組TIF 蛋白在大腸桿菌中表達，并且用鎳柱進行了提純。純化的蛋白被用于處理CHO-K1 細胞與永生化MEF。我們發現高水平的TIF 可以導致CXCR2 下游的Erk 磷酸化水平下降。其可能的機制為CXCR2 在高水平的TIF 作用下的脫敏作用。同時高水平TIF 可以導致永生化MEF 中CD133 水平的下降。因此，CXCR2 脫敏為TIF 導致腫瘤抑制的可能機制。 / Lines of evidence indicate that stromal cell is one of the determinants in tumor formation by providing a favorable microenvironment for the growth of cancer cells. Cancer associated fibroblast (CAF) is a special form of stromal cells which are shown to be derived from bone marrow. Upon reaching the tumor, the bone marrow-derived mesenchymal stem cells differentiate into CAF, which secrets various growth factors and cytokines to promote cancer growth. Furthermore, genetic study shows that CAF displays p53 mutations and other genetic changes. / Tumor induced factor (TIF) is a CXC chemokine that is originally identified from a xenograft tumor. Sequence analysis suggests TIF is a family member of the Gro CXC chemokines, and exerts its cellular function via activating CXCR2 receptors. In order to investigate the functional roles of TIF, a stable cell line over-expressing TIF in hamster CHO-K1 was established. / To explore the cancer-stromal interactions in xenograft, mouse embryonic fibroblast (MEF) were used as a study model for CAF. MEF was sub-cultured by a conventional protocol that was used for developing the NIH3T3 cells. Based on the growth patterns and expressions of cell markers, growth of MEF can be divided into three stages: the early stage, the senescent stage and the immortalized stage. Our results suggested that MEF might mirror the various developmental stages of CAF. / To examine the contributions of MEF in tumorigenesis, CHO-K1 cells and MEF were co-injected into nude mice. Intriguingly, MEF that in senescent and immortalized stages, rather than in early stage, promoted tumor formation. A possibility arose that the contribution of senescent and immortalized MEF in promoted tumorigenesis may due to CD133 and CXCL1, as the expression of CD133 and CXCL1 in senescent and immortalized MEF were higher than that of MEF in early stage. Moreover, as MEF could gradually develop into a fibroblast promoted tumor formation, MEF could be used as a crucial model to illustrate the origination and development of CAF. / Surprisingly, in nude mice co-injected with immortalized MEF with TIF-overexpressing D12 cells, suppression instead of promotion of tumor growth was found. In order to explore the underlined mechanism of tumor suppression, recombinant TIF protein was purified based on a bacterial expression system. Using purified TIF protein to treat CHO-K1 cells and MEF, it was found that low concentration of TIF promoted Erk phosphorylation but high concentration of TIF suppressed it, which might resulted from desensitization of CXCR2 receptors. Reduction of Erk phosphorylation resulted in decreased proliferation in CHO-K1 cells and alleviated expression of CD133 in MEF, which could be the mechanisms for TIF-induced tumor suppression in nude mice. / Taken together, a CAF model was established to examine the function of TIF in tumor-fibroblast interactions. Mechanistic studies indicated that TIF-induced tumor suppression in nude mice was mediated via desensitization of CXCR2 receptors by high concentration of TIF in the tumor microenvironment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qi, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Tumorigenesis --- p.4 / Chapter 1.1.1 --- Virus transformation --- p.4 / Chapter 1.1.2 --- Proto-oncogene and oncogene --- p.5 / Chapter 1.1.3 --- Tumor suppressor gene --- p.7 / Chapter 1.1.4 --- Epigenetic alteration --- p.9 / Chapter 1.1.5 --- Cancer stem cell --- p.11 / Chapter 1.1.6 --- Tumor microenvironment --- p.14 / Chapter 1.2 --- Cancer associated fibroblast (CAF) --- p.17 / Chapter 1.2.1 --- Markers for CAF --- p.17 / Chapter 1.2.2 --- CAF and normal fibroblast --- p.20 / Chapter 1.2.3 --- CAF, a important player in tumor growth --- p.22 / Chapter 1.2.4 --- CAF and angiogenesis --- p.23 / Chapter 1.2.5 --- CAF and tumor invasion --- p.25 / Chapter 1.3 --- Chemokine --- p.27 / Chapter 1.3.1 --- Structure of chemokine --- p.27 / Chapter 1.3.2 --- Chemokine and cell Recruitment --- p.30 / Chapter 1.3.3 --- Chemokine and tumor microenvironment --- p.30 / Chapter 1.4 --- Tumor Induced Factor and its induced tumor suppression --- p.38 / Chapter 1.5 --- The aims of the project --- p.47 / Chapter Chapter Two --- Purification of Tumor Induced Factor / Chapter 2.1 --- Introduction --- p.49 / Chapter 2.2 --- Materials --- p.52 / Chapter 2.2.1 --- Chemical --- p.52 / Chapter 2.2.2 --- Enzyme --- p.52 / Chapter 2.2.3 --- Antibody --- p.52 / Chapter 2.3 --- Method --- p.53 / Chapter 2.3.1 --- Overview of protein expression system --- p.53 / Chapter 2.3.2 --- Purification of Trx-His₆-S-TIF protein --- p.54 / Chapter 2.3.3 --- BCA assay --- p.60 / Chapter 2.3.4 --- SDS-PAGE --- p.60 / Chapter 2.3.5 --- Western blotting --- p.61 / Chapter 2.3.6 --- Preparation of pET28/His₆-Sumo-TIF bacterial expression vector --- p.62 / Chapter 2.3.7 --- Optimization of culture condition for BL21 expressed His₆-Sumo-TIF protein --- p.67 / Chapter 2.3.8 --- Purification of His₆-Sumo-TIF protein --- p.68 / Chapter 2.3.9 --- Homology model of TIF --- p.68 / Chapter 2.4 --- Results --- p.69 / Chapter 2.4.1 --- Purification of Trx-His₆-S-TIF --- p.70 / Chapter 2.4.2 --- Optimization of purification protocol of His₆-Sumo-TIF --- p.71 / Chapter 2.4.3 --- Large scale purification of mature TIF --- p.75 / Chapter 2.4.4 --- Homology modeling of TIF --- p.80 / Chapter 2.5 --- Discussion --- p.83 / Chapter Chapter 3 --- Three Stages Hypothesis / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Material --- p.93 / Chapter 3.2.1 --- Chemical --- p.93 / Chapter 3.2.2 --- Enzyme --- p.93 / Chapter 3.2.3 --- Animal --- p.93 / Chapter 3.2.4 --- Antibody --- p.94 / Chapter 3.3 --- Methods --- p.95 / Chapter 3.3.1 --- Isolate MEF from 13.5 days mouse embryo --- p.95 / Chapter 3.3.2 --- Culture of MEF following 3T3 protocol --- p.96 / Chapter 3.3.3 --- X gal staining --- p.96 / Chapter 3.3.4 --- Analysis of MEF cell size and complexity by flow cytometry --- p.98 / Chapter 3.3.5 --- MTT assay --- p.98 / Chapter 3.3.6 --- Analysis of CD133 by flow cytometry --- p.99 / Chapter 3.3.7 --- ROS detected by DCFH-DA fluorescent probe --- p.99 / Chapter 3.3.8 --- Double staining of cancer stem cell marker and ROS fluorescent probe --- p.100 / Chapter 3.3.9 --- Reverse transcription --- p.101 / Chapter 3.3.10 --- Analysis CXCL1 mRNA expression level by PCR --- p.102 / Chapter 3.3.11 --- Gelatin zymography --- p.103 / Chapter 3.3.12 --- In-vivo tumorigenicity assay --- p.104 / Chapter 3.4 --- Results --- p.106 / Chapter 3.4.1 --- Three Stages of MEF --- p.106 / Chapter 3.4.2 --- X gal staining --- p.106 / Chapter 3.4.3 --- Flow cytometric analysis of cell diameter and cellular complexity of MEF --- p.109 / Chapter 3.4.4 --- MTT assay --- p.109 / Chapter 3.4.5 --- CD 133 expression of MEF detected by flow cytometry --- p.110 / Chapter 3.4.6 --- Reactive oxygen species of MEF detected by flow cytometry --- p.118 / Chapter 3.4.7 --- The level of ROS and CD133 of MEF detected by flow cytometry stimultaneously --- p.121 / Chapter 3.4.8 --- TIF treatment reduces the small CSC subpopulation in senescent stage MEF --- p.124 / Chapter 3.4.9 --- Increased CXCL1 expression in senescent stage and immortalized stage MEF --- p.125 / Chapter 3.4.10 --- Matrix metalloproteinase 2 activities in different stages of MEF . --- p.129 / Chapter 3.4.11 --- In vivo tumorigenicity assay --- p.130 / Chapter 3.5 --- Discussion --- p.133 / Chapter Chapter Four --- Biphasic Effect of TIF in Cancer-Fibroblasts Interaction / Chapter 4.1 --- Introduction --- p.140 / Chapter 4.2 --- Material --- p.143 / Chapter 4.2.1 --- Chemical --- p.144 / Chapter 4.2.2 --- Kit and Instrument --- p.144 / Chapter 4.2.3 --- Antibody --- p.144 / Chapter 4.3 --- Method --- p.145 / Chapter 4.3.1 --- Purification of TIF-His₆-Flag --- p.145 / Chapter 4.3.2 --- Western blotting to detect purified TIF-His₆-Flag --- p.145 / Chapter 4.3.3. --- Measurement of cell proliferation by cell counting --- p.145 / Chapter 4.3.4 --- MTT assay --- p.146 / Chapter 4.3.5 --- Western blotting to detect pErk and total Erk --- p.146 / Chapter 4.3.6 --- Soft agar assay --- p.148 / Chapter 4.3.7 --- Gelatinase detection --- p.148 / Chapter 4.3.8 --- Wound healing assay --- p.149 / Chapter 4.3.9 --- Colony formation assay --- p.149 / Chapter 4.3.10 --- Detection of CD133 by flow cytometry --- p.150 / Chapter 4.4 --- Results --- p.151 / Chapter 4.4.1 --- Purification of TIF-His₆-Flag --- p.151 / Chapter 4.4.2 --- Reduced cell proliferation of D12 in long time culture --- p.153 / Chapter 4.4.3 --- Reduced metabolic activities of D12 cells in time culture --- p.155 / Chapter 4.4.4. --- TIF-CXCR2-pErk signal axis in CHO cells --- p.155 / Chapter 4.4.5 --- Bigger colonies formed by D12 cells in soft agar assay --- p.161 / Chapter 4.4.6 --- TIF-CXCR2-pErk-MMP9 signal pathway in D12 cells --- p.162 / Chapter 4.4.7 --- Reduced migration of D12 cells --- p.164 / Chapter 4.4.8 --- Reduced cell invasion of D12 cells --- p.165 / Chapter 4.4.9 --- Reduced colony number of D12 cells in colony formation assay --- p.168 / Chapter 4.4.10 --- Bi-phasic “bell shape“ bi-phasic response on Erk activation of TIF in CHO-K1 cells --- p.169 / Chapter 4.4.11 --- Bi-phasic “bell shape“ effect of TIF to pErk in immortalized MEFs --- p.172 / Chapter 4.4.12 --- Reduced CD133 in immortalized MEF by high concentration of TIF --- p.173 / Chapter 4.5 --- Discussion --- p.177 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Project Summary --- p.183 / Chapter 5.2 --- Significances of the project --- p.185 / Chapter 5.3 --- Future work --- p.188

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。 / 方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。 / 結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。 / 結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。 / Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied. / Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis. / Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality. / Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Liangliang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 128-144). / Abstract also in Chinese. / Declaration --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Chapter 1 --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells --- p.2 / Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2 / Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4 / Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5 / Chapter 1.2 --- Adipogenesis of MSCs --- p.6 / Chapter 1.3 --- Chondrogenesis of MSCs --- p.7 / Chapter 1.4 --- Osteogenesis of MSCs --- p.8 / Chapter 1.4.1 --- Regulators of osteogenesis --- p.9 / Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11 / Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13 / Chapter 1.5.1 --- ERK signaling pathway --- p.14 / Chapter 1.5.2 --- Wnt signaling pathway --- p.15 / Chapter 1.5.3 --- BMP signaling pathway --- p.17 / Chapter 1.6 --- Migration of MSCs --- p.20 / Chapter 1.7 --- Fracture healing --- p.22 / Chapter 1.8 --- Clinical application of MSCs --- p.23 / Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24 / Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25 / Chapter 1.9 --- Scope of the present study --- p.26 / Chapter 1.9.1 --- CRBP1 --- p.26 / Chapter 1.9.2 --- N-cadherin --- p.27 / Chapter 1.9.3 --- SOX11 --- p.27 / Chapter 1.10 --- Experimental scheme --- p.29 / Chapter 2 --- p.31 / Comparison between PB-MSCs and BM-MSCs --- p.31 / Chapter 2.1 --- Chapter introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.33 / Chapter 2.2.1 --- Cell culture --- p.33 / Chapter 2.2.2 --- Flow cytometry --- p.33 / Chapter 2.2.3 --- Adipogenic differentiation --- p.34 / Chapter 2.2.4 --- Osteogenic differentiation --- p.34 / Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35 / Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36 / Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38 / Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39 / Chapter 2.4 --- Discussion and future work --- p.40 / Chapter 3 --- p.41 / Role of CRBP1 in Differentiation and Migration of MSCs --- p.41 / Chapter 3.1 --- Chapter introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.46 / Chapter 3.2.1 --- Chemicals --- p.46 / Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46 / Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47 / Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48 / Chapter 3.2.5 --- Osteogenic differentiation --- p.50 / Chapter 3.2.6 --- Adipogenic differentiation --- p.50 / Chapter 3.2.7 --- Western blot --- p.51 / Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52 / Chapter 3.2.9 --- Cell migration assay --- p.52 / Chapter 3.2.10 --- Ectopic bone formation assay --- p.52 / Chapter 3.2.11 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53 / Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56 / Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58 / Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61 / Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63 / Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64 / Chapter 3.4 --- Discussion --- p.66 / Chapter 3.5 --- Future work --- p.73 / Chapter 4 --- p.74 / Role of N-cadherin in Differentiation and Migration of MSCs --- p.74 / Chapter 4.1 --- Chapter introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.78 / Chapter 4.2.1 --- Chemicals --- p.78 / Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78 / Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79 / Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81 / Chapter 4.2.5 --- Western blot --- p.81 / Chapter 4.2.6 --- Ectopic bone formation assay --- p.82 / Chapter 4.2.7 --- Statistical analysis --- p.82 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83 / Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84 / Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86 / Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87 / Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89 / Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89 / Chapter 4.4 --- Discussion --- p.91 / Chapter 4.5 --- Future work --- p.94 / Chapter 5 --- p.96 / Role of SOX11 in Differentiation and Migration of MSCs --- p.96 / Chapter 5.1 --- Chapter introduction --- p.97 / Chapter 5.2 --- Materials and methods --- p.105 / Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105 / Chapter 5.2.2 --- Cell culture --- p.106 / Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106 / Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106 / Chapter 5.2.5 --- Adipogenic differentiation --- p.107 / Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107 / Chapter 5.2.6 --- Western blot --- p.108 / Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108 / Chapter 5.2.8 --- Cell migration --- p.110 / Chapter 5.2.9 --- Ectopic bone formation --- p.110 / Chapter 5.2.10 --- Fracture healing model and analysis --- p.111 / Chapter 5.2.11 --- Statistical Analysis --- p.112 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112 / Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113 / Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114 / Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115 / Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117 / Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118 / Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122 / Chapter 5.4 --- Discussion --- p.126 / Chapter 5.5 --- Future work --- p.131 / Appendix --- p.153

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75