京都大学 / 新制・課程博士 / 博士(医学) / 甲第24503号 / 医博第4945号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 椛島 健治, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The aging phenomena is a process to which all organisms eventually succumb. The universality of this phenomena suggests that there may be one overwhelming factor involved. The exact biochemical basis of aging is still unclear. Free radicals such as the superoxide radical (O₂⁻) and the hydroxyl radical (OH⁻), formed in biological oxidation reactions may be responsible for cellular aging. Because of the high reactivity of the O₂⁻ and the OH⁻ they can produce extensive damage to lipids, proteins, and nucleic acids. In this study we have developed an <i>in vitro</i> quiescent model using density dependent bovine embryonic lung fibroblast (BELF). The effect of this process on the antioxidant defense enzymes such as, the superoxide dismutases, catalase, glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, was studied. We have also extensively monitored the levels of free radical in the cell by both direct and indirect methods. The results indicate that no significant (p<0.05) changes in the activity of any of the major antioxidant enzymes in our quiescent model. Significant increases were observed in the intracellular levels of lipofuscin (age pigment) with time, but no changes in the generation of free radicals were observed using electron spin resonance spectrometry, cytochrome <i>c</i> reduction or spectrofluorometric techniques (caution should be placed in statistical interpretation of the data because of the small sample size in some experiments). The transcriptional and translational controls of the one of the major antioxidant defense enzymes (manganese superoxide dismutase) in bovine embryonic lung fibroblasts, human pulmonary artery endothelial cells (HPAE) and bovine PAE cell lines were also studied. Our preliminary data suggest that inhibitors of protein and RNA synthesis both cause a significant decrease in the induction of the manganese SOD in bovine pulmonary endothelial cells. / Master of Science

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.