神經膠質瘤 (Glioma) 是最常見也是最惡性的原發性中樞神經系 統腫瘤,其強大的侵襲性使其在臨床上 90%-95% 的患者在發現罹患 此疾病後平均存活率僅兩年[1],而該腫瘤強大的侵襲力與腫瘤血管 新生能力、腫瘤相關巨噬細胞及腫瘤缺氧區分佈有密切關係。研究指 出腫瘤所分泌的血管新生相關基因 sdf-1 會影響腫瘤生長速率並改 變腫瘤的侵襲能力[2, 3]。本研究以小鼠星狀細胞 (ALTS1C1) 作為研 究的模型,過表達 sdf-1 基因來探討其對腫瘤微環境的影響。首先, 利用 liposome 轉殖 DNA 的方式建立 ALTS1C1-SDF-1 細胞株。觀 察 SDF-1 對細胞分裂與增生能力的影響,再以組織免疫染色法探討 腫瘤微血管密度、巨噬細胞及缺氧區域的病理狀態,最後藉由流式細 胞儀分析腫瘤相關巨噬細胞 (TAMs) 與微膠細胞 (Microglia) 在腫 瘤生長各時期的比例分佈。實驗結果發現新建立的 ALTS1C1-SDF-1 腦腫瘤模式小鼠最短生存天數僅 21 天,相較於 ALTS1C1 腫瘤小數 平均存活天數(29天)大幅的縮短,且 ALTS1C1-SDF-1 腫瘤具備許多 臨床上高度侵襲性的腦腫瘤特性,包括表現量較高的腫瘤血管密度、 分佈密集的腫瘤相關巨噬細胞和較低的缺氧區;從不同時期的腫瘤相 關巨噬細胞比例分佈結果再次証實 Sdf-1 的分泌會增加腫瘤內巨噬 細胞的分佈數量。本論文研究提供了一個俱有高度侵襲能力的臨床腦 腫瘤小鼠模式,可以用來研發設計腦腫瘤治療的新策略。

Gliomas are the most common primary neoplasms of the central nervous system (CNS) in adults. The powerful tumor invasiveness is
closely related to tumor angiogenesis, tumor-associated macrophages and hypoxia. Studies indicate that stroma-derived factor -1 (SDF-1), one of tumors-secreted angiogenesis-related factors, affects tumor growth rate and tumor invasion capability. In this study, we used mouse astrocytoma cells, ALTS1C1, as a research model to over-express sdf-1 gene in order to establish an invasive tumor model and study the tumor microenvironments of an invasive astrocytoma. Firstly, the ALTS1C1-SDF-1 cell line was established by using transgenic DNA liposome method. Secondly, the relationship between SDF-1 and tumor cell migration, proliferation, tumor microvessel density, or tumor -associated macrophages was studied by immunohistochemical (IHC) apprach. Finally, the relative change of microglia and macrophage during tumor progression was examined by flow cytometery. This study has successfully established an invasive brain tumor model and provided evidences to demonstrate that SDF-1 acts as a chemoattractant or survival factor for TAM tropism toward hypoxic tumor region. This brain tumor model serves an important tumor model for the design of new treatment protocol against the invasive glioma.

中文摘要................................................................................................. I Abstract ..................................................................................... II 致謝 ........................................................................................................III 目錄 ........................................................................................................IV 圖 目 錄 ................................................................................................. VIII
第一章 緒論 ............................................................................................1 1.1 神經膠質瘤.......................................................................................................1 1.1.1 神經膠質瘤簡介..........................................................................................1 1.1.2 神經膠質瘤的分級......................................................................................1 1.1.3 神經膠質瘤的症狀......................................................................................2 1.1.4 神經膠質瘤的治療......................................................................................3 1.2. 腫瘤微環境......................................................................................................4 1.2.1 腫瘤血管新生..............................................................................................4 1.2.2 腫瘤缺氧區(Tumor Hypoxia) .....................................................................5
1.2.3 微膠細胞(Microglia)與腫瘤相關巨噬細胞(Tumor associated
macrophages, TAMs).............................................................................................6
1.3 小鼠腦腫瘤模型..............................................................................................7 1.3.1 動物腦腫瘤模型簡介..................................................................................7 1.3.2 ALTS1C1 小鼠腦腫瘤模式........................................................................8
1.4 基質細胞衍生因子-1 (stromal-cell-derived factor -1, SDF-1).....................8
第二章 實驗材料與方法 ....................................................................11 2.1 分子生物實驗- pIRES-SDF-1 overexpression 質體建構 .........................11 2.1.1 聚合酶素連鎖反應(Polymerase Chain Reaction) ....................................11 2.1.2 限制酶酵素切割反應(Restriction Enzyme Digestion).............................12 2.1.3 DNA 的接合作用(Ligation)......................................................................13 2.1.5 微量純化製備質體(PD100, Geneaid, Taiwan) ........................................14 2.1.6 洋菜膠電泳................................................................................................15 2.1.7 中量純化製備質體(12145, QIAGEN, USA)............................................15 2.2 細胞培養.........................................................................................................16 2.2.1 配製 DMEM(Dulbecco's Modified Eagle Medium)細胞培養液 .............16 2.2.2 細胞培養液配方........................................................................................17 2.2.3 細胞繼代....................................................................................................17 2.3 質體在轉染細胞中的表現...........................................................................17 2.3.1 細胞的轉染(Transfection).........................................................................17 2.3.2 RT PCR.......................................................................................................18 2.3.3 酵素連結免疫吸附法(Enzyme-linked Immuno-sorption Assay).............21 2.3.4 細胞生長曲線............................................................................................22 2.4 動物實驗.........................................................................................................22 2.4.1 動物來源....................................................................................................22 2.4.2 動物分組....................................................................................................23 2.4.3 原位腦瘤植入模型....................................................................................23 2.4.4 小鼠腦組織收成........................................................................................24 2.4.5 免疫組織化學染色(IHC) ...........................................................................24
2.4.6 流式細胞儀分析........................................................................................26 2.5 數據分析.........................................................................................................27
第三章 實驗結果 .................................................................................28 3.1 IRES-SDF-1 質體的構築與確認 .................................................................28 3.2 質體 IRES-SDF-1 DNA 在細胞 ALTS1C1 中的表現 ..........................29
3.2.1 IRES-SDF-1 質體送入 ALTS1C1 細胞 .................................................29 3.2.2 轉染細胞中基因 mRNA 的表現情形....................................................29 3.2.3 轉染細胞中蛋白質的表現情形................................................................30 3.2.4 細胞分裂增生速率測試 (Cell Doubling Time).......................................30
3.3 動物實驗.........................................................................................................31 3.3.1 Sdf-1 對 ALTS1C1 顱內腫瘤生長的影響.............................................31 3.3.2 SDF-1 蛋白分泌對腫瘤血管的影響........................................................32 3.3.3 SDF-1 蛋白分泌對 ALTS1C1 腫瘤中腫瘤相關巨噬細胞 (TAMs) 的 影響......................................................................................................................34 3.3.4 SDF-1 分泌對腫瘤缺氧區 (hypoxia area) 的影響.................................35 3.3.5 以流式細胞儀分析 SDF-1 分密對 ALTS1C1 腫瘤在不同時期腫瘤相 關巨噬細胞的比例分佈......................................................................................37
第四章 討論 ..........................................................................................40 4.1 ALTS1C1-SDF-1 可用於模擬高侵襲力的神經膠質瘤 ...........................40 4.2 SDF-1 促進 ALTS1C1 顱內腫瘤生長 ......................................................40 4.3 SDF-1 使腫瘤微血管功能更趨成熟 ..........................................................41 4.4 TAMs 在 ALTS1C1-SDF-1 模型中扮演的角色......................................42
4.5 ALTS1C1-SDF-1 模型中的 TAMs 增加腫瘤侵襲力 ..............................43
4.6 ALTS1C1-SDF-1 腫瘤的缺氧區域縮小 .....................................................45 4.7 ALTS1C1-SDF-1 腫瘤有更多 M2 巨噬細胞分佈於缺氧區.................46 4.8 TAMs 隨癌細胞生長而進入腫瘤 ...........................................................47
第五章 圖表 ..........................................................................................49 參 考 文 獻 .................................................................................................83

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