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系統識別號 U0026-1707201623484600
論文名稱(中文) 探討介白素八在建立口腔癌幹細胞微環境的角色
論文名稱(英文) The Roles of Interleukin-8 in the Establishment of Oral Cancer Stem Cell Niche
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 104
學期 2
出版年 105
研究生(中文) 翁睿貝
研究生(英文) Jui-Pei Weng
學號 S16021028
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 指導教授-袁國
召集委員-陳玉玲
口試委員-王仰高
中文關鍵字 介白素八  腫瘤相關纖維母細胞  口腔癌幹細胞微環境 
英文關鍵字 Interleukin-8  Cancer-associated fibroblasts  Oral CSC niche 
學科別分類
中文摘要 口腔鱗狀細胞癌(OSCC)是台灣癌症發生率的第五位,近年來發生率與死亡率逐年攀升,並且趨於年輕化。儘管診斷與治療已有一些改進,病患仍因為復發和轉移,導致不良預後和低存活率。癌幹細胞 (CSCs)是癌細胞中占少數的次細胞群,具有幹細胞的特性,包括自我更新與多樣分化的能力,被認為可能是導致抗藥性的原因。此外,CSCs常位於靠近腫瘤血管的浸潤前緣,此區域稱為血管周圍環境。最近研究表示腫瘤微環境藉由自分泌或旁分泌的訊息助於CSCs形成和腫瘤血管生成。因此,本篇研究目的是探討口腔癌細胞和基質細胞之間的相互作用如何促進癌症的發展。首先,我們利用直接與間接接觸共培養舌鱗狀細胞癌株(SAS)與不同的基質細胞,發現SAS與人類正常的齒齦纖維母細胞 (HGF)共培養下會大量增加表現介白素八 (IL-8)。我們進行細胞分選發現SAS與HGF的IL-8表現皆顯著上升,且HGF的IL-8表現更為強烈。在異種移植的實驗,抑制IL-8表現會影響腫瘤形成的體積。我們也發現IL-8在人類口腔癌樣本中有高量表現。免疫組織化學染色的結果顯示:腫瘤組織的微血管和嗜中性白血球的密度高於正常組織。另外,SAS與HUVEC共培養下,促使SAS表現CD44與OCT4兩種重要的幹細胞標記。總而言之,我們的結果暗示口腔癌細胞和纖維母細胞的相互作用,誘導纖維母細胞分泌促血管生成與發炎的因子IL-8,吸引其他的基質細胞至腫瘤組織,創建更複雜的微環境以利於CSCs形成。這些結果指出在利於CSCs的微環境中,腫瘤相關纖維母細胞扮演重要的角色。
英文摘要 Oral squamous cell carcinoma (OSCC) is the fifth most common cancer in Taiwan. Emerging evidences show that cancer stem cells (CSCs) have resistance to chemoradiation therapy and lead to tumor relapse. Moreover, there are studies suggesting that tumor microenvironment contributes to supporting CSCs and tumor vasculature. The aim of this study was to investigate how interaction between oral cancer cells and stromal cells promote cancer progression. We performed the direct and indirect contact co-culture model of SAS cells and various stromal cells. We observed that fibroblasts predominantly and strongly expressed interleukin-8 (IL-8) through contact with SAS cells. We also found that IL-8 was highly expressed in human oral cancer specimens. Immunohistochemistry analysis demonstrated that tumor tissues had higher density of microvessel and neutrophils than normal tissues. Additionally, endothelial cells up-regulated the expression of CSC-associated genes in SAS cells. Taken together, our results implied that interaction between oral cancer cells and fibroblasts may play an important role in creating the oral CSC niche via IL-8.
論文目次 中文摘要 I
Abstract II
誌謝 VI
圖目錄 IX
縮寫檢索表 X
第一章 緒論 1
一、口腔癌概論 1
二、癌症幹細胞 (Cancer stem cell, CSC) 2
三、腫瘤微環境 (Tumor microenvironment) 3
四、血管新生(Angiogenesis) 6
五、介白素八 (Interleukin-8) 7
六、研究動機 9
第二章 材料與方法 10
一、材料 10
二、方法 16
(一)細胞培養 16
(二)磁性細胞分選 (Magnetic-activated Cell Sorting, MACS) 20
(三)RNA 萃取 21
(四)反轉錄聚合酶連鎖反應 (Reverse Transcription PCR, RT-PCR) 23
(五)即時定量聚合酶連鎖反應 (Real-time Quantitative PCR, q-PCR) 24
(六)酵素連結免疫吸附分析法 (Enzyme-linked Immunosorbent Assay, ELISA) 25
(七)免疫組織化學染色 (Immunohistochemistry, IHC) 26
(八)蛋白質萃取 28
(九)蛋白質濃度測定 29
(十)SDS-PAGE蛋白質電泳 (SDS-PAGE Protein Electrophoresis) 30
(十一)西方墨點法 (Western blot, WB) 32
(十二)質體製備 33
(十三)建立穩定細胞株 34
(十四)動物實驗之腫瘤生長分析 36
(十五)統計方法 36
第三章 結果 37
一、血管新生因子在口腔癌細胞與不同間質細胞直接接觸共培養的表現 37
二、血管新生因子在口腔癌細胞與不同間質細胞間接接觸共培養的表現 37
三、比較直接接觸共培養的條件培養液中的介白素八 38
四、分析表現IL-8的細胞來源 38
五、癌幹細胞標記在口腔癌細胞與纖維母細胞或內皮細胞直接接觸共培養的表現 39
六、IL-8在影響生物體形成腫瘤的能力 39
七、IL-8在小鼠腫瘤組織的功能 40
八、IL-8在人類口腔癌與正常齒齦組織的表現和功能 41
九、EphA4參與促進IL-8表現的機制 41
第四章 討論 43
第五章 結論 49
參考文獻 50
附圖 57


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