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系統識別號 U0026-1908201512083900
論文名稱(中文) 口腔癌前病變相關纖維母細胞的外胞體對癌進程的影響
論文名稱(英文) Role of Exosomes from Oral Precancer Associated Fibroblasts in Oral Cancer Progression
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 103
學期 2
出版年 104
研究生(中文) 張雅涵
研究生(英文) Ya-Han Chang
學號 T46021033
學位類別 碩士
語文別 中文
論文頁數 78頁
口試委員 指導教授-陳玉玲
指導教授-王東堯
召集委員-洪澤民
口試委員-洪建中
中文關鍵字 外胞體  Galectin-1蛋白  口腔癌前病變之纖維母細胞 
英文關鍵字 exosomes  Galectin-1  pre-cancer associated fibroblasts 
學科別分類
中文摘要 口腔癌在台灣的發生率與死亡率逐年上升,目前是台灣男性十大癌症死亡率排名第四位。腫瘤的組成除了癌細胞外還含有許多腫瘤微環境相關細胞皆對腫瘤進程有影響,先前研究已知腫瘤相關的纖維母細胞會影響癌細胞的幹細胞特性、移行、與轉移,我們實驗室也證實腫瘤相關纖維母細胞的Galectin-1表現量增加會促進口腔癌轉移。口腔癌發生的過程中經常伴隨癌前病變的產生,而口腔癌前病變中的纖維母細胞是否參與癌變機制及其如何調控癌變進程則目前甚少研究。外胞體(exosome)是奈米大小的細胞衍生小泡,帶有蛋白質,RNA,線粒體DNA和大片段的DNA,在腫瘤微環境中扮演重要訊息溝通的角色,但外胞體在癌前病變過程的探討極為有限,而galectin-1是否透過外胞體影響癌轉移也未知。在本研究,我們分離與培養來自相同口腔癌病患正常、癌前病變、及癌組織中的纖維母細胞,發現癌前病變相關纖維母細胞比正常的牙齦纖維母細胞生長速度較快且較為活化,我們也發現癌前病變纖維母細胞的條件培養基(conditioned media)有較強的口腔癌細胞移行的誘導功能,純化各種纖維母細胞所分泌出的外胞體在電子顯微鏡下觀察,發現癌前病變及癌相關纖維母細胞的外胞體體積較大,而在癌相關纖維母細胞中的Galectin-1含量較多,且Galectin-1會藉由外胞體的包覆而進入到口腔癌細胞中,進而影響口腔癌細胞移行的能力。綜合以上結果,癌前病變以及癌相關纖維母細胞所釋放至微環境的因子以及外胞體傳送的機制在口腔癌的進程中扮演著重要的角色,透過了解口腔癌癌前病變微環境對口腔癌的影響,期望未來可以做為口腔癌診斷及治療標靶之參考。
英文摘要 In our previous study, we isolated and cultured several fibroblast sets from oral cancer patients’ normal, pre-cancer, cancer tissues. We found proliferation rate and α-SMA expression level, a marker of activated fibroblasts, were higher in pre-cancer associated fibroblasts than in normal fibroblasts. The conditioned media from pre-cancer associated fibroblasts have higher cancer cell migration promoting abilities than those from normal fibroblasts. Moreover, we purified exosomes from the conditioned media of fibroblasts and found the size of exosomes derived from cancer- or precancer-associated fibroblasts is larger than that from normal fibroblasts. We also found that the levels of galectin-1 in the exosomes from cancer-associated fibroblasts are higher, and galectin-1 can be package by exosomes and delivered into oral cancer cells, thereby affecting the cell function. Taken together, pre-cancer-associated fibroblasts can increase cancer cell migration. Exosomes from pre-cancer- and cancer-associated fibroblasts are very important in oral cancer progression. Galectin-1 can be packaged and delivered by exosomes of cancer-associated fibroblasts to regulate oral cancer development. Through understanding of the impact of oral precancerous fibroblasts in oral cancer microenvironment, we hope new prognosis markers and therapeutic targets in more early stage of oral cancer could be discovered.
Key Words: exosomes、Galectin-1、pre-cancer associated fibroblasts
論文目次 中文摘要 I
英文延伸摘要(EXTENDED ABSTRACT) II
誌謝 VI
目錄 VIII
英文縮寫檢索表 XIV
緒 論 1
一、口腔癌(oral cancer) 1
二、口腔癌前病變(oral pre-cancer) 2
三、腫瘤微環境(Tumor microenvironment) 3
四、腫瘤相關纖維母細胞(Cancer associated fibroblast ) 4
五、外胞體(Exosomes) 5
六、Galectin-1蛋白(Galectin-1) 6
七、Galectin-1蛋白與腫瘤相關纖維母細胞 7
研究動機 9
材料與方法 10
一、細胞培養 Cell culture 10
1-1初代培養 10
1-2繼代培養細胞 10
1-3冷凍保存細胞 11
1-4解凍細胞 12
1-5細胞計數 12
二、細胞蛋白質表現分析(Protein expression assay) 13
2-1細胞蛋白質樣本的製備 13
2-2蛋白質定量 13
2-3西方墨點法 14
2-3-3轉印(Eletrotransfer) 16
2-3-4免疫轉漬法(Immunobloting) 17
三、細胞內RNA表現分析(RNA expression assay) 18
3-1 RNA萃取(RNA extraction ) 18
3-2 RNA定量(RNA quantitative) 18
3-3反轉錄酶反映(Reverse transcription) 19
3-4聚合錄酶連鎖反應(Polymerase Chain Reaction,PCR) 20
3-5洋菜膠體電泳分析(Agarose gel electrophoresis) 21
3-6即時定量PCR(qRT-PCR) 21
四、細胞增殖分析(Cell proliferation assay) 22
五、細胞移行能力分析(Cell migration assay) 23
六、細胞群落分析(Cell colony formation) 24
七、上皮間質型的轉換(Epithelial-mesenchymal transition) 24
八、細胞螢光染色(Immunofluorescence) 25
九、免疫組織化學染色(Immunohistochemistry) 26
十、細胞激素蛋白質晶片分析(Proteome ProfilerTM Human Cytokine Array Panel A Array Kit) 28
十一、外胞體純化(Exosomes purification) 29
十二、穿透式電子顯微鏡拍攝(Transmission Electron Microscopy) 31
十三、抑制外胞體內吞機制(Inhibition of exosomes endocytosis mechanism) 31
十四、病毒感染(virus infection) 32
十五、轉染(Transfection) 33
十六、材料 34
16-1抗體 34
16-2 Primer 35
16-3 shRNAs 35
16-4 Plasmid 35
十七、儀器 36
十八、統計分析 37
實驗結果 38
一、人類纖維母細胞的初代培養 38
二、肌纖維母細胞指標蛋白α-Smooth muscle actin(α-SMA)與纖維連接蛋白fibronectin在OSF表現量會較HGF高 38
三、OSF conditioned medium(CM)比起HGF conditioned medium(CM)更能夠有效促進口腔癌細胞移行的能力 39
四、OSF conditioned medium(CM)比起HGF conditioned medium(CM)更能夠有效促進口腔癌細胞形成細胞群落 39
五、Conditioned medium(CM)不影響口腔癌細胞上皮變間質型的轉換 40
六、Cytokines array證明HGF、OSF與CAF conditioned medium內的cytokines的表現會有所差異 40
七、.來自不同纖維母細胞conditioned medium(CM) 的外胞體(exosomes)型態有所差異 41
八、纖維母細胞的exosomes可以進入口腔癌細胞內並且影響口腔癌細胞的功能 41
九、口腔癌細胞OEC-M1藉由內吞的機制使exosomes進入細胞內進而影響細胞功能 42
十、由CAF所分離出來的exosomes中帶有Galectin-1的表現 43
十一、CAF中的Galectin-1會藉由exosomes的包覆傳送至口腔癌細胞中 44
十二、將CAF中的Galectin-1 knockdown後會使周邊口腔癌細胞的移行能力受到抑制 44
討論 46
結論 51
參考文獻 52
圖表 57
表一、口腔組織檢體的來源及臨床病理資料 57
圖一、人類纖維母細胞的初代培養 58
圖二、來自口腔癌不同進程階段手術檢體所培養出來的纖維母細胞的生長速度 59
圖三、肌纖維母細胞指標蛋白α-Smooth muscle actin(α-SMA)與纖維連接蛋白fibronectin在OSF的表現量比HGF高 60
圖四、利用免疫組織化學染色分析肌纖維母細胞指標蛋白α-Smooth muscle actin(α-SMA) 在臨床口腔癌進程中病人檢體的表現 61
圖五、口腔癌相關纖維母細胞的conditioned medium能夠增加誘導口腔癌細胞OEC-M1的移行能力 62
圖六、口腔癌相關纖維母細胞的conditioned medium能夠增加誘導口腔癌細胞OECM-1形成細胞群落 63
圖七、觀察conditioned medium(CM)對口腔癌細胞上皮變間質型轉換的影響 64
圖八、Cytokines array分析HGF與OSF conditioned medium內cytokine表現的差異 65
圖九、觀察由纖維母細胞conditioned medium(CM)分離出來的exosomes 66
圖十、西方墨點法分析Flottlin-1在exosomes上的表現量 67
圖十一、纖維母細胞的exosomes可被內吞入口腔癌細胞內 68
圖十二、纖維母細胞的exosomes對口腔癌細胞移行能力的影響 69
圖十三、抑制口腔癌細胞OEC-M1的內吞機制會對exosomes進入細胞內造成影響 70
圖十四、抑制口腔癌細胞OEC-M1的內吞機制會影響exosomes誘導的細胞移行 71
圖十五、由CAF所分離出來的exosomes中帶有Galectin-1的表現 72
圖十六、CAF中的Galectin-1會藉由exosomes的包覆傳送至口腔癌細胞 73
圖十七、將CAF中的Galectin-1 knockdown後不影響CAF釋放exosomes的數量 74
圖十八、將CAF中的Galectin-1 knockdown後不影響exosomes進入口腔癌細胞的數量 75
圖十九、將CAF中的Galectin-1 knockdown後exosomes進入口腔癌細胞會抑制細胞的移行能力 76
圖目錄 77
附圖一 77
附圖二 78
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