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研究生:閻思尹
研究生(外文):Ssu-Yin Yen
論文名稱:以EZH2和AXL-1為標的基因探討藥物EF-001在人類腦膠質腫瘤特性、生長與其轉移和侵入之調控機制
論文名稱(外文):Investigating the mechanism of human glioma cells proliferation, migration and invasion by targeting EZH2 and AXL-1
指導教授:邱紫文邱紫文引用關係
指導教授(外文):Tzyy-Wen Chiou
學位類別:博士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
論文頁數:83
中文關鍵詞:多型性神經膠母細胞瘤CD133+細胞EZH2Axl遷移侵襲
外文關鍵詞:glioblastomaCD133+ cellsAxl receptor tyrosine kinaseenhancer of zeste homolog 2migrationinvasion
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多形性膠質母細胞瘤是最常見和最具侵略性的惡性腦瘤。由於高復發率和預後性差,患者整體的存活期約為12-15個月。研究指出,Axl酪胺酸激酶在許多癌症中大量表達,並在腫瘤的生長、血管新生、轉移與侵襲等病理過程中扮演重要角色。在先前研究中已發現EF-001在惡性腦瘤細胞可明顯抑制Axl酪胺酸激酶的表現,並且透過Axl酪胺酸激酶調控,進而抑制惡性腦瘤細胞增生、遷移與侵襲的效果。在本論文研究中,進一步探討並發現EF-001透過Axl酪胺酸激酶而抑制惡性腦瘤細胞進行上皮細胞間質轉化,並且證明EF-001是經由ERK1/2訊息傳遞路徑來調控Axl酪胺酸激酶。此外,在動物實驗方面,利用兩種動物模式(皮下腫瘤模式與原位腦瘤模式)分別證明經由皮下注射給予EF-001或經由藥物控制釋放裝置(EF-001 wafer)局部給藥皆可有效抑制腫瘤生長並延長動物存活期。同時組織切片結果顯示,隨著治療藥物濃度提高,Axl酪胺酸激酶受體表現量明顯被抑制,並且有效抑制惡性腦瘤細胞的遷移與侵襲能力。然而大多數研究學者認為造成腫瘤復發的原因可能是因腫瘤幹細胞的存在,其具有高侵襲性以及對於化學藥物治療、放射線治療具有抗性。因此,在本研究中進一步評估小分子EF-001抗惡性腦瘤CD133+細胞生長的潛力,並評估其抑制惡性腦瘤CD133+細胞遷移與侵襲能力,探討影響遷移與侵襲相關基因調控。在研究中發現EF-001可具有抑制惡性腦瘤CD133+細胞生長能力、群落形成能力,並使細胞停滯於G2/M期進而誘導細胞凋亡之反應。我們進一步發現,EF-001可抑制惡性腦瘤CD133+細胞腫瘤幹細胞相關基因,包括CD133、EZH2、Sox2及Oct4表現降低,其與腫瘤幹細胞的存活、生長、特性及細胞群落形成能力有關。此外,腫瘤細胞進行上皮-間質轉化,會使腫瘤幹細胞幹性相關基因表現的增加,使得腫瘤幹細胞的活化。在本研究結果也發現,EF-001可抑制惡性腦瘤CD133+細胞的上皮-間質轉化,並且抑制細胞遷移與侵襲相關的基因Axl酪胺酸激酶。在體外細胞遷移模式結果顯示,隨著 EF-001 藥物濃度提高,可抑制細胞遷移,並抑制可降解細胞外基質的水解酶的基質金屬蛋白酶2的表現。最後進一步利用質體轉染,探討Axl、EZH2及Sox2在惡性腦瘤CD133+細胞的轉移與侵入機制中的角色。由實驗結果發現,Axl及EZH2可能是為EF-001調控惡性腦瘤CD133+細胞遷移與侵襲的重要關鍵基因,此調控機制,可作為未來開發EF-001對抗惡性腦瘤的臨床應用之基礎。
Glioblastoma (GBM) is the most common and most aggressive brain malignant tumor. Due to the high recurrence rate and poor prognosis of GBM, the patients’ median survival rate is 12-15 months. Most researchers suggest that the recurrence is due to the presence of cancer stem cells (CSCs), which has radioresistant, chemoresistant, highly invasive, self-renewal and tumorigenic potentials. Axl receptor tyrosine kinase has been shown to be linked to various high-grade cancers, and Axl is also correlated with tumor growth, angiogenesis, and tumor cell invasion. In our previous studies, we demonstrated that EF-001 could inhibit GBM cell migration and invasion in vitro. In this study, it showed that EF-001 not only inhibited GBM tumor growth, prolonged the survival rate in an orthotopic tumor model, but inhibited tumor cell invasion in vivo also. Furthermore, we used EF-001 to assess the anti CD133+ cell potential, and then tried to find out the associated gene regulation in invasion and migration. Our results indicated that EF-001 could induce CD133+ cell apoptosis through G2 / M phase arrest, and reduce the colony formation ability of CD133+ cell. Moreover, we found that EF-001 could inhibit the expression of CD133+ cells associated genes, including CD133 and enhancer of zeste homolog 2 (EZH2), which are involved in CSCs maintenance, growth and colony formation ability. Also, EF-001 reduced the stemness related genes of CD133+ cells as well. In addition, EF-001 inhibited the expression of Axl in a dose dependent manner and reduced the epithelial-to-mesenchymal transition (EMT), migratory and invasive capabilities of GBM CD133+ cells. EMT is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression. When tumor cells are under EMT process, it will increase the CSCs stemness related gene expression and induce CSCs activation. Finally, we used the overexpression of Axl, EZH2 and Sox2 in GBM CD133+ cells to prove that Axl and EZH2 genes are crucial target in the inhibition of GBM CD133+ cells EMT, migration, and invasion. We found that Axl/EZH2, not Sox2, might be the key regulator in tumor invasion, migration and EMT. The results of this study may contribute to the development of EF-001 for clinical applications in the future.
摘要.............................................................i
Abstract.......................................................iii
目錄.............................................................v
表目錄..........................................................vii
圖目錄.........................................................viii
前言..............................................................1
研究背景..........................................................3
惡性腦瘤(Malignant brain tumor)...................................3
多形性膠質母細胞瘤(Glioblastoma, GBM)..............................3
腦瘤治療現況與困境.................................................4
癌症幹細胞........................................................6
惡性腦瘤CD133+細胞................................................8
CD133/Prominin-1.................................................9
EF-001...........................................................9
EZH2 (enhancer of zeste homolog 2)..............................10
Axl酪胺酸激酶受體................................................11
材料與方法.......................................................15
抗體(Antibodies)種類與來源.......................................15
細胞來源與培養...................................................15
流式細胞儀分析...................................................16
體外細胞毒性測試.................................................16
細胞週期分析.....................................................17
細胞轉染.........................................................17
細胞 total RNA 之抽取............................................18
反轉錄聚合酶連鎖反應(RT-PCR)與膠體分析.............................18
全細胞之蛋白質抽取及蛋白質濃度測定.................................19
西方墨點分析法...................................................19
體外細胞遷移性試驗(Wound healing assay)..........................20
體外細胞侵入性試驗...............................................20
聚酸酐高分子製備.................................................21
動物實驗........................................................21
(i) Xenograft model............................................21
(ii) Orthotic model............................................21
免疫組織染色分析................................................22
統計分析.......................................................23
結果...........................................................25
Part I:探討EF-001對腦瘤細胞之生長、遷移與侵襲機制之分析...........25
EF-001抑制人類惡性腦瘤細胞Axl表現................................25
EF-001經由Axl的調控影響惡性腦瘤細胞的上皮-間質轉換.................25
EF-001經由ERK訊息傳遞路徑調控Axl.................................27
動物實驗-皮下腫瘤動物模式及原位腦瘤動物模式........................28
EF-001可抑制皮下腫瘤生長並降低Axl和MMP2的表現.....................28
EF-001可延長大鼠存活期並抑制腫瘤細胞遷徙與侵襲.....................29
Part II:探討EF-001對腦瘤CD133+細胞之生長、遷移與侵襲機制之分析....30
惡性腦瘤CD133+細胞之特性分析.....................................30
EF-001經由G2/M抑制腦瘤CD133+細胞1XM生長..........................32
EF-001抑制癌症幹細胞相關基因表現.................................33
EF-001透過Axl影響腦瘤CD133+細胞遷徙與侵襲........................34
EF-001透過Axl/EZH2調控腦瘤CD133+細胞遷徙與上皮間質轉換............35
討論...........................................................37
結論與未來展望..................................................41
圖與表.........................................................43
參考文獻.......................................................75
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陳雪容,中草藥萃取物抑制Axl酪胺酸激酶受體與對抗惡性腦瘤遷移與侵入之研究,2009,國立東華大學生物技術研究所,碩士論文。
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