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系統識別號 U0026-2008201319593900
論文名稱(中文) 離胺基氧化酶透過膠原蛋白/整合素相關聯的訊號對細胞硬性及移動的調控
論文名稱(英文) Lysyl oxidase modulates cell stiffness and migration through collagen/integrin-associated signaling
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 101
學期 2
出版年 102
研究生(中文) 呂瑜珍
研究生(英文) Yu-Jhen Lyu
學號 S46004103
學位類別 碩士
語文別 英文
論文頁數 52頁
口試委員 指導教授-楊倍昌
口試委員-蔡曜聲
口試委員-王仰高
中文關鍵字 離胺基氧化酶  細胞移動  細胞硬度 
英文關鍵字 lysyl oxidase  cell migration  cell stiffness 
學科別分類
中文摘要 細胞周圍的微環境主要是由各種間質細胞和胞外基質所組成,他們在調控細胞上扮演重要角色。過去的研究發現腫瘤的微環境與一般組織有很大的不同,例如過多的間質細胞和胞外基質累積,甚至比一般組織來的硬;而這些改變都能促進癌細胞的存活與轉移。離胺基氧化酶(lysyl oxidase, LOX)為一種胞外基質的調控者,藉由氧化膠原蛋白與彈性纖維,促使他們產生交聯(cross-linking)反應,以維持胞外基質的硬性及結構的穩定性。在許多腫瘤中發現離胺基氧化酶有過度表現的情況;文獻中也指出離胺基氧化酶所導致的過多膠原蛋白交聯會使膠原蛋白的受體-整合素(integrin)過度活化,進而影響下游蛋白的表現,促進癌細胞的惡化。為進一步探討離胺基氧化酶在癌化細胞中所扮演的角色,在本篇研究中,我們建立了離胺基氧化酶過度表現的腦癌細胞株 (U118-MG)。實驗發現離胺基氧化酶能夠促進U118-MG單細胞遷移及傷口癒合的能力;同時過度表現離胺基氧化酶U118-MG的培養液 (LOX/CM) 也能有效提高MCF7及HepG2的傷口癒合的速度。且這些現象在加入了β-amino-propionitrile (離胺基氧化酶抑制劑)後,就能夠抑制。原子力學顯微鏡測試也發現過度表現離胺基氧化酶U118-MG及經過LOX/CM處理的細胞都具有較高的細胞硬度;但利用攜帶膠原蛋白靜默RNA之慢病毒轉染降低U118-MG的膠原蛋白表現後,就能抑制離胺基氧化酶的作用。這些結果顯示膠原蛋白的交聯在細胞硬度上扮演一個重要的角色。我們近一步發現利用抗體使整合素失去作用後,也能有效阻止離胺基氧化酶所導致的細胞硬度上升現象。離胺基氧化酶也會去促進整合素下游蛋白的磷酸化,例如FAK、SRC、ERK;相同的,此種活化情形在抑制膠原蛋白及整合素的情況下就不會發生。綜合以上的結果顯示離胺基氧化酶可能藉由調控膠原蛋白/整合素相關的訊息路徑去促進細胞的移動及硬度。我們在未來會近一步去探討離胺基氧化酶對基質修飾後所導致的整合素訊息傳遞在腫瘤生長上的影響。
英文摘要 The microenvironment surrounding the cancer cells is composed of stromal cells and a variety of extracellular matrix (ECM) proteins. Increases in stromal cells and ECM deposition promote tumor survive and invasion. Besides, tumor nodules are stiffer than normal tissues. Lysyl oxidase (LOX) is a copper-dependent enzyme, and catalyzes the cross linkage of collagen and elastin, which contributes to matrix stiffness. Crosslinking of collagen results in integrin activation and new proteins expression, which are positively correlated with tumor malignancy. Elevated expression of LOX has been detected in many types of tumors. To study the molecular mechanism underlying the tumor promoting activity of LOX, LOX gene has been ectopically expressed in U118-MG (human glioma cells). LOX-overexpression significantly increased single cell migration and wound healing ability. Besides, 48-h conditioned media from LOX-overexpression U118-MG cells (LOX/CM) also accelerated the polarized migration of MCF7 and HepG2. Treating cells with β-amino-propionitrile, an irreversible LOX antagonist, effectively diminished this augmented cell motility in tumor cells tested. A higher level of cell stiffness was observed in LOX-overexpression cells and LOX/CM-treated cells detected by atomic force microscopy as compared with those of their respective control groups. Down regulation of collagen IV by lentiviral-mediated transduction inhibited the LOX-induced stiffness elevation of U118-MG indicating that collage crosslinking played a role in cell stiffness. Blockage of integrin, the major receptor of collagen, averted the LOX-associated change of stiffness in U118-MG, MCF7, and HepG2. In addition, LOX-overexpression and LOX/CM-treatment induced the activation of several downstream kinases of integrin signaling pathway, such as FAK (Y397, Y861), Src (Tyr416), and Erk, but the induction was not found in collagen-deficient U118-MG and anti-1-treated cells. In summary, our study suggested that LOX increased the cell migration and stiffness by stimulating the collagen/integrin signal pathway. In the future, we will analyze the contribution of integrin signal transduction initiated by LOX-dependent ECM crosslinking to tumorigenesis.



論文目次 考試合格證明.........................................I

中文摘要............................................II

Abstract...........................................III

Acknowledgement....................................V

Contents...........................................VII

Figure index.......................................IX

Introduction.......................................1

Tumor-associated microenvironment..................1

Alteration of ECM in tumor progression.............1

The role of lysyl oxidase (LOX) in cell stiffness and migration..............2

Rationale of this study............................3

Materials and Methods..............................4

Materials..........................................4

Media and Buffer preparation.......................8

Methods............................................13

Mini plasmid isolation.............................13

Transformation.....................................14

Cell culture.......................................15

Transfection.......................................15

Reverse-transcription polymerase chain reaction (RT-PCR)............16

Western blot.......................................17

Cell proliferation assay...........................18

Single cell motility assay.........................19

In vitro wound healing assay.......................19

Stiffness analysis.................................19

LOX activity.......................................20

Results ...........................................21

Overexpression of LOX gene in human glioma cells............21

Overexpression of LOX gene increased the cell motility of U118-MG cells............21

Conditioned medium of U118-LOX (LOX/CM) enhanced cell migration of various cancer cells............22

LOX increased cells stiffness through collagen/integrin-associated pathway............23

LOX induced the phosphorylation of FAK, SRC and ERK...................24

Discussion.....................26

LOX in cancer cells migration.......................26

The role of LOX in cell stiffness...................27

References..........................................29

Figures and figure legends..........................33


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