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系統識別號 U0026-0812200913451122
論文名稱(中文) 探討VEGFR1和VEGFR2在內皮細胞和口腔癌細胞的次細胞位置
論文名稱(英文) Study of VEGFR1 and VEGFR2 subcellular localization in endothelial and oral cancer cells
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 95
學期 2
出版年 96
研究生(中文) 林奕宇
研究生(英文) Yi-Yu Lin
電子信箱 rafaellyy@gmail.com
學號 t1694103
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 指導教授-吳梨華
指導教授-周楠華
口試委員-蔣輯武
口試委員-張文粲
中文關鍵字 次細胞位置  口腔癌  血管內皮細胞生長因子 
英文關鍵字 subcellular localization  VEGF  oral cancer  VEGFR1 
學科別分類
中文摘要 血管內皮細胞生長因子A (vascular endothelial growth factor-A, VEGF-A)能經由它的兩種位於細胞表面的受體來刺激血管新生的進行,這兩種受體,分別是VEGFR1和VEGFR2,它們都是屬於具有酪氨酸激酶活性的受體 (receptor tyrosine kinase, RTK),這些受體原先是被認為只會表現在內皮細胞,但是近年來發現有越來越多的癌細胞會表現這兩種受體,並且在VEGF-A的刺激下,能增加此類癌細胞諸如入侵(invasion)、轉移(migration)以及形成新的癌細胞聚落(colony-forming)的能力。其他的RTK,例如上皮細胞生長因子受體第一型(epidermal growth factor recaptor-1, EGFR-1)在經由它的配體(ligand)活化之後,能夠進到細胞核內,扮演轉錄因子(transcription factor)的角色,進而調控特定基因的表現。另外,存在於細胞核的EGFR在乳癌的預後診斷中具有參考的價值。我們進一步的使用細胞核質分離的方式來追蹤VEGFR1和VEGFR2是否會進入細胞核,結果,我們發現在經由VEGF-A刺激過的人類臍帶內皮細胞(human unbilcal vein endothelial cells, HUVEC),VEGFR1會出現在核中,但VEGFR2卻不會,而且,在口腔癌細胞株中,即使未經過VEGF-A的刺激,VEGFR1也能在核中被偵測到,更進一步地,VEGFR1的表現和口腔癌細胞的侵襲能力是正相關的。藉由免疫沉澱(immunoprecipitation),我們也證明VEGFR1會和importinβ結合,形成聚合體,而importinβ在物質向細胞核的運輸中,扮演了重要的角色。藉著使用GFP作為報導基因( reporter gene)和點突變(site-directed mutagenesis)技術,我們辨識了一段座落在VEGFR1的kinase insert domain中,具有潛在的入核訊號( nuclear signal)的蛋白質序列。經由點突變(site-directed mutagenesis)技術,我們也辨識出再調節此段NLS功用的關鍵氨基酸。綜合以上各點,我們提供了一條新的VEGFR1的訊號傳遞路徑,並且它可能和口腔癌細胞的侵襲能力有關。至於次細胞位置(subcellular localization)VEGFR1的確切機轉,則需要更多研究來闡
明。
英文摘要 Vascular endothelial growth factor A (VEGF-A) stimulates angiogenesis and vasculogenesis via two cell surface receptors, VEGF receptor 1 (VEGFR1 or flt1) and VEGF receptor 2 (VEGFR2 or flk1/KDR). Both receptors belong to receptor tyrosine kinase (RTK). Originally they were thought to be expressed on endothelial cells only, but more and more studies showed that many kinds of cancer cells also express VEGFR1 and VEGFR2. Following stimulation with VEGF-A, the cancer cells which express VEGFR would increase invasion, migration and colony-forming ability. In addition to being a cell surface receptor, that ligand-bound epidermal growth factor receptor-1 (EGFR-1), one of RTK, could enter nucleus as a transcription factor to modulate target gene expression. Nuclear EGFR also offers a prognosis value in breast cancer. We thus examined by subcellular fractionation whether VEGFR1 or VEGFR2 could enter into nuclei. Here, we demonstrate that VEGFR1 but not VEGFR2 was localized in the nuclei after treatment with VEGF-A in HUVEC, and VEGFR1 is also observed in nuclei of oral cancer cells even in the absence of VEGF stimulation. The VEGFR1 expression was correlated to the invasion ability of oral cancer cell line. With immunoprecipitation, we also demonstrated that VEGFR1 interacted with importinβ1, which plays an important role in nuclear transport. Using GFP as a reporter gene and site-directed mutagenesis, a putative nuclear signal was identified in the kinase insert domain of VEGFR1. We also identified the critical residues for NLS function regulating by mutagenesis. Together, we offer a novel VEGFR1 signaling transduction pathway and it may correlate to the invasion ability of oral cancer. The exact function of distinct subcellular localization of VEGFR1 will be studied in the context of oral cancer cells.
論文目次 Contents

中文摘要…………………………………………………………………I
Abstract ……………………………………………………………II
Contents………………………………………………………………VIII
List of Tables……………………………………………………….X
List of Figures………………………………………………………XI
Abbreviation…………………………………………………………XII

1. Introduction………………………………………………………1
1.1 Mechanism of neovasculogenesis ………………………..1
1.2 The role of VEGF-A and its receptors in angiogenesis……….....1
1.3 VEGFR1 and VEGFR2 in cancers…………………………3
1.4 Signal transduction pathway of receptor tyrosine kinase………4
1.5 The presence of nuclear localization sequence in receptor tyrosine kinase……………………………………………5
1.6 Importin in nucleocytoplasmic transport………………………6
1.7 Subcellular localization of VEGFR1 and VEGFR2…………7
1.8 Hypothesis……………………………………………………7
1.9 The specific aim……………………………………………….8
2. Materials and Methods…………………………………………….9
2.1 Materials…………………………………………………………9
2.2 Cell culture……………………………………………………….9
2.3 Cell treatment…………………………………………………….10
2.4 Nuclear/ cytosol fraction isolation………………………………..10
2.5 Immunoprecipitation and Immunoblotting……………………….11
2.6 Plasmid Constructs………………………………………………..11
2.7 Transient transfection……………………………………………..12
2.8 Endosome inhibitor treatment…………………………………….12
2.9 Immunoflurorescence microscopy………………………………..13
3. Results………………………………………………………………....14
3.1 The presence of VEGFR1 in the nuclei of VEGF-A-treated HUVEC…………………………………………………………… 14
3.2 VEGFR1 was observed in the nuclei of some oral cancer cells without VEGF stimulation………………………………………………...…14
3.3 VEGFR1 interacts with importin β1……………………………..15
3.4 Subcellular localization of GFP-VEGFR1-IC or GFP-VEGFR2-IC
in non-EC……………………………………………………………16
3.5 A putative NLS is conserved in the kinase insert domain of VEGFR1 during evolution………………………………………………….17
3.6 VEGFR1 harbor a functional NLS…………………………………17
3.7 Lysine residues in the putative NLS are critical for the subcellular localization of VEGFR1-GFP fusion protein………………………..18
3.8 The nuclear transport of GFP-VEGFR1 recombinant protein is cell
type specific ………..…………………………………………..…….19
3.9 VEGFR1 expression was correlated to the invasion ability of OECM1
of certain oral cancer cell line…….…………………………………20
4. Discussion…………………………………………………………….….21
5. Reference…………………………………………………….…………..25
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