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研究生： 彭秋瑩
Peng, Chiu-Ying
論文名稱： 探討VEGFR1在口腔癌細胞內的次細胞分佈現象與其所扮演的角色
The study of vascular endothelial growth factor receptor 1 (VEGFR1) subcellular localization and its functional implication in carcinogenesis
指導教授： 吳梨華
Wu, Li-Wha
蔡森田
Tsai, Sen-Tian
學位類別： 碩士
Master
系所名稱： 醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2008
畢業學年度： 96
語文別： 英文
論文頁數： 48
中文關鍵詞： 口腔癌 、importin β1 、nuclear localization signal (NLS) 、VEGFR1 、VEGF-A
外文關鍵詞： importin β1, nuclear localization signal (NLS), VEGF-A, VEGFR1, oral cancer
相關次數： 點閱：153  下載：1 
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    • 血管內皮細胞生長因子受器第一型 (VEGFR1) 原先被認為只表現在內皮細胞、單核球細胞、巨噬細胞、造血幹細胞中，參與調控血管新生、單核球細胞/巨噬細胞與造血幹細胞的爬行到他們受吸引的來源。可是近年來的研究都證實血管內皮細胞生長因子受器第一型也表現在各型各類的癌症中，且能用它的表現量來預測癌症惡性程度與病人的癒後情況。最近研究顯示這個原本認為坐落在細胞膜上的受器，在B細胞慢性淋巴球性白血病(B-CCL)的癌細胞裡被發現在活化的情況下會被細胞吞噬進入核內，與調控基因表現的轉錄因子STAT3結合。因此，本研究目的為探討VEGFR1是如何進入核內的，還有它在癌症的進程中扮演什麼樣的角色。首先，我們利用定點突變(site-directed mutagenesis)與免疫螢光染色探討VEGFR1中一段推測的NLS是否有功能。結果我們確認這段推測的NLS中有會促使細胞質中的蛋白入核，也會與大部分會入核的大蛋白分子都需會用到的核輸入受器(importin)結合，而NLS中的第954個位置的離氨基酸更是有絕對存在的必要。只是全長VEGFR1的入核還需要其他入核訊號的幫忙。利用免疫組織染色發現在口腔癌病人的檢體中， VEGFR1布景會表現於核內，且在組織入侵的前線有表現量較高的趨勢。口腔癌細胞常會大量表現VEGF-A，可是我們發現外加的VEGF-A並不會促使VEGFR1在細胞內的次位置改變。剔除口腔癌細胞株內的VEGFR1後，會抑制癌細胞的生長與爬行能力。最後總結來說，VEGFR1確實含一段有效能的NLS，只是口腔癌細胞核中的VEGFR1有什麼功能、是否具有癒後指標意義，還必須待更進一步研究闡明。

    Vascular endothelial growth factor receptor 1 (VEGFR1) is a receptor tyrosine kinase (RTK) predominantly expressed on the plasma membrane of endothelial cells. Recent studies have shown that VEGFR1 is also expressed in several types of human cancer cells, and its expression correlates with cancer progression and predicts the outcome of cancer patients following cancer treatments. Studies in chronic B cell leukemia suggest that VEGFR1 could be internalized from cell membrane and associated with phosphorylated STAT3 in response to VEGF-A stimulation. We hypothesize that VEGFR1 may work in similar fashion like EGFR, which employs activated receptor internalization to act as a transcription mediator in nucleus. We first examined the function of a putative nuclear translocataion signal (NLS) segment within VEGFR1 by site-directed mutagenesis and immunofluorescence (IF) microscopy. Although additional sequence may be required, VEGFR1 indeed harbored a functional NLS segment, in which lysine 954 residue played a dominant role. The nuclear localization of VEGFR1 was in part mediated by its association with importin β1 (impB1), normally required for nuclear transport of proteins. Exogenous VEGF-A, frequently overexpressed in oral cancer cells, had no effect on the subcellular localization of VEGFR1. Knocking down the expression of VEGFR1 decreased the proliferation and migration abilities of oral cancer cells. Nuclear expression of VEGFR1 was also observed in the invasion front of oral cancer specimens. Together, VEGFR1 indeed harbored a functional NLS to enter cell nucleus, the exact function of nuclear VEGFR2 in oral cancer cells and the prognostic value of its expression in nuclei remains to be elucidated.

    I. 中文摘要 I II. Abstract II III. 誌謝 III IV. Table of context IV V. List of figures VI VI. Abbreviation VII VII. Introduction 1.1 VEGF-A and angiogenesis 1 1.2 The role of VEGFR1 in angiogenesis 1 1.3 VEGFR1 in cancers 2 1.4 VEGFR1 and oral cancers 3 1.5 Traditional signal transduction pathways of receptor tyrosine kinases (RTK) 4 1.6 RTK internalization 4 1.7 Mechanism of importin-mediated nuclear translocation 5 1.8 Previous studies on VEGFR1 6 VIII. Hypothesis 7 IX. Specific aims 7 X. Materials and methods 2.1 Materials 8 2.2 Primer list 9 2.3 Cell culture 10 2.4 Plasmid constructs 10 2.5 Transient transfection 11 2.6 shRNA knockdown 12 2.7 Immunofluorescence 12 2.8 Immunohistochemistry 13 2.9 Immunoblotting (Western blotting) 13 2.10 Immunoprecipitation 14 2.11 Nuclear-cytosolic fractionation 14 2.12 Proliferation 15 2.13 Wound healing 15 XI. Results 3.1 A stretch of functional NLS, located in the kinase insert domain, is responsible for VEGFR1 nuclear translocation 16 3.2 VEGFR1 utilized importin β1 (impB1) for its nuclear import. 17 3.3 Subcellular localization of VEGFR1 in NIH3T3 cells ectopically expressing VEGFR1, and different cancer cell lines 18 3.4 Additional signal might be also required for full-length VEGFR1 nuclear localization 18 3.5 VEGFR1 specific shRNA clone 632 and 633 could successfully knockdown VEGFR1 in 3T3/flt1, and OC2 but not OECM1 cells 19 3.6 VEGFR1 positively regulated proliferation and migration in oral cancers 19 3.7 VEGFR1 expression was intense at the invasive front of late-stage oral cancer patient specimens 20 XII. Discussion 21 XIII. Reference 23 XIV. Figures 28 XV. Appendix 46

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