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系統識別號 U0026-2901201921483200
論文名稱(中文) 探討EMP1在泌尿上皮癌化療藥物抗性中所扮演的角色
論文名稱(英文) The Potential Role of Epithelial Membrane Protein 1 in Chemoresistance of Urothelial Carcinoma
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 107
學期 1
出版年 108
研究生(中文) 盧昱融
研究生(英文) Yu-Jung Lu
電子信箱 waxdwalf@hotmail.com
學號 T16031040
學位類別 碩士
語文別 英文
論文頁數 31頁
口試委員 指導教授-周楠華
口試委員-蔡育賢
口試委員-蔡弘文
中文關鍵字 泌尿上皮癌  化學抗性  EMP1基因  表皮生長因子受體  細胞週期蛋白D1 
英文關鍵字 Urothelial carcinoma  chemoresistance  EMP1  EGFR  cyclin D1 
學科別分類
中文摘要 在台灣男性癌症排名中,膀胱癌名列第九。而其中百分之九十以上的膀胱癌屬於泌尿道上皮癌。現行對於UC的治療包含手術切除、放射療法和化療。於初期的泌尿道上皮細胞癌化療中,CG結合使用為一線藥物,而EGFR抑制劑為標靶治療中的主流藥物。然而癌細胞對於化療藥物使用所產生的抗藥性為一大治療障礙,且其機制目前尚未明瞭。EGFR家族的信息傳遞途徑活化已被發現和泌尿道上皮癌所產生的抗藥性有關。因此,找尋和抗藥性的生物標記基因,對於有效治療泌尿道上皮癌會有甚大助益。EMP1,是一種位於細胞膜上的穿膜蛋白,生理上參與細胞間的緊密連結。先前報導指出,EMP1在非小細胞肺癌的表達量,與gefitinib的抗藥性呈現相關性。因此本研究要探討EMP1是否參與膀胱癌治療用的一線化療藥物之抗藥性,以及其可能機制。我們利用NTUB1細胞株建立穩定表達EMP1蛋白的細胞株,以及用TSGH8301細胞株建立EMP1蛋白表達下降的細胞株。並觀察EMP1對是否影響上述細胞株對常用一線化療藥物 (如 cisplatin, doxorubicin, methotrexate, gemcitabine, vincristine, sorafenib or gefitinib)的敏感性。儘管EGFR/ERK相關的信息傳遞途徑被抑制,我們發現EMP1蛋白表達,對於sorafenib 或gefitinib的敏感性並無顯著變化。在EMP1蛋白表達,對於cisplatin和doxorubicin的敏感性增加,反之對methotrexate和gemcitabine的敏感性下降。為了解釋這個反向效果,我們測試了會導致抗藥性產生的蛋白表達量,發現在施與cisplatin後,cisplatin抗性蛋白ERCC1在EMP1蛋白表達穩定上升細胞株中的表達量會顯著下降。而在施予gemcitabine或methotrexate後,對gemcitabine抗性蛋白RRM1與methotrexate抗性蛋白DHFR的表達量都會顯著上升。為了測試EMP1在已產生抗藥性的細胞株中是否有相同的效果,我們在cisplatin抗藥細胞株NP14以及gemcitabine抗藥細胞株NG1.5這兩株細胞株中,建立穩定表達EMP1蛋白的細胞株。上述兩種細胞在分別施予cisplatin或gemcitabine後,EMP1蛋白穩定表達對於cisplatin的敏感性上升,反之對gemcitabine的敏感性下降。這說明了EMP1穩定上升表達,不論是在一般泌尿道上皮細胞癌抑或對cisplatin或gemcitabine已經產生抗藥的細胞株中,皆會提高對cisplatin的敏感性。本篇研究發現了EMP1對膀胱癌抗藥性的影響以及其分子機制,對於未來泌尿道上皮癌的治療提供了新的研究方向。
英文摘要 Bladder cancer is the ninth important male cancer in Taiwan. More than 90% of bladder cancer is urothelial carcinoma (UC). Currently, standard treatment for UC includes surgery, radiotherapy and chemotherapy. Combination of gemcitabine and cisplatin is the first-line chemotherapy for advanced UC patients, and EGFR inhibitor represents the mainstream of targeted therapy. However, resistance to chemotherapy or targeted therapy is a major obstacle to treatment; but the underlying mechanisms are still unclear. EGFR family related signal pathways have been demonstrated to involve in chemoresistance of UC. Therefore, discovery of biomarker(s) associated with chemoresistance is imperative in the design of treatment of UC patients. EMP1, a transmembrane glycoprotein, was recently reported to correlate with gefitinib resistance in non-small cell lung cancer. This study was designed to investigate the potential of EMP1 involved in chemoresistance of UC and its underlying mechanism(s). EMP1 overexpression and knockdown stable cell lines were first established from NTUB1 cells and TSGH 8301 cells, respectively. Then, sensitivity to cisplatin, doxorubicin, methotrexate, gemcitabine, vincristine, sorafenib or gefitinib treatment was measured in vitro. EMP1 overexpression enhances the sensitivity to cisplatin and doxorubicin, but suppresses the sensitivity to methotrexate and gemcitabine. When EGFR/ERK pathway was inhibited, EMP1 overexpression stable cells cell showed no significant difference of sensitivity to sorafenib or gefitinib treatment. To explain for the dichotomous effect, additional drug resistance related pathways were analyzed in vitro. The ERCC1, biomarker of cisplatin resistance, is significantly down-regulated after cisplatin treatment. In contract, DHFR and RRM1, biomarker of methorexate and gemcitabine resistance, are significantly up-regulated after treatment with methorexate or gemcitabine. Then, effects of EMP1 expression on native or resistant cell lines with/without targeted therapy agent were examined in vitro. EMP1 overexpression stable cell line was established from NP14 (cisplatin resistant) and NG1.5 (gemcitabine resistant) cells, respectively. We showed that EMP1 enhances the sensitivity of UC cells to cisplatin, but suppresses the sensitivity to gemcitabine in vitro. The results of our experiments suggest that EMP1 may have potential as a biomarker in the design of treatment planning for UC patients and modulating EMP1 deserves further investigation as a novel chemosensitizer of cisplatin. Together, we have identified a new avenue to contest chemoresistance in human UC patients.
論文目次 Contents
Abstract in English I
Abstract in Chinese III
Contents V
Index of Figures IX
Introduction
-Urothelial carcinoma………………………………………………..…1
- Chemotherapy………………..………………………………..……….1
- Chemoresistance………………………………………………………..2
-Targeted cancer therapy..……………………………………………….3
- Epithelial Membrane Protein 1………..………………………...…….3
Material and Methods
-Cell lines……………………………………………………………….4
-Construction of EMP1..……………………………………………….4
-Mini preparation……………………………………………………….4
-Midi preparation……………………………………………………….5
-Transfection……………………………………………………………5
-Western blot (WB)……………………………………………………..6
-3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) Assay…………………………………………………………………….6
-Immunofluorescent assay (IF)…………………………………………7
Results
Expression pattern of EMPs in human uroepithelial cells…………...8
Establishment EMP1 stable overexpression and knockdown cells…...8
The effect of EMP1 on cell growth and cell cycle progression……….8
Response to chemotherapy in vitro in relation to EMP1 overexpression………………………………………………………….9
The effects of EMP1 on DNA repair system……….……...………….10
The effects of EMP1 on chemoresistance markers…………...……...10
Discussion………………………………………………………………12
References………………………………………………………………13

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