臺灣博碩士論文加值系統

中文摘要
Ubiquitin-specific protease 2 (USP2)是deubiquitinases成員之一，主要有三個isoforms， USP2a、USP2b以及USP2c。已知最大的USP2a 可經由deubiquitinate目標蛋白(FASN、MDM2、MDM、以及CCND1等)使其穩定，避免其被運送至proteasome分解，而參與在細胞生長調控或藥物作用上。此外，近期在前列腺癌中也發現USP2a可透過調控redox的機制造成抗藥性產生。在此，本實驗利用cisplatin (CDDP)處理NTUB1人類泌尿上皮細胞癌細胞，以MTT細胞毒性測試得知，過量表現USP2a會造成NTUB1細胞對於CDDP的敏感度下降，並以siRNA靜默(knockdown) USP2a表現量時，回復細胞對CDDP的敏感性，證實USP2a參與在細胞對CDDP的敏感性作用。進一步以細胞週期分析觀察到WT-USP2a可將由CDDP所引起之細胞凋亡族群轉移至G0/G1。利用Western blot觀察到內源性的USP2a表現量下降，且c-Myc與Mcl-1穩定性增加。同時在免疫螢光染色實驗中初步觀察到外源性的USP2a表現在細胞內的特殊位置。為更了解USP2a在CDDP作用下參與角色，以二維電泳與質譜儀分析，初步實驗結果顯示USP2a在CDDP作用下影響特定分子的表現差。綜合以上，USP2a可能透過特定的機制造成NTUB1對於CDDP藥物敏感度上升，本研究結果將有利於未來癌症之應用評估。

英文摘要
Ubiquitin-specific protease 2 (USP2) belongs to deubiqutinase family and composes three isoforms. USP2a, the largest form, exerts its oncogenic function through deubiqutinating target proteins (FASN, MDM2, MDM and CCND1, etc.) and preventing them from proteasome degradation. USP2a protected prostate cancer cells from drug-induced oxidative stress and led to chemoresistance. However, the underlying molecular mechanism by which USP2a-mediated cisplatin (CDDP) response is unclear. In this study, we first observed that (CDDP) reduced the USP2a expression in human urothelial carcinoma NTUB1 cells. Overexpressed USP2a reduced CDDP sensitivity in NTUB1 cells. In addition, knocking-down USP2a by siRNA sensitized NTUB1 cells to (CDDP). Furthermore, USP2a caused the G0/G1 arrested instead of the apoptosis population which induced by (CDDP). Potential USP2a target proteins were then examined. USP2a increased c-Myc expression and prevented the degradation of Mcl-1 upon (CDDP) treatment. The cellular localization of USP2a was examined by immuno-fluorescence staining. The results indicated that USP2a expressed in specific compartments in cytoplasm and may relate to its cellular function. Lastly, 2-D electrophoresis and MS-MS were performed to analyze USP2a-mediated potential targets in CDDP responses. In conclusion, USP2a reduces CDDP sensitivity through stabilizing specific molecules in NTUB1 cells.

目錄
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.4
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.5
第一章：緒論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.6
第一節：泌尿系統和人類泌尿上皮細胞癌的介紹‧‧‧‧‧P.6
第二節：順鉑 ( Cisplatin; CDDP ) 的介紹‧‧‧‧‧‧‧‧P.11
第三節：Deubiquitinases ( DUBs ) 酵素蛋白的介紹‧‧‧‧P.13
第四節：Myeloid Cell Leukemia 1 (Mcl-1) 抗凋亡分子的介紹P.14
第二章：研究目的‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.15
第三章：材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.16
第一節 : 實驗材料‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.16
第二節 : 實驗方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.20
第四章：實驗結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.34
第五章：討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.38
第六章：圖表‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.42
附錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.55
參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧P.60

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