指導教授：陳進庭臺灣大學：生化科技學系賴亭薇Lai, Ting-WeiTing-WeiLai2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261590癌症為人類疾病的三大死因之一，癌症為由不正常增生的細胞所形成的腫瘤 (tumor)，而其中惡性腫瘤會隨著淋巴或血液系統轉移到其他正常器官。現今癌症不論在診斷或治療上都已有非常大的改善，雖然癌症初期的治療可透過手術切除、物理治療搭配化療藥物達到良好的治療效果，但造成癌症無法根治、復發及高死亡率的最大原因則是癌症的轉移。過去研究已知基質金屬蛋白酶matrix metalloproteinases 9 (MMP9) 在腫瘤的發展與轉移中扮演非常重要的角色，其可分解細胞外間質，促使癌細胞的轉移。除此之外，過去研究亦顯示一氯離子通道蛋白chloride intracellular channel 4 (CLCI4)會經由調控MMP9進而影響癌細胞的侵襲能力。此氯離子通道CLIC4不論在細胞凋亡、血管新生、腫瘤發展與訊息傳導路徑皆扮演相當重要的角色，因此本研究之目的即為找出CLIC4為透過何訊息傳導路徑或分子影響MMP9表現量。本論文使用了不同的乳癌細胞株MDA-MB-231及 MCF7，並欲利用siCLIC4以及自行建構的CLIC4 overexpression plasmid調控細胞內CLIC4表現量，利用抑制以及過量表現CLIC4觀察其對MMP9之影響，發現只有當CLIC4被抑制時，MMP9 mRNA表現量會跟著下降。同樣的現象也能在MMP9的活性分析中觀察到，當CLIC4被抑制時，MMP9 活性會隨之下降。因此，為了進一步分析CLIC4的下降是如何影響MMP9表現量與活性，便於MDA-MB-231細胞中轉染了帶有不同長度MMP9 promoter的luciferase reporter plasmid，發現當CLIC4表現量被抑制時，可能是影響MMP9 promoter位置-545~ -578的區域上的轉錄因子而改變MMP9的轉錄活性。更利用了免疫共沈澱法分析了與CLIC4共同結合的蛋白質找出可能與CLIC4結合並共同調控MMP9的分子。Cancer is caused by abnormal growth or division of cell and it can mainly be divided into two types: benign neoplasms and malignant neoplasms. The characteristics of malignant neoplasms are tumor cells with metastatic ability. It has been shown that matrix metalloproteinases 9 (MMP9) plays an important role in tumor progression and metastasis. MMP9 can help the digestion of extracellular matrix and is an important marker of invasion and metastasis. Chloride intracellular channel 4 (CLIC4) is a chloride intracellular channel protein, which involved in many cellular functions like apoptosis, angiogenesis, tumor progression and invasion. Previously, we have found that CLIC4 will affect the invasion ability through the regulation of MMP9 expression. The purpose of this study is to investigate how CLIC4 regulate MMP9 expression. We first use CLIC4 siRNA to knockdown the expression level of CLIC4 in MDA-MB 231 cells. The mRNA expression level and the activity of MMP9 decrease when the expression level of CLIC4 was knockdowned. These results indicated that CLIC4 might play a role in regulating the MMP9 expression. Furthermore, luciferase reporter with different MMP9 promoter region was used to examine the possible transcriptional factor involved in CLIC4-mediated MMP9 expression. Finally, immunocomplex pull-downed by antibody against CLIC4 was analyzed by LC-MS/MS. Some candidates of possible transcriptional factors and signal transduction pathways have been found.摘要................................................................................................................................. I Abstract ......................................................................................................................... II 目錄............................................................................................................................... III 圖目錄........................................................................................................................... VI 表目錄......................................................................................................................... VII 第一章 緒論 .................................................................................................................. 1 1.1 癌細胞的轉移 (METASTASIS) ................................................................................ 1 1.1.1 癌細胞轉移的進程 ......................................................................................... 1 1.1.2 癌轉移的基因表現 ......................................................................................... 2 1.2 基質金屬蛋白酶 (MATRIX METALLOPROTEINASES, MMPS)。 .............................. 4 1.2.1 基質金屬蛋白酶之調控 ................................................................................. 4 1.2.2MMP9 ............................................................................................................... 5 1.3 胞內氯離子通道蛋白 (CHLORIDE INTRACELLULAR CHANNEL 4, CLIC4) ............. 6 1.4 研究動機與目的 .................................................................................................... 8 1.5 研究架構 ................................................................................................................ 9 第二章 材料方法 ........................................................................................................ 10 2.1 藥品與儀器 .......................................................................................................... 10 2.1.1 藥品 .............................................................................................................. 10 2.1.2 抗體 ............................................................................................................... 12 2.1.3 儀器 .............................................................................................................. 12 2.2 細胞株與培養液 .................................................................................................. 14 2.3 細胞培養與繼代 .................................................................................................. 14 2.4 細胞解凍與冷凍 .................................................................................................. 14 2.5 細胞計數 .............................................................................................................. 15 IV 2.6 質體建構 .............................................................................................................. 15 2.6.1 目標基因序列的PCR 放大 ......................................................................... 16 2.6.2 TA cloning ..................................................................................................... 17 2.6.3 質體建構 ...................................................................................................... 17 2.7 細胞轉染 .............................................................................................................. 17 2.8 MRNA 定量分析 .................................................................................................. 18 2.8.1 RNA 萃取 (RNA extraction) ....................................................................... 18 2.8.2 反轉錄 (Reverse Transcription, RT) ........................................................... 19 2.8.3 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) ................................ 19 2.8.4 洋菜膠體電泳 (Agarose electrophoresis) ................................................... 20 2.9 西方點墨法 (WESTERN BLOT) ............................................................................. 20 2.9.1 蛋白質萃取 (Protein extraction) ................................................................. 20 2.9.2 蛋白質定量 (Protein quantification) ........................................................... 21 2.9.3 膠體電泳與轉印 (SDS-PAGE electrophoresis and Transfer) .................... 22 2.9.4 化學冷光免疫分析法 (Chemiluminescence Immunoassay, CLIA) ........... 22 2.10 明膠蛋白酶活性電泳分析法 (ZYMOGRAPHY) ................................................ 22 2.11 細胞之免疫染色 (IMMUNOCYTOCHEMISTRY, ICC) .......................................... 23 2.12 螢光素酶報導分析 (LUCIFERASE REPORTER ASSAY) ........................................ 24 2.13 免疫共沈澱 (IMMUNOPRECIPITATION) ............................................................... 24 2.14 蛋白質體學分析 (PROTEOMICS ANALYSIS) ....................................................... 25 2.15 統計分析 ............................................................................................................ 25 第三章 結果 ................................................................................................................ 27 3.1 改變細胞中CLIC4 之表現量對細胞型態之影響 ............................................ 27 3.2 增加細胞中CLIC4 之表現量後，CLIC4 之MRNA 表現量、蛋白質表現量與 細胞內的分佈位置 .................................................................................................... 27 V 3.3 改變細胞內CLIC4 表現量對MMP9 MRNA 表現量與轉錄活性之影響 ....... 29 3.4 改變細胞中CLIC4 表現量對MMP9 活性之影響 ........................................... 30 3.5 改變細胞中CLIC4 表現量對NF-ΚB 之入核現象之影響 ............................... 31 3.6 抑制細胞中CLIC4 表現量，不同長度MMP9 PROMOTER 之活性改變 ......... 32 3.7 蛋白質體學分析 ............................................................................................... 33 第四章 討論 ................................................................................................................ 34 4.1 SHCLIC4 與SICLIC4 ........................................................................................... 34 4.3 OVEREXPRESS CLIC4 對細胞的影響 ................................................................... 36 4.4 細胞MMP9 表現量之改變 ................................................................................ 38 第五章 結論 ................................................................................................................ 42 第六章 附錄 ................................................................................................................ 43 第七章、參考資料 ...................................................................................................... 7412856298 bytesapplication/pdf論文公開時間：2017/08/11論文使用權限：同意有償授權(權利金給回饋學校)氯離子通道蛋白基質金屬蛋白酶訊息傳導路徑[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261590/1/ntu-103-R01b22003-1.pdf