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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳進庭(Chin-Tin Chen) | |
dc.contributor.author | Shin-Nan Lee | en |
dc.contributor.author | 李昕南 | zh_TW |
dc.date.accessioned | 2021-06-16T05:36:51Z | - |
dc.date.available | 2017-08-17 | |
dc.date.copyright | 2014-08-17 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56596 | - |
dc.description.abstract | 腫瘤的進程中,腫瘤細胞會誘導周遭其他細胞的轉型,例如使纖維母細胞(fibroblast)轉型為成肌纖維細胞(myofibroblast)。這些轉型的細胞最後會形成腫瘤基質(tumor stroma),提供腫瘤細胞生長所需的環境,使腫瘤進一步的發展。Fibroblast-myofibroblast transition發生的可能機制之一是透過腫瘤細胞分泌轉型生長因子β ( Transforming Growth Factor β, TGF-β) 去調控fibroblast細胞中CLIC4的基因表現,使其上升進而影響細胞週期和轉形,最終被誘發轉形成為myofibroblast的細胞群,可提供有利於腫瘤細胞生存相關的激素及基質,藉由調控微環境因素而促使癌症發生。但由於此路徑中CLIC4基因的上游調控機制尚不明瞭,所以本研究中,利用TGF-β處理fibroblast細胞作為研究模式,希望能藉此找出調控CLIC4基因表現的作用分子。於確立細胞實驗模式之後,分析CLIC4 啟動子區域中可影響基因表現的片段,再經由軟體分析比對出可能對該片段具有調控能力的轉錄因子 (putative transcriptional factors),並以RNA interference的方式進一步確認特定分子與CLIC4之間的關聯性,期望能藉此找出可調控CLIC4基因表現的上游轉錄因子,釐清此基因的調控機制。由報導基因實驗結果中,本研究發現小鼠CLIC4啟動子於(-2000 ~ -1200)的片段上有可能的轉錄抑制者(repressor),而(-234 ~ -56)上則有促進表現的轉錄因子(activator)的結合位置。同時在利用shRNA抑制可能的轉錄因子E2F1與SP1時也發現抑制此二因子時並不會對CLIC4的表現造成影響。未來希望能再將可能與轉錄因子結合的啟動子區域做進一步的縮小範圍,並透過反向染色質免疫共沉澱分析(reverse chromatin immunoprecipitation)找出可能與CLIC4啟動子區域結合的轉錄因子,希望此研究成果對於將來探討惡性腫瘤的進程能夠有進一步的幫助。 | zh_TW |
dc.description.abstract | Fibroblast-myofibroblast transition plays a crucial role in tumor development. During tumor progression, TGF-β secreted by tumor cells stimulates nearby fibroblast to transform into myofibroblast, which further involves in tumor malignancy by producing growth factors and extracellular matrices. Chloride intracellular channel 4 (CLIC4) protein has been shown involving in the transformation of fibroblasts into myofibroblasts. However, the molecular mechanisms remain unclear. In this study, we intend to address the possible transcription factors involved in TGF-β-mediated CLIC4 expression. To fulfill this goal, we first constructed various reporter constructs with different promoter regions of mouse CLIC4 and measured their activity changes after TGF-β treatment. After confirming the promoter region affected by TGF-β, we further analyzed the DNA sequence to find out the possible transcriptional factors, which were further confirmed by shRNA knockdown. Our results showed that a transcriptional inhibitor binding site was located at -2000 bps to -1200 bps of the mouse CLIC4 promoter (+1 being the transcriptional start site). Meanwhile, we also identified an activator binding site at the range of -234 to -22 of CLIC4 promoter. Further studies showed that the putative transcriptional factors E2F1 and SP1 were not involved in the TGF-β-mediated gene expression of CLIC4. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:36:51Z (GMT). No. of bitstreams: 1 ntu-103-R01B22024-1.pdf: 5390178 bytes, checksum: d5eecca94395fd1c0a4eae92a8a2386f (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 中文摘要 i
Abstract ii 圖目錄 vi 表目錄 vii 第一章、緒論 1 1.1 癌症之發展與惡化 1 1.1.1惡性腫瘤之形成 1 1.1.2 腫瘤與微環境之互動 1 1.1.3 Fibroblast/ myofibroblast transition 3 1.2 TGF-β 調控機制與相關分子 4 1.2.1 TGF-β誘發之訊息傳遞路徑 4 1.2.2 TGF-β於myofibroblast transition中所扮演的角色 5 1.3 氯離子通道蛋白 (Chloride intracellular channel 4, CLIC4) 5 1.3.1 CLIC4基本介紹 5 1.3.2 CLIC4 相關調控機制 6 1.3.3 CLIC4於myofibroblast transition中所扮演的角色 7 1.4 基因表現調控機制 7 1.4.1 轉錄因子 7 1.4.2 表觀遺傳修飾 8 1.5 動機與目的 9 1.6 研究架構 10 第二章、材料與方法 11 2.1 藥品與儀器 11 2.1.1 藥品 11 2.1.2 儀器 13 2.2 細胞株 14 2.3 細胞培養、計數與繼代 15 2.4 細胞冷凍與解凍 15 2.5 TGF-β處理細胞 16 2.6 mRNA定量分析 16 2.6.1 RNA萃取 16 2.6.2反轉錄 17 2.6.3聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 17 2.6.4洋菜膠體電泳分析 19 2.7報導基因(reporter assay)質體建構 19 2.7.1 目標基因序列的PCR放大 19 2.7.2 TA cloning 20 2.7.3 質體建構 20 2.8 以SEAP assay測定promoter片段活性 21 2.8.1 轉染預測定之SEAP reporter plasmid 21 2.8.2 SEAP活性測定 21 2.8.3 Promoter活性計算 21 2.9 RNAi gene knockdown 22 2.10 細胞存活率分析:粒線體去氫酶活性分析 22 2.11 統計分析 23 第三章、結果 24 3.1 TGF-β處理對fibroblast細胞之影響 24 3.1.1細胞型態之改變與細胞生長速度的減緩 24 3.1.2 ACTA2基因的表現量變化 24 3.1.3 CLIC4 基因的表現量變化 25 3.1.4 甲基轉移酶DNMT1可能不參與此路徑 25 3.1.5 MRC-5並不適合進行基因轉染表現實驗 25 3.2 可能參與TGF-β調控CLIC4表現上升之啟動子片段分析 26 3.2.1 無處理下啟動子片段活性分析 26 3.2.2啟動子片段於TGF-β處理後之活性分析 27 3.2.3影響CLIC4表現量之啟動子片段之軟體分析 27 3.3 參與TGF-β調控CLIC4表現上升之相關轉錄因子 27 3.3.1 E2F1與SP1於TGF-β處理後之表現量變化 28 3.3.2 Knockdown E2F1、SP1後CLIC4表現量於TGF-β處理下的變化 28 3.4 CLIC4啟動子片段的進一步分析 29 3.4.1無處理下啟動子片段活性分析 29 3.4.2啟動子片段於TGF-β處理後之活性分析 29 第四章、討論 30 4.1 TGF-β對細胞的影響 30 4.1.1 NIH/3T3細胞 30 4.1.2 MRC-5細胞 31 4.2 CLIC4 promoter分析與可能的調控因子位置 31 4.2.1 甲基化因素 31 4.2.2 轉錄repressor 32 4.2.3 轉錄activator 33 4.3 轉錄因子E2F1與SP1對CLIC4表現量的影響 34 4.4 進一步分析CLIC4可能轉錄因子的方法 34 第五章、結論與未來展望 36 圖表 37 附表 61 附圖 64 參考文獻 70 | |
dc.language.iso | zh-TW | |
dc.title | 探討TGF-β調控CLIC4基因表現路徑中可能的轉錄因子 | zh_TW |
dc.title | Investigation of putative transcription factors involved in TGF-β mediated CLIC4 expression | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張麗冠(Li-Kwan Chang),林晉玄(Ching-Hsuan Lin),李銘仁(Ming-Jen Lee) | |
dc.subject.keyword | CLIC4,TGF-β,纖維母細胞,成肌纖維細胞,轉錄因子, | zh_TW |
dc.subject.keyword | CLIC4,TGF-β,fibroblast,myofibroblast,transcription factors, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-103-1.pdf 目前未授權公開取用 | 5.26 MB | Adobe PDF |
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