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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 廖憶純(Yi-Chun Liao) | |
dc.contributor.author | Wei-Ming Wu | en |
dc.contributor.author | 吳偉銘 | zh_TW |
dc.date.accessioned | 2021-05-19T17:44:45Z | - |
dc.date.available | 2023-08-16 | |
dc.date.available | 2021-05-19T17:44:45Z | - |
dc.date.copyright | 2018-08-16 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7488 | - |
dc.description.abstract | C-terminal tensin like (CTEN) 蛋白質是 tensin 家族的一員,主要存在於細胞的 focal adhesions,CTEN 於前列腺和胎盤等少數器官中有較高的基因表現,而 CTEN 同源蛋白質僅存在於哺乳動物。CTEN 蛋白質於前列腺上皮細胞的含量明顯高於其他型態的前列腺細胞,因此我們推測 CTEN 可能參與前列腺上皮組織的發展。為了探討 CTEN 在前列腺上皮組織中扮演的角色,我們使用非惡性前列腺上皮細胞株 RWPE-1 作為研究平台,以 siRNA 抑制 CTEN 基因表現或誘導外源表現 CTEN,分別於二維和三維培養環境中探討CTEN 對細胞行為與型態的影響。我們發現於 RWPE-1 細胞中,缺乏 CTEN 會導致細胞停滯於 G1/S 期,並使 CDK 的活性抑制蛋白質 p21 和 p27 含量增加;CTEN 的減少也會抑制 RhoA 活化以及細胞移動能力。此外,我們發現 CTEN 基因座上有許多 ΔNp63 的結合位置,ΔNp63 為影響前列腺發展的重要轉錄因子,能夠正向調控 CTEN 基因表現,並藉由 CTEN 幫助細胞貼附於細胞外基質。透過線上資料庫分析發現 CTEN 在前列腺基底細胞的表現量高於管腔細胞,於是我們將 RWPE-1 培養於以 Matrigel 為基底的三維環境,以模擬前列腺上皮細胞分化與腺泡生成,我們發現隨著時間的增加,CTEN 的表現會受到抑制,而進一步在腺泡形成的過程中誘導外源表現 CTEN,則會破壞腺泡結構與細胞分化,可能是透過維持 FAK Y397 的磷酸化所造成。另一方面,雖然正常前列腺上皮細胞中的 CTEN 表現量很高,然而在多數前列腺癌組織中 CTEN 表現量非常低,為了解 CTEN 於前列腺癌中的角色,我們外源表現 CTEN 於轉移性前列腺癌細胞株 DU-145,結果顯示過度表現 CTEN 雖然不影響 DU-145 的增生能力,但卻會抑制細胞遷徙能力。整體而言,本研究證實 CTEN 有助於非惡性前列腺上皮細胞的增生、移動和貼附,卻抑制轉移性前列腺癌細胞的移動能力,在分化為管腔細胞的過程中,CTEN 表現受到抑制,此負向調控 CTEN 是維持正常腺泡生成所必需,於前列腺上皮組織中,CTEN 的分布與其表現量的調控對於前列腺上皮組織的成熟與恆定具有重要的生理意義。 | zh_TW |
dc.description.abstract | C-terminal tensin like (CTEN) protein is a member of tensin family, mainly localized in focal adhesions. CTEN is enriched in a few tissues, such as prostate and placenta, and CTEN homologies are found only in mammals. The levels of CTEN protein is significantly higher in prostate epithelial cells than other types of prostatic cells, suggesting that CTEN might participate in the development of prostate epithelium. In this study, to elucidate the role of CTEN in prostate epithelium, a non-malignant prostate epithelial cell line, RWPE-1, was used. We manipulated CTEN level by siRNA-mediated gene silencing or inducible ectopic gene expression to examine the effect of CTEN on cell behaviors and morphology in 2D or 3D culture system. Our study shows that loss of CTEN in RWPE-1 leads to accumulation of CDK inhibitors, p21 and p27, and hindrance to cell cycle G1/S transition. Depletion of CTEN also suppresses RhoA activation and cell migration. Additionally, we reveal the ΔNp63 binding region on CTEN gene locus and verified ΔNp63, a crucial transcription factor of prostate development, mediates cell-extracellular matrix adhesion through up-regulation of CTEN. Moreover, by analyses of publicly available online databases, we found that the level of CTEN expression is higher in prostate epithelial basal cells than in luminal cells. RWPE-1 cells in Matrigel-based 3D cultures were used to investigate the role of CTEN in prostate epithelial cell differentiation and acinar morphogenesis. We demonstrate that CTEN is inhibited during the course of luminal differentiation. Ectopic expression of CTEN maintains FAK pY397 level and disturbs the acini formation and luminal differentiation. On the other hand, although CTEN is highly expressed in prostate epithelial cells, it is significantly down-regulated in prostate cancer. To decipher the role of CTEN in prostate cancer, CTEN expression was induced in DU-145, a metastatic prostate cancer cell line. CTEN overexpression in DU-145 cells suppressed cell migration but made little impact on cell proliferation. In summary, our study demonstrates that CTEN facilitates proliferation, migration and adhesion in non-malignant prostate epithelial cells whereas inhibits cell migration in metastatic prostate cancer cells. Our work also strongly suggested that down-regulation of CTEN during luminal differentiation is required for acinar morphogenesis. The distribution and the expression regulation of CTEN in prostate epithelium is physiologically relevant for the development and homeostasis of prostate epithelium. | en |
dc.description.provenance | Made available in DSpace on 2021-05-19T17:44:45Z (GMT). No. of bitstreams: 1 ntu-107-F99b22054-1.pdf: 3169261 bytes, checksum: 3d70a0a0344c405821c7ac72fd0512bc (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 摘要 I
ABSTRACT II 目錄 III 圖目錄 V 表目錄 VI 縮寫表 VII 第一章 研究背景 1 1.1 C-terminal tensin-like (CTEN) 蛋白質 1 1.1.1 蛋白質結構與功能性區塊 1 1.1.2 基因表現 3 1.1.3 生物功能 4 1.2 前列腺上皮組織生成與癌化 6 1.3 研究緣起與目標 9 第二章 研究材料與方法 11 2.1 研究材料 11 2.1.1質體與保存菌株 11 2.1.2 細胞株與培養條件 13 2.1.3 抗體 13 2.2 研究方法 14 2.2.1 質體製備 14 2.2.2 以siRNA 進行基因靜默 (gene silencing) 16 2.2.3 蛋白質含量分析 (Western blotting) 16 2.2.4 細胞增生能力檢測 (WST-1 assay) 17 2.2.5 細胞週期分析 18 2.2.6 Real-time RT-PCR 18 2.2.7 Chromatin Immunoprecipitation-PCR (ChIP-PCR) 19 2.2.8 細胞貼附能力檢測 21 2.2.9 細胞核細胞質分離 22 2.2.10 細胞移動能力檢測 23 2.2.11 RhoA 活性檢測 23 2.2.12 生物資訊分析 24 2.2.13 慢病毒顆粒製備與穩定細胞株篩選 25 2.2.14 三維空間細胞培養 (腺泡生成) 25 2.2.15 免疫螢光染色 26 第三章 CTEN 缺失對人類前列腺上皮細胞增生、貼附和移動能力的影響 27 3.1 CTEN 缺失會阻礙細胞週期 G1/S 期間的轉換 27 3.2 CTEN 參與 ΔNp63α 所調控之細胞貼附 29 3.3 CTEN 缺失對 EMT 相關生物標記分子的影響 31 3.4 CTEN 缺失抑制細胞遷徙能力與 RhoA 活性 32 第四章 CTEN 於前列腺腺泡生成過程中扮演的角色 34 4.1 CTEN 高度表現於基底型前列腺上皮細胞 34 4.2 CTEN 缺失對前列腺上皮細胞分化的影響 35 4.3 過度表現 CTEN 破壞前列腺腺泡生成 36 第五章 過度表現 CTEN 對前列腺癌細胞株的影響 39 第六章 討論 40 參考文獻 45 圖與表 60 附錄 88 | |
dc.language.iso | zh-TW | |
dc.title | 探討 CTEN 於人類前列腺上皮細胞所扮演的角色 | zh_TW |
dc.title | Study on the Role of CTEN in Human Prostate Epithelial Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 張世宗,沈湯龍,蔡淵欽,賴韻如,謝淑貞 | |
dc.subject.keyword | CTEN,前列腺,增生,貼附,移動,腺泡生成,前列腺癌, | zh_TW |
dc.subject.keyword | CTEN,prostate,proliferation,adhesion,migration,acinar morphogenesis,prostate cancer, | en |
dc.relation.page | 90 | |
dc.identifier.doi | 10.6342/NTU201803093 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2018-08-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
dc.date.embargo-lift | 2023-08-16 | - |
顯示於系所單位: | 生化科技學系 |
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