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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55999完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 廖憶純 | |
| dc.contributor.author | Derrick Lee | en |
| dc.contributor.author | 李昌恆 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:12:39Z | - |
| dc.date.available | 2015-08-26 | |
| dc.date.copyright | 2014-08-26 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-18 | |
| dc.identifier.citation | 高芸歆,(2013) 大腸癌細胞中 Cten 蛋白質在 Beta-catenin 介導訊息路徑中所扮演角色及其進入細胞核之機制探討，國立臺灣大學生命科學院生化科技學系
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55999 | - |
| dc.description.abstract | C-terminal tensin-like (Cten) 蛋白質位於集中附著點並參與細胞附著和移動。CTEN 在各個時期的大腸癌中皆有過量表現的情形，於細胞核中也能偵測到高含量的 Cten，顯示細胞核中的 Cten 可能參與大腸癌細胞癌化的過程。本論文發現在大腸癌細胞株 SW480 中，Cten 與 β-catenin、α-actinin4 在細胞核中有交互作用，Cten 和β-catenin 無直接的交互作用，而 Cten 和α-actinin4 則是會直接結合，α-actinin4 同時利用其 N 端和 C端與 Cten 的 C 端 (327~715) 結合。Cten、β-catenin 和 α-actinin4 之間的交互作用，並不影響彼此於細胞質和細胞核的分佈。本論文由 qPCR 的結果發現 Cten不影響 α-actinin4 所參與調控的 NFκB 下游基因表現。在探討 Cten 於細胞質和細胞核間穿梭的機制方面，實驗結果發現 SH2-PTB domain 是 Cten 進核的重要區塊。Cten 出細胞核的機制是透過 CRM1 所運送，利用線上軟體預測出可能的 NES 序列位於 102~118 胺基酸，將 Cten 的 102~118 胺基酸剔除後，發現 Cten 會於細胞核中累積。說明 Cten於細胞質與細胞核間可能的穿梭機制。 | zh_TW |
| dc.description.abstract | C-terminal tensin-like protein (Cten) locates in focal adhesion complex and participates in regulating cell adhesion and migration. Elevated Cten level has been detected in all stage of colon cancers. Furthermore, a high population of Cten is located in the nucleus. Therefore, nuclear Cten may play an important role in colon cancer cell progression. In this study, we demonstrated that Cten interacts with β-catenin and α-actinin4 in the nucleus. Cten indirectly interacts with β-catenin. Whereas α-actinin4 associates with C-terminal half of Cten (327~715) through its N- and C-terminal domain. The interactions of Cten, β-catenin and α-actinin4 may not be required for the nuclear targeting of each protein. Using the qPCR, we showed that nuclear Cten may not participate in regulating α-actinin4 -mediated NFκB downstream genes expression.
In elucidating the molecular mechanism underlying nucleocytoplasmic shuttling of Cten, we found that Cten may translocate to the nucleus through its SH2-PTB domain and is exported by the chromosome region maintenance-1 (CRM-1) dependent pathway. In silico analysis by two NES database shows that Cten contains nuclear export signal (NES) between 102 to 118 amino acid residues. Deletion in putative NES of Cten results in its nucleus retention. These findings provide possible molecular mechanisms for nucleocytoplasm shuttling of Cten. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:12:39Z (GMT). No. of bitstreams: 1 ntu-103-R01b22007-1.pdf: 5562480 bytes, checksum: 0085b6e89a38524e99ae5f5e88269c09 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄 I
縮寫表 IV 摘要 VII Abstract VIII 一、本論文之研究基礎 1 1.1 Focal adhesion 1 1.2 Tensin 2 1.3 Cten 蛋白質的功能研究 3 1.4 α-actinin4 4 1.5 Nuclear protein import / export 5 1.5.1 Nuclear protein import 6 1.5.2 Nuclear protein export 6 二、材料與方法 9 2.1 細胞培養 9 2.2 細胞繼代 9 2.3 細胞轉染 (Transfection) 10 2.4 Leptomycin B (LMB) 處理 10 2.5 DNA 分析與質體建構 11 2.5.1 聚合酶連鎖反應 (polymerase chain reaction, PCR) 11 2.5.2 DNA 片段純化 11 2.5.3 以限制酶截切質體與 PCR 產物 11 2.5.4 DNA 接合法 (DNA ligation) 12 2.5.5 重組質體之轉型與篩選 12 2.5.6 質體純化 12 2.5.7 以限制酶進行質體分析 12 2.5.8 DNA 瓊脂糖膠體電泳法 13 2.6 RNA分析 13 2.6.1 RNA純化 13 2.6.2 cDNA 製備 13 2.6.3 Quantitative PCR (qPCR) 14 2.7 全細胞之蛋白質萃取 14 2.8 細胞質與細胞核萃取液分離 (fractionation) 14 2.9 免疫沉澱與免疫共沉澱 (Immunoprecipitation, IP and Co-immunoprecipitation, Co-IP assay) 15 2.10 蛋白質分析 16 2.11 共軛焦顯微鏡 17 2.11.1 細胞樣本製備 17 2.11.2 影像拍攝 18 2.12 IN Cell 2000 Analyzer 定量分析 18 2.13 酵母菌雙雜合系統 (Yeast two hybrid assay) 19 2.13.1 AH109 酵母菌勝任細胞製備 19 2.13.2 SD 固態培養基製備 19 2.13.3 酵母菌雙雜合法實驗步驟 19 2.14 篩選穩定表現細胞株 (stable cell line) 20 2.15 Cell adhesion assay 21 2.16 Cell proliferation assay 21 2.17 Soft agar assay 21 三、 研究結果 23 3.1 利用免疫共沉澱法證實 Cten、β-catenin 和α-actinin4 於細胞核中有交互作用 23 3.2 利用酵母菌雙雜合法探討 Cten和 β-catenin 之結合模式 24 3.3利用酵母菌雙雜合法和免疫共沉澱法探討 Cten和α-actinin4 之結合模式與結合區域 25 3.4 Cten、β-catenin 和α-actinin4並不影響彼此在細胞質和細胞核間的分佈 26 3.5 癌症與非癌症細胞株中 Cten 分布之差異 28 3.6 探討 Cten 進出細胞核之機制 29 3.7 利用 CRM1 抑制劑 leptomycin B 探討 Cten 序列上是否有 nuclear export signal 30 3.8 探討 Cten 的 nuclear export signal 之序列位置 30 3.9 探討大腸癌細胞株中 Cten 的 NES 序列是否發生缺失 32 3.10 分析 Cten 與 NES deletion Cten 對於細胞生理的影響 32 四、討論與未來方向 34 五、參考文獻 41 六、圖與表 44 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討 Cten 與 β-catenin 和 α-actinin4 之間的交互作用及 Cten 於細胞質和細胞核間穿梭的機制 | zh_TW |
| dc.title | Studies on the interactions of Cten with β-catenin and α-actinin4 and the molecular mechanism underlying nucleocytoplasmic shuttling of Cten | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王愛玉,張麗冠,冀宏源,謝淑貞 | |
| dc.subject.keyword | Cten 交互作用,細胞質和細胞核間穿梭的機制, | zh_TW |
| dc.subject.keyword | Cten,β-catenin,α-actinin4, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-19 | |
| 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 | |
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