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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Ya-Ya Chang | en |
| dc.contributor.author | 張雅雅 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:18:18Z | - |
| dc.date.available | 2014-07-30 | |
| dc.date.copyright | 2009-07-30 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-27 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42634 | - |
| dc.description.abstract | 微型核醣核酸microRNA（miRNA）為一群內生型，長度在20-24個鹼基之間的核酸，對於其標靶基因能夠藉著與其訊息RNA (mRNA)的3’-非編碼區 (3’-UTR) 結合來進行負向調控，以抑制蛋白質的合成。最近的研究中發現微型核醣核酸除了與細胞的生長發育有密切關係之外，與癌症的發生與發展有密切的關係。此外，微型核醣核酸在細胞訊息傳遞路徑中所扮演的角色也逐漸被發現，但其確切的功能尚未完全解開。李文雄院士、施純傑博士與我們組成的團隊於2008發現並證實一個新的微型核醣核酸P-27-5p，我們以數個人類癌細胞株為材料來證實它的功能。在細胞存活率實驗中發現P-27-5p會導致細胞生長減緩，進一步細胞週期分析發現P-27-5p會使細胞停留在G0/G1時期。經由預測結果得知v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3)為P-27-5p的標靶基因，我們經由冷光酶活性測定分析以及西方墨點法驗證ErbB3確實為P-27-5p的標靶基因。在前人的研究中發現，ErbB3會藉著活化phosphatidylinositol 3-kinase (PI3K)/AKT訊息傳遞路徑來促使細胞增生，並且在很多癌細胞中其ErbB3為大量表現，導致癌細胞擁有快速的增生能力。從西方墨點法中證實P-27-5p會抑制ErbB3的表現，並抑制AKT的磷酸化。總而言之，本項研究發現P-27-5p會抑制ErbB3的表現，進而抑制其下游的訊息傳遞路徑，導致細胞週期停留在G0/G1時期，而使癌細胞生長受到抑制 。 | zh_TW |
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| dc.description.tableofcontents | 目錄
口試委員會審定書..........................................Ⅰ 誌謝......................................................Ⅱ 中文摘要..................................................Ⅳ Abstract..................................................Ⅴ 縮寫表....................................................Ⅵ 第一章 前言.............................................. 1 1-1 miRNAs的介紹........................................1 1-2 miRNA與siRNA的比較 ..................................1 1-3 miRNA與發育的關係.....................................2 1-4 miRNA與癌症的關係.....................................2 1-5 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3)的介紹...................................3 1-6 ErbB3與癌症的關係.....................................4 1-7 phosphatidylinositol 3-kinase (PI3K)/ v-akt murine thymoma viral oncogene homolog 1 (AKT)訊息傳遞路徑與癌症........................................................5 1-8 P-27-5p的發現........................................6 第二章 實驗材料與方法....................................7 2-1、細胞培養........................................... .7 2-2、細胞總核糖核酸(含微型核糖核酸)萃取...................10 2-3、反轉錄反應 (Reverse Transcription) ..................11 1. miRNAs表現使用.........................................11 2. 基因表現使用...........................................11 2-4、即時定量聚合酶鏈鎖反應 (Real-Time PCR) ..............12 2-5、轉殖作用(transfection) ..............................12 2-6、細胞存活率實驗 (MTT assay) .'..........13 2-7、以流式細胞儀進行細胞週期分析 (cell cycle analysis)...13 2-8、以流式細胞儀進行細胞凋亡分析 (cell apoptosis analysis) ................................................13 2-9、西方墨點法 (Westen Blotting) ........................14 1. SDS聚丙烯醯胺膠體製備..................................14 2. SDS聚丙烯醯胺膠體電泳分析..............................15 3. 膠體電泳轉漬...........................................15 4. 抗體作用呈色分析.......................................15 2-10、 pMIR-REPORT-ErbB3 3’UTR expression vector質體構築.16 1. 引子黏合...............................................16 2. DNA限制酵素反應........................................16 3. 引子純化...............................................17 4. Luciferase 質體去鹽純化................................17 5. 接合作用...............................................17 6. 轉型作用...............................................18 7. 小量質體萃取...........................................18 8. 洋菜膠體置備...........................................19 9. 膠體電泳分析...........................................19 2-11、冷光酶活性測定分析(Luciferase reporter assay) ......19 1. 轉殖作用..............................................20 2. Luciferase與β-galactosidase訊號偵測...................20 第三章 結果..........................................22 3-1 利用顯微鏡觀察miRNA P-27-5p對乳癌細胞的影響...........22 3-2 利用real-time PCR方式探討miRNA P-27-5p在不同乳房細胞株的表現量差異................................................22 3-3 利用real-time PCR來驗證miRNA P-27-5p在轉殖後的基因表現量........................................................23 3-4 以MTT assay分析miRNA P-27-5p對細胞存活率的影響........24 3-5 以流式細胞儀觀察miRNA P-27-5p是否造成乳癌細胞凋亡.....24 3-6 以流式細胞儀分析miRNA P-27-5p對細胞週期的影響.........25 3-7 利用冷光酶活性測定分析驗證 miRNA P-27-5p能負向調控其標靶基因ErbB3.................................................26 3-8 利用西方墨點法來分析miRNA P-27-5p其標靶基因ErbB3的蛋白質產物表現量................................................27 3-9 利用西方墨點法來分析miRNA P-27-5p對ErbB2的蛋白質產物表現量有無影響................................................27 3-10利用西方墨點法來分析miRNA P-27-5p對AKT的磷酸化有無影響28 第四章 討論............................................29 圖.......................................................34 參考文獻.................................................60 附錄......................................................71 圖目錄 圖一、miRNAs的生合成路徑..................................34 圖二、miRNA與siRNA的差異..................................35 圖三、miRNA在癌症發生過程中所扮演的角色可依功能分為似抑癌基因或是似致癌基因..........................................36 圖四、ErbB3調控PI3K/AKT訊息傳遞路徑.......................37 圖五、以real-time PCR偵測P-27-5p在不同乳房細胞株的表現量..38 圖六、於MCF-7中加入precursor P-27-5p 48小時後於顯微鏡下的觀察結果....................................................39 圖七、於MCF-7中加入anti-sense P-27-5p 48小時後於顯微鏡下的觀察結果..... ..............................................40 圖八、以real-time PCR偵測P-27-5p在不同乳房細胞株的表現量..41 圖九、以real-time PCR偵測驗證P-27-5p在轉殖後的基因表現量..42 圖十、以MTT assay分析細胞存活率...........................43 圖十一、以流式細胞儀分析乳癌細胞凋亡現象..................44 圖十二、以流式細胞儀分析P-27-5p對乳房細胞MCF-10A及乳癌細胞MDA-MB-231細胞週期的影響..................................45 圖十三、以流式細胞儀分析P-27-5p對乳癌細胞MCF-7及T-47D細胞週期的影響..................................................46 圖十四、以流式細胞儀分析各乳房細胞株其細胞週期總整理......47 圖十五、以流式細胞儀分析肺癌細胞株H838和肺部纖維母細胞IMR-90細胞週期..................................................48 圖十六、P-27-5p 在ErbB3的標靶區域預測與引子設計...........49 圖十七、ErbB3冷光酶質體構築與定序確認.....................50 圖十八、冷光酶活性測定分析基本原理........................51 圖十九、利用冷光酶活性測定分析驗證P-27-5p能負向調控其標靶基因：ErbB3.................................................52 圖二十、利用西方墨點法來分析P-27-5p其標靶基因ErbB3的蛋白質產物表現量..................................................53 圖二十一、利用西方墨點法來分析P-27-5p對ErbB2的影響........54 圖二十二、利用西方墨點法來分析P-27-5p對磷酸化AKT的影響....55 圖二十三、推測P-27-5p藉著調控PI3K/AKT訊息傳遞路徑抑制細胞生長........................................................56 圖二十四、mTOR訊息傳遞路徑................................57 圖二十五、AKT能抑制GSK3β進而穩定Cyclin D.................58 圖二十六、推測P-27-5p能藉由抑制EP300進而抑制由ErbB2、EP300和ER81所形成的正向回饋路徑進而抑制ErbB2的表現...............59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 微型核醣核酸 | zh_TW |
| dc.subject | 細胞週期 | zh_TW |
| dc.subject | 人類上皮因子接受體 | zh_TW |
| dc.subject | 流式細胞儀 | zh_TW |
| dc.subject | 乳癌 | zh_TW |
| dc.subject | ErbB3 | en |
| dc.subject | miRNA | en |
| dc.subject | cell cycle | en |
| dc.subject | breast cancer | en |
| dc.subject | flow cytometry | en |
| dc.title | 新miRNA P-27-5p影響人類癌細胞週期及其分子機轉之探討 | zh_TW |
| dc.title | Effects of a Novel miRNA P-27-5p on Human Cancer Cell Cycle and the Related Molecular Mechanism | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李文雄(Wen-Hsiung Li),黃宣誠(Hsuan-Cheng Huang),施純傑(Arthur Chun-Chieh Shih),林國儀(Kuo-I Lin) | |
| dc.subject.keyword | 微型核醣核酸,細胞週期,乳癌,流式細胞儀,人類上皮因子接受體, | zh_TW |
| dc.subject.keyword | miRNA,cell cycle,breast cancer,flow cytometry,ErbB3, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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