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研究生: 洪憶茹
Hong, Yi-Ru
論文名稱: 螢光標記揭開成熟與變異之核纖層蛋白對核膜構形的交互影響
Fluorogenic Labeling Probes Reveal the Morphological Interplay between PreLamin and Mature Lamin in Nuclear Envelope
指導教授: 陳貴通
Tan, Kui-Thong
口試委員: 許馨云
Hsu, Hsin-Yun
黃郁棻
Huang, Yu-Fen
學位類別: 碩士
Master
系所名稱: 理學院 - 化學系
Department of Chemistry
論文出版年: 2017
畢業學年度: 105
語文別: 中文
論文頁數: 81
中文關鍵詞: 螢光探針核纖層蛋白
外文關鍵詞: Fluorescent probe, Nuclear lamins
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  •   早期利用抗體抗原法或是螢光蛋白雖然可以偵測核膜構形,但是對於正常核纖層蛋白與變異核纖層蛋白間關係仍未知。本實驗中,我們使用兩種新穎之螢光標記探針來觀察不同轉譯後修飾情況下正常與變異核纖層蛋白間之交互作用造成之核膜構形變化。研究結果表明,正常核纖層蛋白將受變異核纖層蛋白濃度影響,進而使核膜產生聚集或是皺褶之構形變化。此外,我們也探討了變異核纖層蛋白進行轉譯後修飾之核層形態的變化。我們發現法尼基化的變異核纖層蛋白可以在5-7小時內恢復正常核膜形狀,但是在非法尼基化的變異核纖層蛋白實驗中則表現出不同結果,即使追蹤至第24小時,非法尼基化的變異核纖層蛋白仍有聚集於核周圍之現象。
    我們相信,透過了解核纖層蛋白之合成過程以及不同轉譯後修飾的形態變化,將會對相關的疾病的治療或是藥物的發展有非常大的幫助。


      Although nuclear lamina morphology can be clearly visualized using Lamin-specific antibodies and fluorescent proteins, it has remained unclear how the normal lamina morphology would respond to the mutant preLamin in living cells. In this study, we used a pair of new fluorogenic labeling probes to elucidate the morphological interplay between mature and mutant Lamin A under two different posttranslational processing controls. Our results reveal that the shape of mature Lamin can be altered by the mutant preLamin and that this alteration is progressive, cumulative and due to a concentration-dependent dominant-negative effect of preLamin. The second set of our experiments explored the changes in nuclear lamina morphology during the posttranslational processing of preLamin. While farnesylated preLamin can be processed and recovered to its normal shape within 5 – 7 hours, non-farnesylated preLamin remained as aggregates around the nuclear envelope periphery within 24 hours of imaging time course. We believe that our increased understanding of how Lamin undergoes the morphological changes in response to different posttranslational modifications might shed light on the development of new therapeutic drugs for treating lamina-related diseases.

    摘要 ii 謝誌 iv 目錄 v 第一章 緒論 1 1-1 核纖層 1 1-2 核纖層蛋白 2 1-2-1 Lamin B1 / B2 3 1-2-2 Lamin A /C 4 第二章 文獻回顧 7 2-1 蛋白質標記簡介 7 2-2 免疫細胞化學法 8 2-3 螢光蛋白 9 2-4 螢光分子 10 2-4-1 利用化學反應連接標記 10 2-4-2 利用蛋白質或胜肽進行標記 11 2-4-2.1胜肽直接標記法 12 2-4-2.2蛋白質直接標記法 14 2-4-2.3間接標記 20 第三章 螢光探針設計構想 25 3-1 探針選擇 25 3-2 蛋白質結合 27 3-3 螢光探針設計 28 第四章 結果與討論 29 4-1 螢光探針合成 29 4-2 螢光探針性質測試 30 4-2-1 螢光測試 30 4-2-2 選擇性及偵測極限測試 30 4-2-3 動力學測試 32 4-3 細胞實驗 33 4-3-1 探針BGNR之細胞成像 33 4-3-2 核纖層蛋白合成之細胞成像 40 4-3-3 核纖層蛋白間交互作用之細胞成像 42 4-3-4 法尼基化PreA之轉譯後修飾過程 48 4-3-5 非法尼基化PreA之轉譯後修飾過程 50 第五章 結論 54 第六章 實驗部分 56 6-1 實驗藥品及器材 56 6-2 螢光實驗測試 57 6-3 細胞實驗測試 57 6-3.1 培養基及試劑 57 6-3.2 細胞繼代培養 57 6-3.2.1 MCF7 / CHO / Hela / HEK293 / HCT116細胞 57 6-3.2.2 RAW264.7細胞 58 6-3.2.3 活體細胞實驗 58 6-3.2.4 雙色脈衝追蹤細胞影像 59 6-3.2.5 變異之核纖層蛋白轉譯後修飾過程追蹤之細胞成像 59 6-3.2.6 光漂白細胞實驗 60 6-4 有機合成與光譜資料 60 參考文獻 63 附錄 72

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    記,國立清華大學碩士論文,2014.


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