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研究生:林憶珊
研究生(外文):Yi-Shan Lin
論文名稱:微小管相關蛋白質1A在COS7細胞株之功能探討
論文名稱(外文):Functional Studies of Microtubule Associated Protein 1A (MAP1A) in COS7 Cell Line
指導教授:錢宗良錢宗良引用關係
指導教授(外文):C.L Chien
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:解剖學暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:微小管相關蛋白質1A綠色螢光蛋白微小管
外文關鍵詞:Microtubule Associated Protein 1A (MAP1A)GFPMycmicrotubuleTaxolNocodazol
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微小管相關蛋白質1A (Microtubule Associated Protein 1A, MAP1A),是由分子量277kDa重鏈和分子量28kDa輕鏈,所組成的高分子量蛋白質。MAP1A在生物體內伴隨著微小管,廣泛地分佈在成熟的神經細胞和神經膠細胞中;而在生物體外,MAP1A蛋白質可以直接和微小管接合,並且促進微管素 (tubulin) 的聚合 (polymerization);因此,傳統上MAP1A被認為在穩定細胞骨架中扮演相當重要的角色。
為了深入探討MAP1A蛋白質在細胞內和微小管的關聯性,我們轉殖了以下幾種以Myc或是GFP為標籤的DNA架構: 1. 表達完整MAP1A蛋白質的MAP1A DNA; 2. 表達MAP1A蛋白質自體相似區域SS1的DNA片段; 3. 表達MAP1A蛋白質自體相似區域SS2的DNA片段;4. 表達MAP1A重鏈蛋白的DNA片段;5. 表達MAP1A輕鏈蛋白的DNA片段。經由在COS7細胞內的表達,來探討MAP1A蛋白質在COS7細胞體內與微小管的互動關係,以及在微小管相關藥物Taxol或者Nocodazol處理後,MAP1A蛋白質與微小管的變化情形。
本論文的實驗結果發現,只有在COS7細胞體內表現完整MAP1A蛋白質,或是共同轉殖表現MAP1A重鏈與輕鏈蛋白的情況下,MAP1A才能夠和微小管共同分佈 (co-localization) 在細胞質中而呈現絲狀結構。此外,MAP1A蛋白質伴隨微小管均會受到藥物Taxol的作用影響,產生聚集成束的情形;而在藥物Nocodazol的作用下,則造成絲狀結構的瓦解。
綜合本論文研究結果,我們推測MAP1A重鏈與輕鏈蛋白之間的相互作用,將會影響MAP1A蛋白質與微小管的接合;而MAP1A輕鏈蛋白則成為微小管相關蛋白質1A在細胞骨架結構中不可或缺的重要成份。

Microtubule Associated Protein 1A (MAP1A) is a high molecular weight protein that comprises heavy chain (277kDa) and light chain (28kDa) and is widely distributed along the microtubules in both mature neurons and glial cells. MAP1A also binds to microtubule directly and promotes the polymerization of tubulin in vitro. Therefore, MAP1A is regarded as an important protein to stabilize cytoskeleton.
To illustrate the association between MAP1A and microtubule, we prepared five DNA constructs with Myc-Tagged or EGFP-tagged: (1) MAP1A DNA expressing whole MAP1A protein; (2) MAP1A-SS1 DNA segment expressing MAP1A self-similar region 1; (3) MAP1A-SS2 DNA segment expressing MAP1A self-similar region 2; (4) MAP1A-HC DNA segment expressing MAP1A heavy chain; (5) MAP1A-LC2 DNA segment expressing MAP1A light chain. Tagged DNA constructs were delivered into COS7 cells by lipofectamine transfection. We investigated the distribution of MAP1A as well as the interaction with microtubule in the transfected cells via immunocytochemistry. Morphological changes of MAP1A and microtubules after Taxol or Nocodazol treatment were also observed from those transfected cells.
In this study, we showed that a complete MAP1A protein which contains both heavy chain and light chain could be colocalized with microtubule network in the cytoplasm of transfected COS7 cells. Cells expressed MAP1A heavy chain or light chain alone failed to be visualized on the filamentous microtubules. MAP1A heavy chain can be decked onto microtubule only under co-transfection of both heavy chain and light chain. MAP1A together with microtubule becomes bundling in the MAP1A-transfected cells after Taxol treatment. MAP1A was affected prior to the microtubule and disengaged from the depolymerizing microtubule after Nocodazol treatment.
From our observation, we suggest that the interaction between MAP1A heavy chain and light chain is critical in the association with microtubule and its light chain functions as an indispensable component of microtubule-associated protein 1A.

壹、中英文摘要 ---------------------------------------------- 1
貳、緒論 ---------------------------------------------------- 5
微小管相關蛋白質1A ------------------------------------------ 5
參、實驗材料與方法 ------------------------------------------10
肆、實驗結果 ----------------------------------------------- 16
MAP1A DNA架構在細胞體內的表達 ------------------------------ 16
Taxol和Nocodazol處理後MAP1A蛋白質的表現 -------------------- 19
Western Blot的分析 ----------------------------------------- 21
伍、討論 --------------------------------------------------- 23
陸、參考文獻 ----------------------------------------------- 28
柒、圖表說明 ----------------------------------------------- 35

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