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研究生:孫義坤
研究生(外文):Yi-Kun Sun
論文名稱:(I)分析Dyneinheavychain被裂解對細胞的影響(II)以化學交聯法探討細胞核膜蛋白質之成份
論文名稱(外文):Cleavage of dynein heavy chain UV irradiation in the presence of vanadatePull out outer membrane proteins of rat liver nuclei by nano-depth-tagging methodologies
指導教授:張兗君
指導教授(外文):Yen-Chung Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:92
語文別:中文
論文頁數:64
中文關鍵詞:運動蛋白細胞核外膜
外文關鍵詞:Dynein Heavy Chainouter nuclear membrane
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本篇論文分為兩部分,第一部分是為研究在細胞中 Dynein Heavy Chain (DHC) 的生理作用。 Cytoplasmic dynein 是一種運動蛋白,靠 DHC 水解 ATP 產生能量,使 dynein 能在微管 (microtubule) 上移動運輸物質,並且和細胞分裂時胞器的位置有關。當我們在細胞中外加 100 μM vanadate (Vi) 並照射 UV 光使 DHC 裂解,觀察 DHC 被裂解後對細胞生理作用的影響。結果發現照 UV 光會使細胞大量死亡,導致無法繼續研究。
本論文之第二部分是利用大小約 80 μm 的 agarose beads 接上交聯試劑 (cross-linking reagent) 後,來標定 (pulled out) 出細胞核外膜上蛋白質。進一步用二維電泳法分析比較外膜蛋白質與原來細胞核蛋白質的成份差異。選出有顯著差異的膠點進行 in-gel digestion ,再利用 MALDI-TOF 儀器分析鑑定蛋白質的身份。鑑識出來的結果來對照這些蛋白質可能出現在細胞核膜上的位置。最明顯的就是 Lamin A這個蛋白質不會出現在標定之蛋白質裡。由此可證明這個方法不但是成功的標定出細胞核外膜上蛋白質,而且還可以了解外層核膜蛋白質的立體結構,來推測這些蛋白質在核膜上扮演了何種角色。
壹、前言…………………..……….……………………………………1
貳、材料與方法………………….…..…………………………………5
(一)、藥品與材料………………………………….…………………..5
(二)、老鼠的育種…………………………….…...………………….6
(三)、細胞培養………………………………………………….……..6
(四)、細胞加入Vanadate後照UV光………….……………….……….9
(五)、計算細胞存活率……………………….….……………………10
(六)、凝膠電泳分析……………..…………..……………………..11
(七)、西方點漬法……………………………..………………………13
(八)、老鼠肝臟細胞核的標定……...………..…………………….14
(九)、蛋白質濃度的測定…………...………..…………………….15
(十)、奈米標定細胞核膜表面蛋白質………..……………………..15
(十一)、蛋白質沉澱………………………….….……………………16
(十二)、二維凝膠電泳分析………………….….……………………17
(十三)、膠片染色法………………………….….……………………19
(十四)、膠體內蛋白質分解……………..…….…………………...21
參、結果…………..……………………….……………...…………23
(一)、DHC光裂解………………..……………....……….…………23
(二)、DHC光裂解後細胞的存活率………....………………...…..23
(三)、純化細胞核與beads鍵結的細胞核…….…..…………....…24
(四)、不同交聯劑 (cross-linker) 奈米標定細胞核表面蛋白質…24
(五)、一維電泳分析………………………………………....….….25
(六)、二維電泳分析………………………………………..…...….25
(七)、以MALDI-TOF MS 鑑定pulled out 及total nuclear proteins..................................................26
肆、討論…….…...…………………………………………..……..28
(一)、DHC光裂解………..……………………………………….…..28
(二)、細胞核純化……………………..………………………………29
(三)、利用不同cross-linker 標定 Pulled out proteins…..….30
(四)、Pulled out proteins 與 Total nuclear proteins 比較…30
(五)、未來展望…………………………………………..……...….33
伍、 參考文獻………………………………………………….………35
陸、 圖表集………………………………………………….…………40
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