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研究生:邱奕潔
研究生(外文):Yi-Chieh Chiu
論文名稱:粒線體Lon蛋白酶藉由結合熱休克蛋白60/70複合體調節細胞的凋亡
論文名稱(外文):Mitochondrial Lon Protease Regulates Apoptosis via the Interaction with Hsp60/mtHsp70 Complex
指導教授:高婷玉高婷玉引用關係
指導教授(外文):Ting-Yu Kao, Ph.D.
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學檢驗生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:59
中文關鍵詞:粒線體Lon蛋白酶熱休克蛋白60熱休克蛋白60細胞凋亡
外文關鍵詞:MitochondrialLon ProteaseHeat shock proteins 60Heat shock proteins 70Apoptosis
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人類Lon蛋白酶是一種存在在粒線體基質的ATP依賴型的多功能蛋白質,參與分解遭氧化損壞的粒線體蛋白質、重新摺疊損傷蛋白質的錯誤結構、調節細胞凋亡、參與細胞壓力反應途徑。在我們之前的研究發現,人類Lon蛋白酶在各種癌症中都發現會過量的表現,而且會抑制癌細胞的凋亡。然而人類Lon蛋白酶如何調節粒線體參與的細胞凋亡機制仍不清楚。Hsp60 (熱休克蛋白60)和mtHsp70 (線粒體熱休克蛋白70 )已被證明是參與蛋白質裝配和折疊、並在壓力下調控線粒體凋亡。之前的蛋白質體研究結果發現,人類Lon蛋白酶可能和Hsp60/mtHsp70結合形成複合體。在本研究中,我們經由免疫共沉澱(co-immunoprecipitation),GST pull down和免疫螢光共定位實驗驗證人類Lon蛋白酶和Hsp60/mtHsp70複合體的交互作用。我們的結果發現Hsp60和mtHsp70的蛋白質穩定性取決於人類Lon蛋白酶。此外,線粒體Lon蛋白酶在壓力下調控細胞凋亡是與Hsp60有關。
Mitochondrial Lon is a multi-function protein that contains protease, ATPase, DNA binding, and chaperone activities. Lon is a stress protein and associated with protein quality control, apoptosis, and stress response pathways. Our previous study showed that overexpression of Lon, found in various cancers, prevents cancer cell from apoptosis under stress. However, the mechanism of how Lon regulates mitochondria-mediated apoptosis remains unclear. We have identified Hsp60/mtHsp70 complex as Lon-associated proteins by proteomics approach using the Lon-overexpressing cells. Hsp60 (heat-shock protein 60) and mtHsp70 (mitochondrial Hsp70) have been shown to be involved in protein assembly and folding and apoptosis regulation under stress in mitochondria. In the present study, the association between Lon and Hsp60/mtHsp70 complex was confirmed by co-immunoprecipitation (Co-IP), GST pull down assay, and immunofluorescence co-localization assay. Our results showed that the protein stability of the Hsp60 and mtHsp70 is dependent on Lon protein. Moreover, mitochondrial Lon-regulated apoptosis is dependent on Hsp60 under stress.
誌謝 VI
中文摘要 VII
英文摘要 VIII
目錄 IX
圖目錄 XI
第一章 緒論………………………………………………….…………………1
1.1 研究背景與動機……………………………….………………….1
1.2 研究問題及目的……………………………….………………….2
第二章 介紹………………………………………………….…………………3
2.1 粒線體
2.2 人類Lon蛋白酶
2.3 Molecular chaperones
2.4 熱休克蛋白
2.5 熱休克蛋白60與粒線體熱休克蛋白蛋白70
2.6 細胞凋亡
2.7 細胞凋亡訊息傳遞路徑
2.8 粒線體細胞凋亡
2.9 粒線體途徑相關的凋亡因子
第三章 材料方法……………………………….…………………………….12
3.1 實驗細胞培養
3.2 配置培養液
3.3 細胞解凍與培養
3.4 冷凍細胞
3.5 Transformation
3.6 抽取質體
3.7 細胞蛋白質萃取
3.8 蛋白質定量
3.9 SDS-PAGE
3.10 蛋白質轉漬
3.11 細胞轉染(Cell Transfection)
3.12 TUNEL 染色法(TUNEL assay)
3.13 免疫共沉澱(Co-Immunoprecipitation)
3.14 GST pull-down assay
第四章 結果……………………………….…………………………………..18
第五章 討論與結論……………………………….…………………………..20
參考文獻……………………………….…………………………………..…..41
補充資料
(第二十九屆生物醫學聯合學術年會參加證明、第一屆醫護福祉學院專題製作比賽得獎獎狀)

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