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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹智強(Chih-Chiang Chan) | |
| dc.contributor.author | Yu-Chien Hung | en |
| dc.contributor.author | 洪瑜謙 | zh_TW |
| dc.date.accessioned | 2022-11-25T05:35:12Z | - |
| dc.date.available | 2027-01-03 | |
| dc.date.copyright | 2022-01-18 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-01-04 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82070 | - |
| dc.description.abstract | UQCRC1 (Ubiquinol-Cytochrome C Reductase Core Protein 1)是粒線體中電子傳遞鍊上的complex III其中一個次單元蛋白，先前多將其視為粒線體標記，偶有研究提及UQCRC1和神經退化性疾病間的關聯，但多為DNA/RNA階段。近年來開始亦有研究探討UQCRC1蛋白於神經細胞中的功能性。與本實驗室合作之林靜嫻醫師在先前針對遺傳性帕金森氏症患者及其家族進行全外顯子定序(whole exome sequence，WES)檢測，試圖發現與帕金森氏症相關之未知基因，而從其中發現UQCRC1單位點變異可能與帕金森氏症有關。先前對於UQCRC1於細胞之功能了解甚微，因此本研究希望以果蠅與人類神經細胞為模式，探討UQCRC1於粒線體或於神經細胞中所扮演之角色。從實驗中發現，於果蠅神經系統減少UQCRC1同源異構物uqcrc1，會導致果蠅爬行能力降低和多巴胺分泌神經數量減少，與人類帕金森氏症之病之類似；且發現完全剔除uqcrc1會導致果蠅於發育階段即死亡，可能是由於細胞凋亡(apoptosis)的發生。這些因缺少uqcrc1而導致的現象，都可因重新表現UQCRC1而減緩，表示人類與果蠅於該蛋白有些許功能方面之保守性。此外，我們亦發現減少uqcrc1會導致cyt-c移動至細胞質中，在先前研究也認為cyt-c從粒線體移至細胞質會與細胞凋亡的活化起始相關。後續實驗中，減少cyt-c表現量亦或表現細胞凋亡抑制因子p35都可減緩由於失去uqcrc1造成的神經退化現象。由此可知，UQCRC1或有調控cyt-c影響細胞凋亡發生之作用。此外，我們發現失去uqcrc1也會導致autophagy相關蛋白表現增加，且可能會與PINK1共同調控autophagy的進行。最後，我們還看到糖解作用酵素表現會受uqcrc1或cyt-c影響，可能是以此維持細胞中能量的生成。 | zh_TW |
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| dc.description.tableofcontents | "謝辭 I 中文摘要 II 英文摘要 III 目錄 IV 第一章 研究背景 1 1.1 粒線體中電子傳遞鍊之作用 2 1.2 Complex III (CIII)於電子傳遞鍊中之重要性 3 1.3 UQCRC1 (Ubiquinol-Cytochrome C Reductase Core Protein 1) 5 1.4 細胞色素c (cytochrome c)之於細胞凋亡 6 1.5 粒線體功能異常與神經退化性疾病 8 1.6 瓦氏效應 (Warburg Effect) 8 1.7 細胞自噬 (autophagy) 9 1.8 研究目的 11 第二章 研究材料與方法 12 2.1 果蠅飼養條件與方法 13 2.2 實驗用果蠅株 13 2.3 實驗用抗體 14 2.4 製備uqcrc1基因剔除果蠅株(CRISPR/Cas9 KO and primers) 15 2.5 製備UQCRC1基因剔除細胞株(SH-SY5Y KO cell line) 16 2.6 製備anti-uqcrc1和anti-UQCRC1抗體 16 2.7 即時定量聚合酶連鎖反應 (Real-time quantitative PCR, qPCR) 17 2.8 西方墨點法(western blotting) 19 2.9 果蠅爬行能力檢測 20 2.10 組織免疫螢光染色 (Immunohistochemistry) 21 2.11 細胞或果蠅組織ATP含量檢測 21 2.12 經由穿透式電子顯微鏡觀察果蠅飛行肌的粒線體型態(TEM) 22 2.13 神經元軸突和粒線體功能檢測 22 2.14 於幼蟲眼碟細胞剔除uqcrc1並檢測活性氧化物 23 2.15 從果蠅飛行肌分離粒線體(mitochondria isolation) 23 2.16 共同免疫沉澱法(co-IP) 23 2.17 蛋白質交互作用鄰位連接法(Proximity Ligation Assay) 24 2.18 果蠅壽命檢測 24 2.19 影像處理 25 2.20 量化及統計方法 25 第三章 研究結果 27 3.1 敲落果蠅uqcrc1導致神經退化之表徵 28 3.2 剔除uqcrc1導致果蠅個體死亡 29 3.3 剔除uqcrc1導致粒線體功能異常及細胞凋亡蛋白表現增加 31 3.4 敲落uqcrc1導致粒線體型態異常 32 3.5 剔除UQCRC1影響細胞軸突生長和降低呼吸鍊複合體活性 33 3.6 剔除uqcrc1改變粒線體中cyt-c分布和表現量 34 3.7 果蠅uqcrc1和cyt-c的交互作用 35 3.8 細胞凋亡抑制因子p35和cyt-c的調控的救援結果 37 3.9 UQCRC1和各胞器間的共位性 38 3.10 UQCRC1與細胞自噬之相關性 39 3.11 敲落uqcrc1或cyt-c會影響糖解作用活化 41 第四章 研究討論 43 4.1 UQCRC1缺失導致神經退化之探討 44 4.2 神經退化性疾病與cyt-c的相關性 45 4.3 果蠅之cyt-c與人類細胞cyt-c作用之差異性 46 4.4 UQCRC1和細胞自噬的相關性 47 4.5 uqcrc1、cyt-c與糖解作用的相關性 48 第五章 實驗圖表 50 Fig. 1. 於果蠅神經系統中敲落uqcrc1基因會導致爬行能力降低 51 Fig. 2. uqcrc1缺失會導致果蠅DA神經群減少，另外表現UQCRC1有救援效果 53 Fig. 3. 以CRISPR/Cas9基因編輯進行uqcrc1基因剔除，建立uq-KO果蠅株 55 Fig. 4. 以果蠅uqcrc1或人類UQCRC1救援因缺少uqcrc1所導致之果蠅表型 57 Fig. 5. uqcrc1缺失導致果蠅幼蟲腦部和肌肉中粒線體型態異常 59 Fig. 6. 失去uqcrc1導致粒線體型態異常和細胞凋亡相關蛋白表現量增加 61 Fig. 7. 於果蠅飛行肌中減少uqcrc1表現量會導致粒線體於功能和形態之異常 62 Fig. 8. 以CRISPR/Cas9基因編輯剔除SHS-Y5Y之UQCRC1並觀察其生長型態和呼吸鏈中各複合體之活性 64 Fig. 9. 野生型細胞和UQ-KO細胞中蛋白表現之差異 66 Fig. 10. 失去uqcrc1會增加cyt-c表現量，重新表現uqcrc1或UQCRC1可調控 67 Fig. 11. 失去uqcrc1使cyt-c與粒線體共位性降低，並活化下游細胞凋亡蛋白酶 68 Fig. 12. 敲落uqcrc1使cyt-c從粒線體中釋出至細胞質中 69 Fig. 13. cyt-c與uqcrc1/UQCRC1之交互作用 71 Fig. 14. 減少cyt-c表現量或大量表現細胞凋亡抑制因子p35可救援由失去uqcrc1所造成之複眼生長狀況 72 Fig. 15. 減少cyt-c表現量或大量表現細胞凋亡抑制因子p35可救援由失去uqcrc1所造成之爬行能力降低 73 Fig. 16. 於果蠅複眼觀察UQCRC1和胞器的共位性 75 Fig. 17. 比較WT和uq-KO細胞中自噬相關蛋白表現量，並觀察兩者的共位性 76 Fig. 18. 在果蠅複眼同時表現HA-PINK1和UQCRC1-V5，觀察兩者的共位性 78 Fig. 19. 於果蠅神經敲落uqcrc1會導致糖解作用相關酵素表現降低，但同時調控cyt-c可以其表現量回復 79 Fig. 20. 檢測果蠅神經細胞中與糖解作用相關的酵素mRNA表現量 80 參考文獻 81 附錄 91" | |
| dc.language.iso | zh-TW | |
| dc.subject | 粒線體 | zh_TW |
| dc.subject | UQCRC1 | zh_TW |
| dc.subject | 果蠅 | zh_TW |
| dc.subject | 神經退化 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 細胞色素c | zh_TW |
| dc.subject | 帕金森氏症 | zh_TW |
| dc.subject | Parkinson’s disease | en |
| dc.subject | mitochondria | en |
| dc.subject | UQCRC1 | en |
| dc.subject | cytochrome c | en |
| dc.subject | apoptosis | en |
| dc.subject | neurodegeneration | en |
| dc.subject | Drosophila | en |
| dc.title | 探討UQCRC1於神經退化性疾病模式之調控機制 | zh_TW |
| dc.title | Molecular investigation of UQCRC1 in a disease model of neurodegeneration | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳俊宏(Hsin-Tsai Liu),林靜嫻(Chih-Yang Tseng),蔡素宜,黃舒宜 | |
| dc.subject.keyword | 果蠅,UQCRC1,粒線體,神經退化,細胞凋亡,細胞色素c,帕金森氏症, | zh_TW |
| dc.subject.keyword | Drosophila,UQCRC1,mitochondria,neurodegeneration,apoptosis,cytochrome c,Parkinson’s disease, | en |
| dc.relation.page | 91 | |
| dc.identifier.doi | 10.6342/NTU202104550 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-01-04 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生理學研究所 | zh_TW |
| dc.date.embargo-lift | 2027-01-03 | - |
| 顯示於系所單位： | 生理學科所 | |
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