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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊榮輝 | |
| dc.contributor.author | Hua-Shan Shih | en |
| dc.contributor.author | 施驊珊 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:20:54Z | - |
| dc.date.available | 2016-08-23 | |
| dc.date.copyright | 2011-08-23 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-15 | |
| dc.identifier.citation | 沈志昱 (2008) 哺乳類動物胚胎著床前關鍵蛋白質之抗體製備及蛋白質體研究。碩士論文。國立臺灣大學微生物與生化學研究所，台北。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41498 | - |
| dc.description.abstract | 動物早期胚胎發育過程中，母源-胚源轉換期 (maternal-to-zygotic transition, MZT)，對於胚胎的正常發展扮演重要角色。本論文利用蛋白質二維電泳，分析斑馬魚 (Danio rerio) 胚胎MZT前後之關鍵調控蛋白質。取得班馬魚256-細胞期及球體期胚胎分別作為MZT前後之標準樣本，萃取此兩時期胚胎的整體蛋白質，進行二維電泳分析，觀察MZT前後之蛋白質體差異。於二維電泳圖譜中比對出17個表現差異之蛋白質，取其中差異最顯著之六個蛋白質色點，進行ESI-QUAD-TOF身份鑑定。以等電點及分子量比對後，鑑定出五種蛋白質，分別為：hypothetical protein LOC768289、mitochondrial processing peptidase alpha (MPP-alpha)、aldehyde dehydrogenase 2b (ALDH2b)、methylmalonate-semialdehyde dehydrogenase (MMSDH) 及ubiquitin C-terminal hydrolase L1 (UCHL1)。這些蛋白質可能和粒線體運輸、支鏈胺基酸代謝或細胞週期調控相關。此外，針對這幾個差異性蛋白質及lactate dehydrogenase (LDH) 上游mRNA，進行半定量RT-PCR，發現在MZT前後其mRNA消長與蛋白質一致，其中UCHL1、MPP-alpha及LDH在統計上具有顯著差異。為了瞭解這些關鍵蛋白質對MZT的影響，利用顯微注射技術，將LDH及UCHL1的專一性抑制劑：oxamate及LDN57 444，注射入斑馬魚胚胎，導致胚胎分別有細胞分裂延遲及體節縮短的現象。另外，測定LDH在MZT前後的酵素活性，在256-細胞期的LDH活性高於球體期者。這些差異性蛋白質可能和早期胚胎粒線體之能量代謝相關，未來可朝早期胚胎代謝體學的方向深入調查，可藉由專一性單株抗體、利用轉殖基因動物或RNA干擾等技術，進一步了解這些蛋白質調控胚胎發育之生理意義。 | zh_TW |
| dc.description.abstract | Maternal-to-zygotic transition (MZT) plays an important role in early embryogenesis. In this study, zebrafish embryos at stages of 256-cell and sphere were used as the standard samples for pre- and post-MZT. Totally, 17 proteins expressed differentially during MZT were picked out by two-dimensional electrophoresis (2-DE). Six of these proteins were identified as hypothetical protein LOC768289, mitochondrial processing peptidase alpha (MPP-alpha), aldehyde dehydrogenase 2b (ALDH2b), methylmalonate-semialdehyde dehydrogenase (MMSDH) and ubiquitin C-terminal hydrolase L1 (UCHL1) by using ESI-QUAD-TOF. These proteins might relate to mitochondrial protein import pathway, branched-chain amino acid metabolism, or cell cycle regulation. Furthermore, we detected the mRNA expression of MPP-alpha, ALDH2b, MMSDH, UCHL1 and LDH (lactate dehydrogenase) by semi quantitative RT-PCR. These results were comparable to 2-DE data. The expression differences of UCHL1, MPP-alpha and LDH during MZT were statistically significant. In an attempt to study the function of these proteins during MZT, we used the specific inhibitors, oxamate and LDN57 444, to inhibit LDH or UCHL1, respectively. The phenotypes of cleavage delay and dorsalis were observed in inhibitors treated embryos. Additionally, we found that LDH activity in 256-cell was higher than that in sphere by LDH activity assay. In conclusion, our results demonstrated that the change of the protein profile during MZT may relate to mitochondrial energy metabolism. These proteins can be further studied by using specific monoclonal antibody, transgenic animal model, or RNA interference. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:20:54Z (GMT). No. of bitstreams: 1 ntu-100-R98b47207-1.pdf: 2936404 bytes, checksum: 03938aa318584fe470cef240475f94ba (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 縮寫表 iii 第一章 緒論 1 1.1斑馬魚早期胚胎發育概述 1 1.2母源-胚源轉換期 2 1.2.1胚源RNA合成與母源RNA降解 3 1.2.2細胞週期延長及細胞分裂速度不一 3 1.2.3 YSL的形成 4 1.2.4細胞移動及細胞命運的決定 4 1.3發育阻障與MZT發生時間一致 4 1.4 MZT前後代謝機制的轉變 5 1.4.1小鼠早期胚胎代謝機制 5 1.4.2硬骨魚類早期胚胎代謝 6 1.5從蛋白質體學角度探討MZT 7 1.6研究動機及實驗材料選定 8 第二章 材料與方法 10 2.1動物材料 10 2.1.1斑馬魚飼養條件及胚胎收集 10 2.2蛋白質樣本製備 10 2.2.1去除卵殼及卵黃 10 2.2.2甲醇-氯仿沈澱 11 2.2.3蛋白質樣本定量 11 2.3蛋白質體實驗 11 2.3.1西方墨點分析 11 2.3.2第一次元：等電焦集法 12 2.3.3第二次元：SDS-PAGE 12 2.3.4膠體內水解及ESI-QUAD-TOF分析 12 2.4蛋白質功能性分析 13 2.4.1蛋白質專一性抑制劑顯微注射 13 2.4.2 LDH在MZT前後活性分析 13 2.5基因表現測定 14 2.5.1斑馬魚胚胎RNA萃取 14 2.5.2 cDNA製備 14 2.5.3半定量RT-PCR 14 第三章 結果 15 3.1斑馬魚胚胎在MZT前後的蛋白質體研究 15 3.1.1去除卵黃效率分析 15 3.1.2二維電泳樣本製備及解析條件 15 3.1.3 MZT前後的二維電泳圖譜 16 3.1.4 MZT前後有五個明顯差異性消長的蛋白質 16 3.2以半定量偵測MZT前後關鍵因子的mRNA表現量 16 3.2.1設計目標基因專一性引子 16 3.2.2 MZT前後關鍵因子mRNA表現情形 17 3.3 MZT前後的關鍵蛋白質功能性分析 17 3.3.1 LDH在MZT前後的活性分析 17 3.3.2專一性抑制LDH對早期胚胎發育的影響 18 3.3.3專一性抑制UCHL1對胚胎發育後期的影響 19 圖與表 20 第四章 討論 36 4.1斑馬魚胚胎MZT前後之蛋白質體研究 36 4.1.1 MPP-alpha 與粒線體蛋白質運輸相關 36 4.1.2 ALDH2b及MMSDH與支鏈胺基酸代謝有關 37 4.1.3 UCHL1調控MZT的細胞週期轉換 38 4.2以基因角度觀察MZT關鍵因子mRNA表現 38 4.3功能性分析 39 4.3.1專一性抑制劑作用原理 39 4.3.2顯微注射結果 39 4.3.3 LDH在MZT前活性較高 40 4.4 MZT前胚胎能量代謝機制之推論 40 4.4.1糖解作用可能在MZT前不活躍 40 4.4.2乳酸可能是MZT前能量代謝來源 41 4.4.3 MAS shuttle可能為調控MZT前能量代謝的因子 41 第五章 結論 49 答問錄 51 參考文獻 56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 母源-胚源轉換期 | zh_TW |
| dc.subject | 蛋白質體學 | zh_TW |
| dc.subject | 二維電泳 | zh_TW |
| dc.subject | 能量代謝 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | Proteomics | en |
| dc.subject | Energy metabolism | en |
| dc.subject | Zebrafish | en |
| dc.subject | Maternal-to-zygotic transition (MZT) | en |
| dc.subject | Two-dimensional electrophoresis (2-DE) | en |
| dc.title | 斑馬魚早期胚胎發育相關蛋白質之研究 | zh_TW |
| dc.title | Studies on Early Embryonic Development-Related Proteins in Zebrafish (Danio rerio) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李士傑,常怡雍,陳翰民,張世宗 | |
| dc.subject.keyword | 母源-胚源轉換期,蛋白質體學,二維電泳,能量代謝,斑馬魚, | zh_TW |
| dc.subject.keyword | Maternal-to-zygotic transition (MZT),Proteomics,Two-dimensional electrophoresis (2-DE),Energy metabolism,Zebrafish, | en |
| dc.relation.page | 66 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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