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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23234完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡懷楨(Huai-Jen Tsai) | |
| dc.contributor.author | Chiu-Chun Lin | en |
| dc.contributor.author | 林秋君 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:48:56Z | - |
| dc.date.copyright | 2009-07-30 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23234 | - |
| dc.description.abstract | Myf5 是肌肉發育 (myogenesis) 調控因子MRFs (myogenic regulatory factors) 的一員，對於肌肉的特化及分化都具有重要的功能。在斑馬魚myf5 intron I 中含有一個 microRNA，命名為miR-In300，它能夠結合dickkopf 3 (dkk3) 3’UTR並抑制其轉錄作用，進而負調控 myf5 表達影響肌肉發育。但 Dkk3 是一種分泌性蛋白質，它是藉由何種調控路徑來影響 myf5 的表達尚不清楚。在本實驗中我們發現抑制 Dkk3 會造成磷酸化的p38a蛋白質下降；注射 p38a 專一轉譯抑制劑 (morpholino, MO) knockdown p38a 的表現後，發現胚胎體節形狀不正常，且 myf5 mRNA表達較少，這些表型和注射dkk3-MO 的胚胎類似，且利用dominant-negative form 的p38a 競爭也得到相同的結果。利用 p38 inhibitor (SB203580) 抑制 p38 磷酸化阻斷MAPK pathway亦會造成myf5下降。當注射p38a-MO到帶有 myf5上游 80kb 啟動子驅動 GFP 的斑馬魚轉殖品系Tg(myf5(80K):GFP) 之胚胎後，會造成myf5啟動子驅動GFP的表現能力下降。若額外添加 p38a mRNA就可以挽救因注射p38a-MO所造成綠螢光表現下降的缺陷，但若額外添加磷酸化位點突變的 p38a mRNA則無法達到挽救的效果。這些結果證明 p38a 是經由磷酸化作用來調控 myf5啟動子的活性。另一方面，加入dominant-negative form 的Smad2及Smad3a 後，胚胎體節發育不正常且 myf5 表現下降，這和注射dkk3-MO及p38a-MO的胚胎有類似的表型。利用 luciferase assay 及注射 smad2/3a/3b/4 mRNA 到Tg(myf5(80K):GFP) 轉殖品系的胚胎，發現過量表現 Smad2 及Smad3a 會增強myf5 啟動子的活性；更進一步地，利用Chromatin immunoprecipitation assay 證實 Smad3a 可直接結合到 myf5 啟動子上，藉由 Smad2/3a complex 開啟 myf5 的啟動子。而當抑制 Dkk3 或 p38a 時，Smad4 蛋白質表現會下降而Smad3蛋白質量不改變；且過量表現 smad4 mRNA可以挽救 dkk3-MO 造成 myf5 下降的情形。綜合上述證據，我們總結 Dkk3 藉由調控 p38a 的磷酸化來維持 Smad4 蛋白質穩定性，進而使 Smad4 能與 Smad2/3a 形成complex 來開啟斑馬魚 myf5 的啟動子。 | zh_TW |
| dc.description.abstract | Myf5, one of the myogenic regulatory factors, plays roles in the specification and differentiation of muscular cells during myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through targeting dkk3. However, the detailed molecular mechanism underlying how the secreted Dkk3 controls the myf5 expression is totally unknown. In this study, we found that knockdown of dkk3 reduced the protein level of the phosphorylated p38a. Injection of p38a-specific morphorlino (MO), which inhibits the translation of p38a mRNA, resulted that the malformed somites and the reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3-MO. These phenotypes were also observed in the embryos injected with the dominant-negative form of p38a. Blocking the MAPK pathway through interfering the phosphorylation of p38 by introducing SB203580 caused the down-regulation of myf5 expression. Furthermore, the GFP fluorescent signal was dramatically decreased when we injected p38a-MO into the embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by myf5 promoter. This p38a-MO-induced defects were rescued by co-injection with p38a mRNA, but were not rescued with the mutated p38a mRNA containing a mutation at phosphorylation domain. This line of evidences suggested that the phosphorylation of p38a is required for activating of myf5 promoter activity. Moreover, the defective phenotypes induced by injection the dominant-negative form of either Smad2 or Smad3 were as same as those of embryos injected with either dkk3- or p38a-MO. Using luciferase assay and injection of samd2/3a/4 mRNA in the embryos derived from Tg(myf5(80K):GFP), we found that overexpression of Smad2 and Smad3a did enhance the myf5 promoter activity. Furthermore, we proved that Smad3a directly bound at the upstream regulatory region of myf5 using chromatin immunoprecipitation assay. Interestingly, knockdown of either dkk3 or p38a resulted in the decrease of Smad4 protein level, but did not change the Smad3 protein level. Injection of smad4 mRNA enabled embryos to rescue the down-regulation of myf5 in the dkk3-MO-injected embryos. Taken together, we concluded that Dkk3 regulates the phosphorylation of p38a to maintain the stability of Smad4 protein, which, in turn, to allow the complex of Smad2/3a/4 to activate the zebrafish myf5 promoter activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:48:56Z (GMT). No. of bitstreams: 1 ntu-98-R96b43009-1.pdf: 18068949 bytes, checksum: 138b48f867df8cc60efc955fc2c6fcbc (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要 ------------------------------------------------------ 1
英文摘要 ------------------------------------------------------ 2 文獻回顧 ------------------------------------------------------ 4 前言 ----------------------------------------------------------- 15 實驗材料與方法 -------------------------------------------- 17 結果 ----------------------------------------------------------- 29 討論 ----------------------------------------------------------- 42 總結 ----------------------------------------------------------- 53 參考文獻 ----------------------------------------------------- 54 圖表 ----------------------------------------------------------- 75 附錄 ----------------------------------------------------------- 96 | |
| dc.language.iso | zh-TW | |
| dc.subject | Dkk3 | zh_TW |
| dc.subject | Smad4 | zh_TW |
| dc.subject | 肌肉發育 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | Myf5 | zh_TW |
| dc.subject | Smad4 | en |
| dc.subject | Myf5 | en |
| dc.subject | Dkk3 | en |
| dc.subject | myogenesis | en |
| dc.subject | zebrafish | en |
| dc.title | Dickkopf 3 (Dkk3)藉由調控p38a的磷酸化來維持Smad4蛋白質穩定性進而影響斑馬魚myf5 啟動子開啟 | zh_TW |
| dc.title | Dickkopf 3 (Dkk3) regulates the expression of zebrafish myf5 promoter via phosphorylated p38a-dependent Smad4 stability | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂勝春,王育民,蔡振寧 | |
| dc.subject.keyword | 斑馬魚,肌肉發育,Dkk3,Myf5,Smad4, | zh_TW |
| dc.subject.keyword | zebrafish,myogenesis,Dkk3,Myf5,Smad4, | en |
| dc.relation.page | 100 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2009-07-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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