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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡懷楨(Huai-Jen Tsai) | |
| dc.contributor.author | Jun-Yu He | en |
| dc.contributor.author | 何郡育 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:49:27Z | - |
| dc.date.copyright | 2016-08-03 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-29 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19222 | - |
| dc.description.abstract | MicroRNA (miRNAs) 為小片段的非轉譯功能的單股RNA，其種子序列 (seed sequence)和目標基因RNA之3’端非轉譯區 (3’-untranslated region, 3’UTR) 互補而抑制目標基因轉錄後層次 (post-transcriptional level) 的表現。MicroRNA-206 (miR-206) 為骨骼肌專一表現之microRNA，可調控肌肉纖維母細胞的增生與分化。藉由 Labeled miRNA pull-down assay (LAMP) ，在斑馬魚胚胎發育早期之16 hpf，篩選出miR-206的標的基因之一為 reticulon4a (rtn4a)。進一步探討miR-206 抑制 rtn4a此一路徑在斑馬魚肌肉發育上的功能，發現當抑制miR-206或者過量表現rtn4a後，發現斑馬魚在48 hpf時，其斑馬魚體節邊界 (somite boundary) 產生缺失，且在缺失區域會有 F-actin 橫跨體節的現象。顯示miR-206抑制rtn4a的路徑影響斑馬魚的體節發育，我們首先利用microarray分析在抑制 miR-206 或過量表現 rtn4a 斑馬魚胚胎中體節細胞的基因表現量，得到在抑制 miR-206 或過量表現 rtn4a mRNA 下，相對於野生種斑馬魚胚胎表現量顯著上升的基因與顯著下降的基因。透過比對，找出抑制 miR-206或過量表現 rtn4a 細胞中，表現量改變趨勢相同的基因，其可能是參與miR-206 抑制 rtn4a此路徑下游的候選基因。利用全胚胎原位雜交染色實驗，從候選基因中篩選出表現量皆下降的 cxcr4a 與表現量皆上升的 thbs3a 基因進行研究。結果發現當抑制 cxcr4a 或過量表現 thbs3a 的體節發育早期 (20 hpf) 的胚胎體節邊界有缺失，而在體節發育成熟時 (48 hpf) 體節邊界的缺失仍存在且 F-actin 會橫跨體節。並發現在調控位階上，cxcr4a為 thbs3a上游基因，在rescue 實驗中，抑制 thbs3a 也能降低胚胎體節邊界缺失的比例。上述結果顯示，miR-206 透過抑制 rtn4a 的表現來影響 cxcr4a 或 thbs3a 藉此調控斑馬魚胚胎體節邊界的正常生成。此外，利用偵測γ-tubulin 或 pFAK 的免疫螢光染色發現在早期胚胎發育的體節邊界缺失的區域中心粒排列異常與 pFAK 訊號的消失，顯示在邊界細胞並未特化成表皮細胞。因此，顯示miR-206抑制 rtn4a 此一路徑透過 cxcr4a 及其下游 thbs3a 參與邊界細胞表皮化的過程。 | zh_TW |
| dc.description.abstract | MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level by targeting the 3’-untranslated region (3’UTR) of mRNAs through a seed sequence. miR-206 is a muscle-specific microRNA and is highly expressed in skeletal muscle. It is able to regulate the differentiation and proliferation in myoblast. That reticulon4a (rtn4a) is one of miR-206 target genes is proved by Labeled miRNA pull-down assay (LAMP) which preformed in 16-hpf zebrafish embryos. Further studying shows that miR-206 knockdown or rtn4a overexpression leads to somite boundary defect where F-actin across somites within 48-hpf zebrafish embryos. It implys that miR-206 plays roles on somite boundary development through silencing rtn4a. To address this issue, we anlazyed the gene expression profile of somite cells of miR-206-knockdown or rtn4a-overexpression embryo by microarray. Compared that with gene expression profile of wild type, we found the candidate genes which may be downstream of miR-206-rtn4a pathway. After microarray analysis, we selected 4 candidate genes which were all up-regulated and 4 candidate genes which were all down-regulated in miR-206-knockdown or rtn4a-overexpression embryo for further study. Using whole-mount in situ hybridization, we further selected two candidate genes ,cxcr4a which is down-regulated and thbs3a which is down-regulated, by its expression patterns and level. Then, we observerd that somite bounday defect within cxcr4a-knockdown or thbs3a-overexrpresion zebrafish embryos. Furthermore, it was proved that cxcr4a was able to repress thbs3a mRNA expression. As mentioned above, rtn4a was silenced by miR-206 inhibated cxcr4a, which repressed thbs3a mRNA expression. In addition, we demonstrated that somite boundary defect as mentioned might result from somite boundary cells wasn’t able to differentiate to epithelium by detecting the arrangement of γ-tubulin or expression of pFAK around the defect area. Evidence thus far accumulated shows that rtn4a was silenced by miR-206 inhibiate cxcr4a and leads to repress thbs3a to regulated the epithelialization of somite boundary cells during the somite development of zebrafish embryo. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:49:27Z (GMT). No. of bitstreams: 1 ntu-105-R01b43020-1.pdf: 5213497 bytes, checksum: 09675e93bd71ad7f303f08a014f9bd6f (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 1
英文摘要 2 文獻回顧 3 前言 17 材料與方法 19 結果 27 討論 33 參考文獻 38 圖說 50 附錄一、Developmental stages in the zebrafish 59 附錄二、miR-206 knockdown 與 rtn4a 過量表現顯著上升的交集基因 63 附錄三、miR-206 knockdown 與 rtn4a 過量表現顯著下降的交集基因 64 | |
| dc.language.iso | zh-TW | |
| dc.title | MicroRNA-206抑制 rtn4a的表現而調節下游基因以維持體節邊界正常的發育 | zh_TW |
| dc.title | MicroRNA-206 Plays Roles on Somite Boundary Development through Silencing rtn4a and Its Downstream Genes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 管永恕(Yung-Shu Kuan),陳曜鴻(Yau-Hung Chen) | |
| dc.subject.keyword | 斑馬魚,體節,rtn4a,miR-206, | zh_TW |
| dc.subject.keyword | somite,rtn4a,miR-206, | en |
| dc.relation.page | 65 | |
| dc.identifier.doi | 10.6342/NTU201601580 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-07-31 | |
| 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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