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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18729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 | Chi-Cheng Hsieh | en |
| dc.contributor.author | 謝其呈 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:22:14Z | - |
| dc.date.copyright | 2014-09-04 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-06 | |
| dc.identifier.citation | 黃薇臻. (2013). 利用有對熱逆境敏銳反應的腦細胞來探討轉譯抑制的機制. 國立臺灣大學生命科學院分子與細胞生物學研究所碩士論文。
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CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes & Development 12, 982-995. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18729 | - |
| dc.description.abstract | C/EBP homologous protein (chop) mRNA上的 5’UTR含有upstream open reading frame (uORFchop)具有抑制下游coding region轉譯的功能。但細胞一旦遭遇逆境時，uORFchop就會失去抑制轉譯的功能，而開啟下游基因的轉譯。之前我們建立含有人類uORFchop (huORFchop) 融合egfp螢光報導基因的斑馬魚品系huORFZ，發現若給予熱休克或缺氧的逆境都可誘導egfp的轉譯，且只表現在中樞神經系統。但是huORFchop在in vivo下如何調控下游基因轉譯的詳細機制，尚未完全明瞭。為了回答此問題，我們先進行下列兩種expression microarray的分析：(1)比較缺氧逆境下脊髓EGFP(+) cells的及未經逆境處理的脊髓GFAP(+) cells；和(2)比較熱休克逆境下腦部EGFP(+) cells及EGFP(-) cells。再從轉錄體變化中找出使huORFchop下游基因表現於中樞神經系統的基因。從這兩種microarray交集的結果，我們得知逆境處理時，魚胚胎內的kinase-enhanced PP1 inhibitor (KEPI)之表現量在EGFP(+) cells中會顯著下降。進一步地，以whole-mount in situ hybridization及qRT-PCR都證實kepi mRNA的表現量在逆境處理後會顯著下降，且得知kepi mRNA與chop於mRNA在表現分布上相似。若於huORFZ一細胞時期之胚胎注射kepi mRNA或DNA，然後在72 hpf時進行缺氧逆境處理2.5小時，並在96 hpf進行觀察，發現huORFZ的螢光表現率會下降，表示kepi會增強huORFchop的轉譯抑制的能力。相反地，若用morpholino (MO)去 knockdown KEPI後，發現即使在沒有逆境處理的huORFZ胚胎也會被誘導表現螢光。同時，我們也發現在缺氧處理後，胚胎內的protein phosphatase 1α(PPP1A)的蛋白質增加、磷酸化程度不變，使PPP1A活性增加，且與uORFchop轉譯抑制能力喪失的時間點一致。但當過量表現kepi時，可以在缺氧逆境處理後，透過PPP1A磷酸化的促進以及PPP1A蛋白質上升的減緩，來降低PPP1A的活性。綜合以上結果，我們總結KEPI參與huORFchop轉譯抑制的機制，而當缺氧逆境下KEPI減少，使PPP1A活性上升促使huORFchop下游基因轉譯。 | zh_TW |
| dc.description.abstract | The upstream open reading frame (uORF) of C/EBP homologous protein (uORFchop ) has been reported that it can direct translational inhibition. When cells encounter stress, uORFchop -mediated translational inhibition is repressed. We generated a transgenic line huORFZ, which harbors an eGFP reporter fused with human uORFchop . Using zebrafish embryos derived from line huORFZ, we found that embryos treated with either hypoxia or heat shock, GFP was exclusively expressed in the brain and spinal cord. However, the molecular mechanism of the uORFchop-mediated translational inhibition is still not completely understood. We performed two microarrays from huORFZ embryos. One microarray was based on that the expression levels of genes which were of the GFP-positive cells from spinal cord after hypoxia were compared with those of genes which were of the glial fibrillary acidic protein (GFAP)-positive cells from spinal cord without being stressed. Another microarray was based on that the expression levels of genes which were of the GFP-positive cells from brain after heat shock were compared with those of genes which were of GFP-negative cells from brain after heat shock. We screened putative genes whose expression levels were significantly decreased shown on both microarrays. Microarray results were farther confirmed by Whole-mount in situ hybridization (WISH), overexpression and knockdown the candidate gene mRNA, DNA or morpholino (MO) into huORFZ embryos. Among candidate genes, kinase-enhance PP1 inhibitor (KEPI) was selected for further study. Using whole-mount in situ hybridization of zebrafish embryos, we found that the expression pattern of the transcripts of kepi was co-localized with that of chop mRNA. Additionally, qRT-PCR results indicated that the mRNA level of kepi was greatly decreased in brain in the hypoxia-treated embryos. Furthermore, to confirm that kepi is involved in the translation inhibition of endogenous uORFchop, we microinjected the kepi mRNA and DNA into huORFZ embryos, and treated the injected embryos with deoxygenated water for 2 hr when embryos developed at 72 hpf. Results showed that the GFP signal was normally expressed in the brain and spinal cord of control embryos in which kepi was not overexpressive. However, unlike the control embryos, we found that the expression of GFP was suppressed in the kepi-overexpressive embryos when we observed embryos at 96 hpf. Moreover, kepi knockdown did induce the translation of uORFchop downstream GFP in brain and spinal cord without stress treatment. We found that the protein level of protein phosphatase 1α (PPP1A) increased at the same time period when GFP was expressed after hypoxia treatment. Moreover, kepi overexpressions suppressed the protein level of PPP1A and promote its phosphorylation. Based on these results, the role of KEPI on regulating the translational inhibition function of uORFchop is PPP1A dependent. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:22:14Z (GMT). No. of bitstreams: 1 ntu-103-R01b43011-1.pdf: 4144358 bytes, checksum: ebf5d4380fee515c7fbbbb6f4f43bc4d (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 中文摘要…………………………………………………1
英文摘要………………………………………………2 文獻回顧…………………………………………………4 前言……………………………………………………12 材料與方法……………………………………………15 結果……………………………………………………22 討論……………………………………………………28 參考文獻………………………………………………33 圖表……………………………………………………41 補充資料………………………………………………50 | |
| dc.language.iso | zh-TW | |
| dc.title | KEPI在chop基因的upstream open reading frame轉譯抑制機制中所扮演的角色 | zh_TW |
| dc.title | KEPI plays role in repressing the translation inhibition mediated by upstream open reading frame of chop gene | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂勝春(Sheng-Chung Lee),鄭子豪(Tzu-Hao Cheng),黃憲斌(Hsien-Bin Huang) | |
| dc.subject.keyword | 斑馬魚,轉譯抑制,內質網逆境, | zh_TW |
| dc.subject.keyword | zebrafish,Danio rerio,translation inhibition,ER stress,CHOP,protein phosphatase 1,KEPI, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-06 | |
| 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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