臺灣博碩士論文加值系統

Upstream open reading frame (uORF) 是一段存在於在特定基因的mRNA 的5’端不轉譯區 (5’UTR) 中，具有對下游所接基因產生調控功能，例如人類CCAAT/Enhancer Binding Protein Homologous Protein (chop) uORF。但是目前人類chop uORF 抑制轉譯能力的研究都在in vitro，而缺乏在in vivo 的探討。因此，本篇利用斑馬魚的胚胎在in vivo 中探討人類chop uORF 的特性。首先我們利用CMVpromoter 驅動chop uORF-GFP (pcDNA-uORF-GFP)，並於一細胞期注射入斑馬魚胚胎中，於30 hpf 時發現chop uORF 片段會抑制下游GFP 的表現。接著合成chopuORF-GFP mRNA 並進行顯微注射後，於30 hpf 觀察發現綠螢光也會被抑制，而且經由western blot 實驗顯示，chop uORF 抑制現象確實在轉譯層次上。為建立一個可以穩定繼代遺傳chop uORF-GFP 斑馬魚基因轉殖品系，以方便將來探討chop uORF片段在斑馬魚體內的作用機制或在發育時期的意義，我們注射質體pcDNA-uORF-GFP 到斑馬魚胚胎，再置於40℃高溫逆境之下，找出當胚胎發育至24hpf 時加熱6 小時胚胎可以呈現正常發育的外觀，而且有60.3%的胚胎都能出現較多的綠螢光(“++”和“+++”二層次)。於是使用此條件進行挑選出6 隻表現型態皆為全身散佈著均勻點狀綠螢光蛋白的G0 chop uORF-GFP 斑馬魚基因轉殖品系。另一方面，當斑馬魚胚胎在浸泡Mitogen-activated protein (MAP) kinases 訊息傳遞路徑中p38 的活化劑Anisomycin 時，會造成 chop uORF 所接下游報導基因的開啟。進一步地，當共同注射質體 pcDNA-uORF-GFP 和專一抑制p38 轉譯之Morpholino (MO) 以及單獨注射pcDNA-uORF-GFP，並浸泡藥物Anisomycin 後作比較，發現共同注射pcDNA-uORF-GFP 和p38 MO的胚胎綠螢光表現量在 “++”和“+++”的二層級總和中下降了23.2% (n=123)；同時，若將注射質體 pcDNA-uORF-GFP 的斑馬魚胚胎共同浸泡在MNK-1 抑制劑CGP57380 及Anisomycin 中，其綠螢光表現量和單獨浸泡在Anisomycin 的胚胎相比，在“++”以及“+++”的二層級總和中也下降了19.5% (n=93)。從上述結果，推測出在in vivo 中Anisomycin 最主要的訊息傳遞途徑會透過活化p38、MNK-1，其次才是透過ER stress 導致chop uORF 失去抑制所接下游綠螢光報
導基因開啟的能力。

Upstream open reading frame (uORF) is an mRNA sequence at 5’ UTR transcripted from a specific gene, regulating its downstream gene and Human CCAAT/Enhancer Binding Protein Homologous Protein(chop) uORF is a good example; however, all the studies at the present time for researching its ability of inhibiting translation are at a level
of only in vitro rather than in vivo. Therefore, here we use embryos of zebrafish to investigate the characters of human chop uORF in vivo. First, we injected chop uORF
fragment with downstream gene, GFP(pcDNA-uORF-GFP) which is driven by CMV promoter into one-cell-stage embryos of zebrafish and found that the expression of GFP was inhibited by chop uORF fragment at 30 hpf. Next, after microinjection of synthesized chop uORF-GFP mRNA, the expression of GFP was inhibited as well at 30 hpf, and the
results of western blot demonstrated that the effect of inhibition occurs during translation. In order to establish an inheritably stable chop uORF-GFP transgenic line of zebrafish which facilitates us to investigate the mechanism of chop uORF fragment in zebrafish or its meaning during development, we injected plasmids (pcDNA-uORF-GFP) into the embryos of zebrafish, heating them for 6 hours under 40℃ adversity of high temperature when they were at 24 hpf and found that there were 60.3% of embryos under normal appearances displaying more GFP expression (“++” and ”+++” levels). Six fish from G0 chop uORF-GFP transgenic line with the same phenotype of dotted GFP being evenly distributed over their bodies have eventually been picked out under such criterion. At one side, we found that the downstream report genes of chop uORF would be activated when the embryos were immersed in Anisomycin, the activator of p38 during Mitogen-activated protein (MAP) kinases signal transduction pathway, and further, when one group after coinjection of plasmids of pcDNA-uORF-GFP and Morpholino(MO), the specific inhibitor of p38 translation was compared with the other group as a control after injection of pcDNA-uORF-GFP only, both immersed in Anisomycin later, the former group was found to have 23.2%(n=123) lower expression in totality of GFP at “++” and ”+++” levels; at the other side, we compared GFP expression in embryos immersed in both MNK-1 inhibitor, CGP57380 and Anisomycin with that immersed in Anisomycin alone, the former had 19.5%(n=93) lower expression in totality of GFP at “++” and ”+++” levels. From the
results discussed above, we speculate that Anisomycin will in the end lead to reduction the ability of chop uORF to inhibit the downstream GFP reporter gene through ctivating p38 and MNK-1.

中文摘要………………………………………… 1
英文摘要………………………………………… 3
前言……………………………………………… 5
材料與方法……………………………………… 11
結果……………………………………………… 22
討論……………………………………………… 33
參考文獻………………………………………… 38
圖表……………………………………………… 44
附錄……………………………………………… 58

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