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研究生:歐瓊雯
研究生(外文):Chiung -Wen Ou
論文名稱:KnockdownADAR2基因對斑馬魚胚胎發育的影響
論文名稱(外文):Effects of ADAR2 knock-down on the zebrafish embryogenesis
指導教授:周姽嫄
指導教授(外文):Wei-Yuan Chow
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:46
中文關鍵詞:斑馬魚細胞凋亡
外文關鍵詞:zebrafishADAR2RNA editingapoptosis
相關次數:
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  • 下載下載:16
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中文摘要
A-to-I RNA editing是真核生物將pre-mRNA adenosine 脫氨成inosine的後轉錄修飾,可增加基因產物的複雜性。ADARs (adenosine deaminase that act on RNA)是負責催化A-to-I (adenosine to inosine)的RNA editing 酵素,脊椎動物表現三種ADARs,ADAR1,ADAR2與ADAR3。先前本實驗室將按ADAR2 5’ 端序列設計的反訊息(anti-sense) morphonino寡核酸顯微注射入斑馬魚受精卵,干擾正常胚體ADAR2蛋白質的表現,觀察到胚體發育過程異常。異常的表現型包括眼睛較小、卵黃囊(yolk sac)發育不全、對觸碰不敏感、受精後72小時胚體發育逐漸遲緩、最遲於胚體發育七天內死亡。為進一步瞭解ADAR2反訊息morphonino寡核酸如何影響斑馬魚胚體的發育,以HuC免疫染色及zNav1.6 mRNA全覆式原位雜合染色追蹤ADAR2 基因被抑制後胚體24至72小時發育過程,發現胚體神經細胞的分佈、數量及表現時間點和野生種胚體不同。以crystallin αA進行mRNA全覆式原位雜合染色觀察水晶體發育,染色結果顯示ADAR2表現受抑制之36至72小時胚體的水晶體發育遲緩。以acridine orange染色與TUNEL assay追蹤細胞凋亡,染色結果顯示ADAR2 基因被抑制後16 hpf胚體發育過程之細胞凋亡無明顯異常,但在胚體發育20至36小時則偵測到過量的細胞凋亡。即時定量PCR分析顯示,ADAR2 基因被抑制後胚體發育16至36小時促細胞凋亡基因zBax表現量顯著增加,促細胞凋亡基因zBad與抗細胞凋亡基因zBcl-xl、zMcl-1a mRNA表現量則無明顯改變。本研究顯示ADAR2在斑馬魚神經細胞、眼睛的發育過程及早期發育過程裡細胞凋亡的調控扮演相當重要的角色。
Abstract
The A-to-I (adenosine to inosine) RNA editing catalyzing deamination of adenosines to inosines on pre-mRNA is a post-transcriptional modification mechanism which increases complexities of a gene product. Vertebrates express three kinds of ADARs (adenosine deaminase that act on RNA), ADAR1, ADAR2, and ADAR3. ADAR2 knock-down zebrafish embryos developed abnormally. The abnormal phenotypes include small eyes, yolk sac defects, touch insensitivity, locomotive defects, growth retardation, and death before 7 dpf. The temporal and spatial expression patterns of HuC and the sodium channel, zNav1.6, of wildtype and morphant zebrafish embryos were compared to delineate a possible involvement of ADAR2 in the neurogenesis. The numbers and distribution patterns of primary neurons in the morphant differed from those of the wildtype embryos. Comparisons of crystallin αA expression patterns between wildtype and morphant showed that the lens development might be retarded. TUNEL assay and acridine orange staining were performed to study the patterns of apoptosis in the embryos. Excessive and widespread apoptosis were detected in the morphant embryos between 20 to 36hpf. Quantitative RT-PCR analysis revealed that the expression level of pro-apoptotic zBax mRNA increased significantly in the 16-36 hpf morphants, whereas the mRNA levels of zBad, zBcl-xl and zMcl-1a remained the same between wildtype and morphant. These results suggest ADAR2 plays an essential role in the embryogenesis and is required for the developments of neuron, lens, and the regulation of apoptosis.
目錄
Abstract 2
中文摘要 3
前言 4
材料與方法11
結果 17
討論 23
參考文獻 28
附表 33
附圖 35
參考文獻
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