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研究生:李柏翰
研究生(外文):Po-Han Lee
論文名稱:HIF2α在視網膜發育的功能
論文名稱(外文):Function of HIF2α on retina development
指導教授:胡清華胡清華引用關係
指導教授(外文):Chin-Hwa Hu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:78
中文關鍵詞:斑馬魚低氧誘發因子視網膜
外文關鍵詞:zebrafishHIF2αretina
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在脊椎動物的胚胎發育中,bHLH/PAS 蛋白扮演重要的角色。其中的低氧誘發蛋白(hypoxia-inducible factor,HIFs),負責調控細胞內供氧的平衡,在低氧的狀況下,會進入細胞核,與ARNT形成異偶合體(heterodimer),結合至下游基因的HRE序列(hypoxia responsible element)調控細胞缺氧時的反應。
過去的研究發現,斑馬魚hif1α/-2α/-3α同時弱化會抑制視網膜兩極細胞 (bipolar cell)的vsx1和桿狀感光細胞 (rod-photoreceptor) 的視紫蛋白 (Rhodopsin) 基因rho表現,顯示視網膜的發育受到抑制。隨後分別弱化hif1α、hif2α及hif3α後觀察發現,hif2α弱化後對rho有明顯的影響。序列分析發現rho上游啟動子含有多個HIF所辨識的HRE序列,顯示HIF2α有可能直接調控rho轉錄。為了證明此點,本實驗中將啟動子上游2400bp片段內的HRE進行定點突變後,發現並不影響報導基因在視網膜上的專一性表現,顯示rho啟動子並未接受HIF的直接控制。先前的研究發現,在斑馬魚中,HIF2α會藉由控制細胞凋亡抑制蛋白(inhibitor of apoptosis proteins) survivin 1與-2的方式影響中樞神經分化。本實驗中其次想證明hif2α弱化後是否也是透過相同的方式抑制視網膜神經分化。在弱化birc5a與5b(survivin1與-2)的實驗中,發現皆會抑制rho的表現,顯示survivin 1與-2和眼睛細胞分化間有密切的關係。其次進一步以視網膜發育各時期的標識基因觀察hif2α和birc5a弱化對斑馬魚視網膜發育所造成的影響。結果顯示,HIF2α及survivin 1弱化均會抑制視網膜中晚期的分化,推論HIF2α可能藉由survivin 1影響視網膜分化。顯示在斑馬魚中HIF2α對於視網膜的分化扮演重要的角色。本實驗的結果顯示HIF2α可能是透過控制survivin基因的表現的方式參與視網膜的發育,但並不直接參與rho基因的調控。

Hypoxia-inducible factors (HIFs) are known for their functions in oxygen homeostasis by regulating the genes for glucose uptake and metabolism, erythorpoiesis, angiogenesis, apoptosis and cell proliferation. The HIFs are heterodimeric basic-helix-loop-helix-PAS transcription factors consisting of an oxygen-sensitive alpha subunit and a constitutively expressed beta subunit, also known as ARNT. When cells are subjected to hypoxia, the HIFα factors are stabilized, associate with ARNT and activate the target genes.
Previously it was revealed that concurrent knockdown of zebrafish hif1α, -2α and -3α inhibited the bipolar cell marker vsx1 and photoreceptor marker rho transcriptions, indicating that the retinal development was abrogated in hif1α/-2α/-3α morphants. Further investigation revealed that the retinal defects occurred hif1α/-2α/-3α morphant embryos was caused by the action of hif2α antisense morpholino. Knockdown of hif2α eliminated rho expression. There are multiple hypoxia-responsible elements in the upstream promoter regions of rho gene. It raises a possibility that rho transcription is controlled by HIF2 directly. Nevertheless, mutating the HRE sequence in the 2.4 kb of rho promoter fragment did not eliminate the promoter activity, indicating that rho is not controlled directly by HIF2-interaction.
It was shown that the differentiation of neural progenitor cells in zebrafish CNS is controlled by HIF2α by means of transcriptional regulation of survivin orthologues Birc5a and Birc5b during embryonic stages. The function of survivin orthologues in retina development is investigated by reverse genetic studies. Knock down of survivin-1/birc5a or survivin-2/birc5b all results in inhibition of rho transcription, suggesting that survivin genes act critical functions in retina development. In hif2α or survivin-1/birc5a morphant embryos, only the transcriptions of mid- and late-stage markers (including huC, neuroD ,vsx1 and rho),but not early stage markers (such as rx1), were affected, indicating hif2α and its downstream target birc5a functions on mid-stages of retina development. In conclusion, this study suggests that HIF2α controls retina development indirectly through its downstream survivin/birc5 genes, but it does not control rho transcription directly.

目錄
中文摘要 i
Abstract iii
目錄 v
壹、前言 1
一、低氧誘發因子(hypoxia-inducible factor) 1
二、低氧誘發蛋白與視網膜 3
三、斑馬魚眼睛的發育 4
四、細胞凋亡抑制蛋白 Survivin 6
五、Tol2 移位子系統(Tol2 transposon) 7
六、研究目的 9
貳、實驗方法 10
一、班馬魚rho基因啟動子HRE定點突變(site-directed mutagenesis) 10
二、斑馬魚胚胎DNA顯微注射 12
三、RNA全覆式原位雜交(RNA whole-mount in situ hybridization) 15
四、石蠟切片及組織染色 22
参、實驗結果 25
一、rho啟動子2.4K之HRE定點突變對rho 啟動子活性的影響 25
二、hif-2α、birc5a、birc5b基因弱化對內生性rho mRNA及rho 啟動子活性的影響 25
三、hif-2α、birc5a基因弱化對視網膜標識基因的影響 26
四、hif-2α、birc5a基因弱化對細胞週期相關基因的影響 30
五、組織切片 33
肆、討論 34
一、HIF-2α未參與rhodopsin基因的直接調控 34
二、hif-2α、birc5a、birc5b 弱化對視網膜的影響 34
三、hif-2α、birc5a弱化後造成視網膜分層的發育受到抑制 35
伍、參考文獻 37
陸、圖表 44
柒、附錄 65
一、實驗材料 65
二、實驗藥品配置 66


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