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研究生:陳宜群
研究生(外文):Yi-Chung chen
論文名稱:斑馬魚arnt2a/b/c對胚胎眼睛發育的影響
論文名稱(外文):Functions of zebrafish arnt2a/b/c in embryonic eye 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:2004
畢業學年度:92
語文別:中文
論文頁數:86
中文關鍵詞:斑馬魚眼睛發育
外文關鍵詞:zebrafisheyedevelopmentarnt2
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ARNT為bHLH/PAS (Per-Arnt-Sim) 家族成員之一,能與其他bHLH/PAS蛋白 (HIF, SIM, CLOCK, AHR) 形成異隅合蛋白,以調控基因的表現(如:缺氧反應、毒物代謝、神經系統發育等)。將斑馬魚之arnt2a/b/c進行基因之減弱 (knockdown),再藉由全覆式原位雜交實驗、組織切片、掃瞄式電子顯微鏡等進行觀察後,發現arnt2a/b/c morphant胚胎在視網膜神經細胞及晶體上皮細胞的分化明顯出現問題,顯示出細胞可能停留於未分化的細胞形態。以早期神經分化有關之標記基因neuroD觀察後,發現arnt2a/b/c morphant胚胎在眼睛的區域未出現任何neuroD的表現,顯示位於眼睛多種受neuroD調控之神經細胞應在分化上出現問題。在視神經的發育上arnt2a/b/c morphant以視神經之專一性標記基因觀察後,發現其表現之形態與正常胚胎相異,而這種不正常的表現形態可能會造成視神經的投射出現問題。至目前為止可證實arnt2a/b/c的確在視覺的神經細胞分化上扮演了非常重要的角色。
ARNT is a member of bHLH/PAS family, which dimerizes with other members of bHLH/PAS family proteins, including HIF, SIM, CLOCK, AHR, to form heterodimers and regulate the transcriptional activities of target genes. To investigate the functions of ARNT in vertebrate development, the arnt2a/b/c-specific morpholino oligonucleotide was injected into fertilized embryo at 1-cell stage to block translation of ARNT2A/B/C and observe the effects on tissue development. Via whole mount in situ hybridization, tissue section and scanning electron microscope, it appeared that the development of neural retina and lens epithelium was disrupted in arnt2a/b/c-knockdown embryo. A large number of primordial retina cells were retained in undifferentiated state. In addition, neuroD, one of the key regulator of neuronal differentiation, did not express in the retinal region of arnt2a/b/c morphant. The optic nerve-specific gene exhibited abnormal expression pattern which cause optic nerve projection with wrong destinations. Accodingly, it appeared that arnt2a/b/c played essential roles in visual neuronal cell differentiation.
目錄………………………………………………………...1
英文摘要(Abstrate)…………………………………….….6
中文摘要……………………………………………….…..7
前言.......................................................8
ARNT簡介.............................................8
斑馬魚的眼睛發育.………………………………………14
與眼睛發育相關之標記基因簡介...……………………..16
一、six3..........................................16
二、pax6............................................16
三、vax...............................................17
四、rx..…...……...….…………........................17
五、pax2.1..............................................18
六、N-cadherin..…………….………......................18
七、vsx.................................................19
八、α A-crystallin......................................19
九、rho.............................................20
十、neuoD..........................................20
十一、isl-1……….................................20
實驗材料
A. 限制酵素..............................................23
B. 其他酵素...........................................23
C. 試劑...................................................23
實驗藥品配方..............................................24
A. 菌株培養..........................................24
B. DNA電泳之相關藥品.................................24
C. 微量質體DNA萃取之藥品配方..............................25
D. 質體轉型之藥品配方.................................25
E. RNA純化之藥品......................................26
F. RNA電泳之相關藥品.................................26
G. RNA全覆式原位雜交藥品配方.......................27
H. SEM藥品................................................28
實驗方法...………………………………………………..30
A、斑馬魚飼養與受精胚胎收集...............................30
B、斑馬魚魚卵RNA萃取......................................30
C、核酸電泳分析.........................................31
D、DNA分子之萃取.....................................32
E、DNA接合反應......................................34
F、質體轉型..........................................34
G、反轉錄聚合酶連鎖反應..................................35
H、RNA全覆式原位雜交.....................................36
I、掃描式電子顯微鏡...................................41
J、顯微注射.........................................45
K、組織切片及組織染色.................................48
實驗結果...………………………………………………..50
一、胚胎外觀的觀察....................................50
二、以眼睛發育相關之標幟基因,研究arnt2a衰減(knock down)對斑馬魚眼睛發育之影響...............................51
1. six3b............................................51
2.pax6b..................................................52
3. rx1...................................................53
4. α A-crystallin....................................53
5. pax2.1.............................................54
6. vax1、vax2.....................................55
7. rho..............................................55
8. vsx1..............................................55
9. N-cadherin......................................56
10. neuroD.........................................56
11.isl-1...............................................56
討論………...……………………………………………..58
一、早期的發育........................................58
二、中後期的發育................................58
1. 視網膜的發育.............................58
2. 晶體的發育.................................60
3. 視神經的發育............................61
參考文獻...………………………………………………...65
附圖…………...…………………………………………..71
圖一、six3b的表現情形..............................71
圖二、斑馬魚胚胎外觀...................................72
圖三、組織切片圖...................................73
圖四、組織切片圖........................................74
圖五、掃瞄式電子顯微鏡下斑馬魚胚胎外觀..............75
圖六、six3b.........................................76
圖七、pax6b..........................................77
圖八、rx1...........................................78
圖九、α A-crystallin...................................79
圖十、pax2.1..........................................80
圖十一、vax1.............................................81
圖十二、vax2..........................................82
圖十三、rho..........................................83
圖十四、vsx1.........................................84
圖十五、N-cadherin...................................85
圖十六、neuroD.....................................86
圖十七、isl-1......................................87
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