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系統識別號 U0002-0608200711171100
中文論文名稱 斑馬魚兩型肌肉調控因子MRF4a與MRF4b的分子結構,基因表現及生物功能
英文論文名稱 Molecular structure,spatiotemporal expression and biological functions of zebrafish muscle regulatory factors MRF4a and MRF4b
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 95
學期 2
出版年 96
研究生中文姓名 李駿凱
研究生英文姓名 Chun-Kai Li
學號 693290362
學位類別 碩士
語文別 中文
口試日期 2007-07-19
論文頁數 62頁
口試委員 指導教授-陳曜鴻
委員-陳銘凱
委員-蔡惠珍
中文關鍵字 斑馬魚  肌肉調控因子  肌肉發育 
英文關鍵字 Zebrafish  MRFs  mrf4  muscle development 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本實驗室先前的研究發現在斑馬魚體內有兩型MRF4 (MRF4 a和MRF4 b)存在,兩者蛋白質序列相似度達97%,接著利用mrf4 a和mrf4 b特異性核酸探針進行原位雜交實驗偵測兩型mrf4表現位置發現mrf4 a表現位置在olfactory placode、三叉神經 (trigeminal nerve) 和RB神經元 ;mrf4 b表現位置在體節上。接著我利用核酸類似物胚胎抑制劑 (morpholino)去分別抑制兩型或同時抑制兩型mrf4,再利用各種標定物以了解兩型mrf4在斑馬魚體內分工情形。首先利用肌肉標定物F59、α-actin、tnnt1、tnnt3b,進行免疫螢光染色和原位雜交實驗,結果發現mrf4 a morphant慢肌纖維排列不受影響,α-actin表現正常,mrf4 b被抑制會造成慢肌纖維排列混亂,α-actin表現下降,tnnt1及tnnt3b則有表現下降及表現情形混亂。接著我使用神經標定物aat (anti-acetylated tubulin)、Znp1、α-bungarotoxin、Zn5和Zn12一級抗體對兩型mrf4 morphant進行染色,發現mrf4 a morphant 上RB 神經元樹突生長數目變少且長度變短,二級運動神經發育受阻礙,而mrf4 b morphant中乙醯膽鹼接受體變少,一級運動神經生長受阻礙。综合以上結果我認為兩型mrf4功能有所不同,其中mrf4 a對於神經生長有較大相關性,而mrf4 b則和肌肉生長有較大相關性。

英文摘要 Our previous study shows that zebrafish has two MRF4 isoforms, MRF4 a and MRF4 b. The two MRF4 proteins exhibited a 97 % identity rate. To detect the spatiotemporal expression pattern of mrf4 a and mrf4 b at mRNA level, we used in situ hybridization. Our data showed mrf4 a expressed in trigeminal nerve, olfactory placode and RB (Rohon-beard) neuron. mrf4 b expressed in somite. To test the biological function of each mrf4, we first used anti-sense morpholino to knockdown mrf4 a and mrf4 b individually.Curved body were observed after knockdown. Then we used muscle markers F59, α-actin、tnnt1、tnnt3b,and neuron markers aat (anti-acetylated tubulin), Znp1, α-bungarotoxin, Zn5, Zn12 to detect morphological change of mrf4 morphant in skeletal muscle, slow muscle, fast muscle, motor neuron, innervations, acetylcholine receptor cluster and RB neuron. Our data shows mrf4 a knockdown led to RB neuron dendrite loss, motor neuron innervation, mrf4 b knockdown led to misalignment of slow muscle, α-actin, tnnt1, tnnt3b down reglation, secondary motorneuron defect. On the basis of our observations, we suggest that mrf4 a expression is involved in neuron development, mrf4 b expression is involved in muscle development.
論文目次 目錄

中文摘要----------------------------------------------------------------------------I
英文摘要---------------------------------------------------------------------------II
目 錄--------------------------------------------------------------------------III
圖表目錄--------------------------------------------------------------------------V
第一章 前言-----------------------------------------------------------------------1
第二章 材料與方法--------------------------------------------------------------7
第一節 斑馬魚飼養及胚胎收集(Danio rerio)-------------------------8
第二節 勝任細胞(Competent cell)製作及大腸桿菌之電轉型
(electroporation) --------------------------------------------------8
第三節 小量質體萃取-----------------------------------------------------8
第四節 胚胎固定及脫水--------------------------------------------------9
第五節 morpholino knockdown實驗-----------------------------------9
第六節 斑馬魚total RNA萃取-----------------------------------------10
第七節 逆轉錄聚合酶鏈鎖反應---------------------------------------10
第八節 Digoxigenin(DIG)標定核酸探針製作-----------------------10
第九節 胚胎原位雜交---------------------------------------------------11
第十節 免疫螢光染色---------------------------------------------------12
第十一節 α-bungarotoxin (α-BTX)及Znp-1雙染色----------------12
第十二節 冷凍切片------------------------------------------------------13
第十三節 顯微照相系統------------------------------------------------13

第三章 結果---------------------------------------------------------------------14
第一節 斑馬魚與其他脊椎動物MRF4ㄧ級結構分析及親緣關係
--------------------------------------------------------------------------------15
第二節 以胚胎原位雜交(whole-mount in situ hybridization )偵測mrf4在斑馬魚體內生性表現時空分佈---------------------15
第三節 Mrf4 morpholino設計並以RT-PCR (reverse transcription
polymerase chain reaction) 確定mrf4 morpholino功能---------------------------------------------------------------------16
第四節 各種Mrf4 morphant 於一般光源下生長型態-------------17
第五節 不同濃度下各種mrf4 morpholino注射後對斑馬魚存活
及畸形比例影響------------------------------------------------18
第六節 mrf4表現下降對斑馬魚骨骼肌α-actin表現造成影響---19
第七節 mrf4 morphant的tnnt 1表現情形----------------------------20
第八節 mrf4 morphant的tnnt 3b表現情形---------------------------20
第九節 mrf4 knockdown對慢肌纖維排列造成影響---------------22
第十節 mrf4 morphant對RB(Rohon-Beard)神經元細胞樹突投射
(RB neuron peripheral process)造成影響-------------------22
第十一節 mrf4 morphant一級運動神經與肌肉接點染色---------24
第十二節 mrf4 morphant之二級運動神經染色---------------------26
第四章 討論---------------------------------------------------------------------27
第一節 斑馬魚mrf4 alternative splicing假說------------------------28
第二節 斑馬魚兩型mrf4時空分佈表現的不同---------------------28
第三節 斑馬魚兩型mrf4表現與慢肌纖維生長發育的關係------29
第四節 斑馬魚兩型mrf4在神經生長發育上造成不同影響------29

參考文獻--------------------------------------------------------------------------31
圖表--------------------------------------------------------------------------------37
附錄--------------------------------------------------------------------------------56
圖表目錄

Fig 1. 與其他脊椎動物MRF4胺基酸序列比對結果--------------------38
Fig 2. 斑馬魚與其他脊椎動物MRF4胺基酸序列演化樹--------------39
Fig 3. 斑馬魚mrf4表現時空上的分佈--------------------------------------40
Fig 4. 藉由反轉錄聚合酵素反應偵測斑馬魚mrf4剪接改變-----------43
Fig 5. 野生種與各種不同mrf4 morphant在可見光下觀察受精後36小時的胚胎型態-----------------------------------------------------------44
Fig 6. 野生種及各種mrf4 knockdown斑馬魚胚胎α-actin原位雜交染色--------------------------------------------------------------------------45
Fig 7. 野生種及各種mrf4 knockdown斑馬魚胚胎Tnnt 1原位雜交染色--------------------------------------------------------------------------46
Fig 8. 野生種及各種mrf4 knockdown斑馬魚胚胎Tnnt 3b原位雜交染色--------------------------------------------------------------------------47
Fig 9. 野生種及各種mrf4 knockdown胚胎F59免疫螢光染色,於斑馬魚胚胎受精後36小時慢肌纖維缺陷----------------------------48
Fig10. 各種mrf4 morphant aat成熟神經染色-----------------------------49
Fig 11. 各種mrf4 morphant Zn12 RB神經元染色------------------------ 51
Fig 12. 各種mrf4 morphant Znp 1及α-bungarotoxin雙染色,運動神經及運動神經肌肉接點分布情形------------------------------------52
Fig 13. 各種mrf4 morphant Zn 5 二級運動神經染色-------------------53
Table1. 各物種MRF4胺基酸序列相似度比較表-------------------------54
Table2. 注射不同濃度各種mrf4 morpholino後24hpf及36hpf斑馬魚存活率及畸形率統計 -----------------------------------------------55
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