臺灣博碩士論文加值系統

摘要
心臟為第一個形成且具有功能性的器官，其由背部兩側的中胚層衍生，而先天性的心臟缺損多為胚胎發育時細胞發育的異常。這些發育的缺陷大多出現在心房及心室的缺損。dHAND則主要表現於右心室(right ventricle)、神經脊衍生(neural crest-divided)之心臟管狀結構(heart tube)及鰓弓動脈的血管(branchial arch arteries)。在人類、老鼠、雞及斑馬魚等皆已選殖出dHAND基因，屬於basic Helix-Loop-Helix 轉錄因子(transcription factor) ，此bHLH區域為主要和DNA接合的位置，其在不同的物種間具有高度的相似性，dHAND 對心臟的發育具有重要的地位主要是促進心室及心血管的發育反應。斑馬魚只具有一心房一心室，在斑馬魚中只選殖出dHAND基因。本研究斑馬魚作為模型(model)魚種，來觀察dHAND基因在胚胎發育的不同時期在何處表現及血管之發育，以dHAND morpholino antisense RNA抑制dHAND基因的表現(knockdown)，並觀察dHAND基因缺失對心臟發育及心血管之影響。以mRNA全覆式原位雜交(whole mount in situ hybridization analysis)分析顯示授精後12小時(12 hpf)dHAND基因會在胚胎兩側中胚層(lateral plate mesoderm)表現。24小時後(24hpf)會在心臟及鰓弓動脈(branchial arch)出現。72小時後(72hpf)只表現於心臟部位，而在鰓弓動脈並無表現。dHAND gene knockdown實驗中胚胎的1-4 細胞期注射dHAND morpholino antisense mRNA 12小時之後，dHAND基因mRNA無法在兩側的中胚層偵測到。但在24hpf期會在心管形成處僅有少許表現，亦無心管的成形及心管跳動，且dHAND gene knock down的39個胚胎約有50%胚胎死亡。至36到48小時約70%胚胎會死亡。推測可能受到dHAND antisense RNA抑制使得多數的胚胎心臟無法正常發育而導致死亡。本研究亦發現24小時(24hpf)後當gene knockdown效果失效後有些胚胎繼續發育的胚胎中dHAND基因逐漸恢復正常表現，心臟亦會逐漸發育，雖然胚胎可繼續發育但是有心包膜水腫及心臟發育不全的現象產生。在48小時(48hpf)以顯微血管顯影術(microangiography)發現在dHAND基因knockdown的斑馬魚其主動脈弓及鰓弓動脈發育有缺失現象。研究結果均顯示dHAND基因於斑馬魚胚胎時期之心臟及心血管發育扮演很重要的角色。

Abstract
The heart, the first functional organ in the developing embryo arises from paired regions of dorsolateral mesoderm. Most of congenital heart diseases were the defect of cell differentiation at the embryo developmental stage and these developmental defect most were appeared in the atrium and ventricle. dHAND (dextral Heart、Autonomic nervous system、Neural crest-derived cell types、Deciduum) gene is mainly expressed in the lateral plate mesoderm, right ventricle, heart tube of neural crest-divided, and aortic arch arteries. In human, mice, chicken and zebrafish (Danio rerio) which dHAND had been cloned and characterized as a basic Helix-Loop-Helix transcription factor. The N-terminal b-HLH region a dHAND gene is high conserved within those species. This transcription factor can promote the development of ventricle and aortic arch in zebrafish. The long term goal of this project is to study the molecular mechanism of dHAND on early developmental process in lower vertebrates; i.e. zebrafish. To fulfill this long term goal, a series of short term goals were developed: 1) Gene expression pattern of dHAND gene during embryonic stages, 2) The impacts of dHAND gene expression on early heart development. 3) The influence of dHAND gene on heart-vessel formation during early embryonic development. The results of whole mount in situ hybridization indicated that the dHAND gene was expressed at lateral plate mesoderm (lpm) at 12 hpf and at heart and aortic arch areas after 24hpf as well. However, the expression of dHAND gene was only detected in heart area but not in aortic arch at 72hpf. When dHAND morpholino antisense RNA was injected into the embryonic yolk, the expression of dHAND gene can not be detected at lpm in 12hpf but only a little dHAND mRNA can be detected at developmental heart region but not in aortic arch area by 24hpf. By 24hpf, the heart beating and heart formation cannot be detected in 24hpf and most of the dHAND gene knockdown embryos died at 24hpf. Microangiography data showed that aortic arch and branchial arch was absent at 48hpf on those survived injected embryos. These results subjected that dHAND antisense RNA might cause abnormal heart development in those survived individuals, Interestingly, it was found that the heart development can be restored in some dHAND gene knockdown injected embryos, but with abnormal pericardium edema. All these results indicated that dHAND play an important role in heart formation and heart-vessel development at zebrafish embryonic stage.

中文摘要………………………………………………………………… I
英文摘要…………………………………………………………………II
壹、前言………………………………………………………………… 1
一、胚胎心臟發育之重要性……………………………………………1
二、脊椎動物的心臟發育………………………………………………2
三、斑馬魚胚胎之心臟發育……………………………………………3
四、轉錄因子(Transcription factors)與胚胎發育……………… 4
五、dHAND基因之作用及表現區域…………………………………… 7
貳、材料與方法…………………………………………………………13
1.魚隻飼養與魚卵收集……………………………………………… 13
1-1.斑馬魚之飼養…………………………………………………… 13
1-2.斑馬魚受精卵之收集…………………………………………… 13
2.dHAND mRNA之定量分析…………………………………………… 13
2-1.萃取斑馬魚胚胎之total RNA……………………………………13
2-2.TRIZOL reagent萃取total RNA…………………………………14
2-3.RNA電泳分析(Electrophoretic analysis)……………………14
2-4.RT-PCR之定量分析……………………………………………… 15
3.全覆式原位雜交(whole mount in situ hydrization analysis)…15
3-1.斑馬魚dHAND基因部分(partial)cDNA序列選殖(selection)…… 15
3-1-1.單步驟反轉錄酶-聚合酶連鎖反應(One-step RT-PCR)…………16
3-1-2.RT-PCR產物電泳分析(Electrophoresis of RT-PCR product)…17
3-1-3.膠體萃取(Gel extraction)………………………………… 17
3-2.定序載體的構築(Contructuion)與選殖(Selection)…………18
3-2-1.銜接反應與轉型作用(Ligation Reaction and Transformation) …18
3-2-2.小量質體的抽取與定序應…………………………………… 19
3-3.複製選殖株體…………………………………………………… 20
3-3-1.勝任細胞之製備(Preparation of competent cell)………20
3-3-2.轉型作用(Transformation)………………………………… 21
3-3-3.小量質體培養與小量質體DNA純化……………………………21
3-4.限制脢(restriction enzyme)剪切…………………………… 22
3-4-1.線性DNA之純化…………………………………………………23
3-5.RNA 探針製備(RNA Riboprobe Preparation)…………………24
3-6.RNA探針純化(RNA Riboprobe Purification)…………………24
3-7.RNA全覆式原位雜交(RNA whole-mount in situ hybridation)…25
3-7-1.斑馬魚胚胎之收集與固定…………………………………… 25
3-7-2.斑馬魚胚胎卵殼（chorion）之剝除…………………………25
3-7-3.斑馬魚胚胎之脫水（dehydration）…………………………26
3-7-4.斑馬魚胚胎之復水（rehydration）…………………………26
3-7-5.Proteinase K之處理………………………………………… 26
3-7-6.預雜交反應(prehybridization)…………………………… 27
3-7-7.雜交反應（hybridization）…………………………………27
3-7-8.雜交後胚胎之洗滌（post-hybridization washing）…… 28
3-7-9.抗-DIG抗體之偵測(detection)………………………………28
3-7-10.呈色反應(coloring)…………………………………………29
4.顯微注射(microinjection)……………………………………… 29
4-1. Morpholino 溶液之製備……………………………………… 29
4-2.顯微注射………………………………………………………… 30
5.血管顯微顯影(microangiography)……………………………… 31
5-1.血管顯影液之製備……………………………………………… 31
5-2.血管顯微顯影…………………………………………………… 31
參、結果…………………………………………………………………33
一、斑馬魚胚胎dHAND基因之定量RT-PCR……………………………33
二、斑馬魚胚胎之心臟發育時期…………………………………… 33
三、斑馬魚正常胚胎dHAND基因之全覆式原位雜交表現……………34
四、注射dHAND antisense RNA (morpholino)胚胎心臟之發育… 36
五、dHAND morpholino抑制之全覆式原位雜交表現……………… 36
六、正常胚胎之顯微血管顯影分析(microangiography)………… 37
七、dHAND antisense RNA(morpholino)抑制胚胎之顯微血管顯影分析38
肆、討論…………………………………………………………………39
伍、參考文獻……………………………………………………………48
陸、圖表…………………………………………………………………53
柒、附錄…………………………………………………………………78

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