臺灣博碩士論文加值系統

斑馬魚（Danio rerio）由於易於飼養、成長快速、繁殖容易、胚胎透明，是作為脊椎動物基礎研究指標性的國際性模式魚種，而相關的基因功能研究技術皆相當成熟，如基因轉殖、基因去活化（morpholinos knock-down）等。類胰島素生長因子（IGF）的反應或由類胰島素生長因子本身的旁分泌、自分泌(paracrine/autocrine)的一連串訊息傳遞反應來調控生長，而IGF基因家族在旁分泌及自分泌調控仍有許多未知待發掘，透過旁分泌和自分泌機制所產生的IGF對個體生長和胚胎發育扮演了非常重要角色。
所以類胰島素生長因子基因家族（IGF-1, IGF-2, IGF-1 receptor(IGF-1R)）是生長系統最重要的成員之一，因此本論文研究主要是找出與類胰島素生長因子相關之基因群組，再進而探討其功能性的研究。
斑馬魚的第一型類胰島素生長因子基因在本研究至少發現有二個，但仍只轉譯出一種相同的蛋白質冪嚆(TM)胰島素生長因子(proIGF-1 Ea-2)，而此二基因轉譯出的蛋白質相同程度達100%，至於蛋白質的成熟機制至今仍尚不清楚，其proIGF-1之E 胜月太究竟是演化上冗長多餘的，還是像原胰島素(proInsulin) 能有研究發現其C peptide有重要功能，尤其最近研究指出C peptide在第一型糖尿病上扮演重要角色，基於上述理由，本實驗假設proIGF-1之E 胜月太有模擬IGF-1之功能，尤其在軟骨生長上，所以本研究闡述了Ea-2 胜月太在斑馬魚和吳郭魚的鰓軟骨上皆具有軟骨硫化之功能，其硫化程度比控制組高達三倍以上，除了硫化功能外Ea-2 胜月太尚有[3H]thymidine嵌合DNA之能力並有荷爾蒙濃度依存之效果。為了更進一步了解其Ea-2 胜月太之作用機制，在本研究中採用了IGF-1之頡抗劑(aIR-3)，此一aIR-3可有效的結合IGF-1接受器，使IGF-1訊息無法傳遞，研究發現以aIR-3並無法抑制Ea-2 胜月太之硫化功能，但郤能抑制IGF-1之硫化功能，在此闡述了Ea-2 胜月太之硫化功能可非經由IGF-1接受器，而可能有另外的特有接受器或其他作用機制而使Ea-2 胜月太擁有軟骨硫化之生化功能。
IGF基因在胚胎中胚層扮演極重要的角色，如骨骼系統，心血管系統及真皮組織和皮下脂肪皆由中胚層衍生出來，而間葉細胞則屬於中胚層的衍生物，其可分化為纖維母細胞、免疫母細胞、軟骨母細胞和成骨母細胞等。IGF-1和IGF-2都是普遍性的細胞生長因子並透過IGF-1R接受器來傳達訊息，並可透過自分泌或旁分泌來抑制或擴增其IGF的生物活性。所以IGF-2和IGF-1R是本論文不可少之成員，本論文亦將IGF-2a cDNA和IGF-2b基因選殖出來，並從斑馬魚網站找到一個高相似度的IGF-1R EST選殖株取得其5UTR區域，透過基因功能研究技術:基因轉殖及基因去活化（morpholinos knock-down）來研究IGF基因嚴重影響斑馬魚胸部的發育。
本論文結論是希望能以斑馬魚IGF基因在生長機制上提供一些新的見解，以促進對生長的瞭解，希望能在未來建立調節生長的工程基因網路。

The characteristics of high growth rate, easy breeding and maintenance have made the zebrafish (Danrio rerio) as world-wide recognized species and important embryo model for functional genomics and proteomics research in the post-genomics era.
This dissertation focused on the zebrafish growth related genes and its function, especially IGF-1, IGF-2 and IGF-1R. In this study, we plan to dissect the mechanisms of paracrine/autocrine modes of IGF action in the thorax development.
We cloned zebrafish (Danio rerio) IGF-1 cDNA and its genomic sequence from zebrafish brain cDNA library and an adult zebrafish genomic library, respectively. These two cDNA sequences differ from with each other in different length of 5- and 3-untranslated region (5UTR and 3UTR) and one nucleotide difference at glutamine (A9, CAG/IGF-1a and CAA/IGF-1b) of the A domain. The results of IGF-1 mRNA expression and genomic Southern blotting, RPA, 5悐ACE suggested that the zebrafish have more than two IGF-1 genes. All these findings suggest that the expression of pro-IGF-1 Ea-2 is not controlled by alternative splicing but alternative gene usage in the zebrafish.
Results of RT-PCR and Southern blot analysis showed that, only one form of IGF-1 Ea-2 mRNA was expressed in all tested adult tissues of zebrafish, even under condition of growth hormone, prolactin or insulin administration and fasting. The encoding proIGF-1a Ea-2 protein is 100% identity to proIGF-1b Ea-2. So, we tested the hypothesis whether E domain can mimic IGF-1 function.
In this research, a synthetic peptide of the predicted zebrafish Ea-2 polypeptide (designated as zfEa-2) was not only increased 3 fold of branchial cartilage specific sulfate uptake in vitro compared with control group but also increased the [3H]thymidine incorporation into DNA on the mouse spleen cell. For further understanding the zfEa-2 polypeptide action mechanism, the aIR-3 was used to block the IGF-1 signal transduction. We demonstrated that aIR-3 couldn掐 inhibit the zfEa-2 signal but inhibit the IGF-1 sulfate uptake capability. These findings strongly suggest that the zfEa-2 can mimic IGF-1 function as a local mediator in zebrafish. These findings strongly suggest that the zfEa-2 polypeptide has another receptor to trigger the sulfation signal in zebrafish.
Finally, The IGFs system exhibits an important role on the zebrafish development. So, we are further cloning the IGF-2a cDNA and IGF-2b gene from library. Meantime, we are search one zebrafish IGF-1Ra EST clone and get the 5UTR sequence for morpholino gene knock-down functional assay.
In conclusion, this dissertation hopes to provide a new vision on the IGF gene family (IGF-1, IGF-2, IGF-1R) to distribute onto the growth regulation mechanism. Based on these researches, it can provide some information to construct the growth gene-chip network in the future.

謝辭 ............................................
CONTENTS ........................................ I
LIST OF FIGURES AND LEGENDS ..................... IV
LIST OF MATERIALS AND METHODS ..................... V
ABSTRACT (English) .............................. 1
ABSTRACT (Chinese) .............................. 3
INTRODUCTION .................................... 5
1. Theory about the growth .......................... 5
a. GH-IGF-1 axis (Somatomedin Hypothesis) ......... 5
b. Modify Somatomedin Hypothesis .................. 6
2. IGF-1 genes, IGF-2 genes and IGF-1 receptor ...... 8
a. The IGF-1 ligand family ........................ 8
b. Functional domains of preproIGF-1 protein ...... 12
c. Regulation network between IGFs ligand and IGF-1R 15
3. Zebrafish development ............................ 22
a. The zebrafish fish as a model system of postfertilization development .................. 22
4. Scope of this thesis ............................. 23
MATERIALS AND METHODS ........................... 25
RESULTS ......................................... 47
1. The novelty of preproIGF-1 genes ................. 47
2. One form proIGF-1 Ea-2 mRNA is expressed ......... 52
3. Biological activity of Synthetic zfEa-2 polypeptide ...................................... 55
4. The novelty of the zebrafish IGF-2 genes ......... 57
5. Spatial and temporal expression pattern of IGF-1, IGF-2a and IGF-2b mRNA during zebrafish embryogenesis and tissue distribution in adult zebrafish ........................................ 58
FIGURES AND LEGENDS ............................. 61
DISCUSSION ...................................... 105
1. More than two IGF-1 genes exist in zebrafish ..... 105
2. The zfEa-2 polypeptide mimic IGF-1 function ...... 109
CONCLUSION ...................................... 114
REFERENCES ...................................... 116
APPENDIX ........................................ 129
1. Publication List ................................. 129
a. Major publication .............................. 131
2. Abbreviations .................................... 140
3. BMI index ........................................ 142
4. Results of IGFs system on the thorax development of zebrafish ......................... 142
VITAE ........................................... 155

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