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研究生:陳書瑋
研究生(外文):Shu-Wei Chen
論文名稱:第一型膠原蛋白在斑馬魚表皮發育中扮演之角色
論文名稱(外文):The role of collagen type I in zebrafish epidermaldevelopment
指導教授:黃鵬鵬黃鵬鵬引用關係
指導教授(外文):Pung-Pung Hwang
口試委員:張清風林豊益曾庸哲
口試委員(外文):Ching-Fong ChangLi-Yih LinYung-Che Tseng
口試日期:2013-07-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:漁業科學研究所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:68
中文關鍵詞:斑馬魚第一型膠原蛋白表皮幹細胞黏液細胞表皮發育
外文關鍵詞:zebrafishcollagen type 1epidermal stem cell (ESC)mucous cellsepidermal development
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膠原蛋白,為細胞外間質中最主要的成分。不僅參與結構組織的形成,也調控細胞極化、固著、黏附、遷移和分化這類型的生理過程。魚類的第一型膠原蛋白已被證實如同哺乳類動物一般,為皮膚中最主要的膠原蛋白。過去在研究表皮發育機制中,除了指出真皮中纖維母細胞合成膠原蛋白外,是否還有哪一類型表皮細胞參與合成,目前鮮少資料提及也尚未有證據證實。到目前為止,關於第一型膠原蛋白在魚類皮膚中的基因表現和功能尚未完全清晰。本實驗目的為闡明第一型膠原蛋白在斑馬魚胚胎表皮上所扮演的角色及功能。斑馬魚的第一型膠原蛋白有三種isoforms,分別為alpha 1a 、alpha 1b與alpha 2。我們以斑馬魚作為實驗材料,利用RT-PCR的技術檢測發現,col1a1a mRNA大量表現在皮膚中並且在受精後2小時的階段就有表現產生,另外兩種isoforms則是受精後8小時才有基因表現的訊號產生。定位雜交(in situ hybridization)技術顯示三種isoforms在胚胎表皮上有著類似的分布型態。利用反義核酸(morpholino oligos)抑制三種第一型膠原蛋白isoforms短肽的合成後,發現表皮細胞中的表皮幹細胞(ESCs)、離子細胞、角質細胞和黏液細胞數量都有顯著性下降。另外也觀測到col1a1a mRNA與表皮幹細胞的免疫染色共定位於一起,第一型膠原蛋白的抗體免疫染色也顯示第一型膠原蛋白表現在黏液細胞中。在此研究中,我們證明了第一型膠原蛋白於早期的胚胎發育時期表現,並且通過表皮幹細胞來生成。而根據第一型膠原蛋白表現在黏液細胞中的結果,我們推測或許第一型膠原蛋白也參與了皮膚上的防禦機制。綜合以上所述,這篇研究對於第一型膠原蛋白在皮膚的發育中所扮演的角色提供了不同的理解和新穎的知識。

Extracellular matrix majorly composed of collagen. Collagens not only function as structural component but also participate in many physiological processes such as cell polarity, anchorage, adhesion, migration, and differentiation. Similar to mammals, teleosts collagen type 1 has been identified as major collagen in skin. Previous studies linked collagen type 1 in epithelial development but none can provide the specific mechanism behind such as the type of epidermal cell synthesizing collagen type 1 besides fibroblast is yet to be unidentified. So far, the knowledge of collagen type 1 isoforms gene expression and function in fish skin is not completely clear. The purpose of the present study is to examine the role of collagen type 1 in zebrafish embryos epidermal development. There are 3 isoforms of collagen type 1 in genome database, the collagen type 1 alpha 1a (col1a1a), alpha 1b (col1a1b) and alpha 2 (col1a2), all were detected by RT-PCR and cloned. Tissue scan indicated that col1a1a mRNA expressed abundantly in the skin and the expression begins to be endogenously transcribed from 2 h post fertilization (hpf) followed by other isoforms from 8 hpf. Expression of collagen type 1 isoform mRNAs showed similar spatial patterns via in situ hybridization. The knockdown of col1a1a, col1a1b, and col1a2 results showed that all of the ESCs, ionocytes, keratinocytes, and mucous cells number were decreased. mRNA in situ hybridization of col1a1a co-localized with ESC and immunostaining showed collagen type 1 is present only in agr2/mucous cells. The presence of collagen in ESCs and keratinocytes was not established in this study due to the limitation of commercial antibodies used. Nevertheless, we demonstrated that collagen type 1 is present very early during the embryogenesis and is being synthesized by the ESCs, clarifying the timing and location of expression. The presence of collagen in mucous cells may indicate possible role in this defense mechanism of the skin. Hence, this work provides new knowledge and additional understanding of the collagen type 1 role in skin development.

口試委員審定書............................................................................................................... i
謝辭.................................................................................................................................. ii
摘要................................................................................................................................. iii
Abstract............................................................................................................................ iv
Table of Contents............................................................................................................. vi
Introduction...................................................................................................................... 1
Collagen gene expression mechanism...................................................................... 1
Collagen classifications and functions..................................................................... 2
Collagen linked to organ development and diseases................................................ 3
Collagen involvement in skin development............................................................. 4
Collagen in teleost fish............................................................................................. 6
Recent platform of zebrafish epidermal cells classifications and functions............. 8
Purpose of the study................................................................................................ 11
Materials and methods.................................................................................................... 13
Experimental animals............................................................................................. 13
Preparation of total RNA........................................................................................ 13
Reverse-transcription polymerase chain reaction (RT-PCR) analysis................... 14
Morpholino oligonucleotide (MO) knockdown..................................................... 14
Whole-mount in situ hybridization......................................................................... 15
Whole-mount immunohistochemistry.................................................................... 16
Whole-mount Alcian blue staining......................................................................... 17
Transgenic zebrafish (anterior gradient 2 homologue:GFP) ................................. 18
Western blot analysis.............................................................................................. 18
Statistical analysis................................................................................................... 19
Results............................................................................................................................ 20
The tissue distribution of fibril-forming collagens in adult zebrafish.................... 20
The timing and pattern of collagen type 1 expression during early development
of zebrafish embryos.............................................................................................. 20
Effect of collagen type 1 knockdown in epidermal stem cells number.................. 21
Effect of collagen type 1 isoforms knockdown in ionocytes number.................... 22
Effect of collagen type 1 isoforms knockdown in keratinocyte and mucous cells number.................................................................................................................... 23
Localization of collagen mRNA and antibody with epidermal stem cells............. 23
Localization of collagen type 1 protein in ionocytes and keratinocytes of zebrafish.................................................................................................................. 24
Collagen type 1 patterns are co-localized with mucous cells patterns................... 25
Effect of collagen type 1 knockdown in agr2 mRNA expression and agr2:GFP patterns number...................................................................................................... 25
Effect of collagen type 1 knockdown in mucous cells number after cortisol treatment................................................................................................................. 26
Immunohistochemistry and western blot demonstrated the function of antibody of collagen type 1........................................................................................................ 26
Discussion....................................................................................................................... 28
The role of collagen type 1 in zebrafish epidermal development........................... 28
The function of collagen type 1 in ECM................................................................ 28
Collagen type 1 in fish embryonic development.................................................... 29
The relationship of collagens and cell physiological processes............................. 30
Collagen synthesis in zebrafish basal epidermal layer........................................... 31
The presence of collagen type 1 in mucous cells................................................... 32
The role of collagens in skin with ascorbic acid and cortisol................................. 34
Summary......................................................................................................................... 35
Perspectives.................................................................................................................... 36
References...................................................................................................................... 37
Table............................................................................................................................... 49
Figures............................................................................................................................ 50



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