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研究生:王榕萱
研究生(外文):Jung-Hsuan Wang
論文名稱:內皮素對斑馬魚適應酸性環境時排酸功能的急性調節
論文名稱(外文):The role of Endothelin-1 in acute regulation of acid secretion upon acid challenge in zebrafish
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:林豊益曾庸哲張清風
口試委員(外文):Li-Yih LinYung-Che TsengChing-Fong Chang
口試日期:2014-06-30
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:54
中文關鍵詞:內皮激素斑馬魚離子細胞酸適應
外文關鍵詞:endothelinzebrafish(Danio rerio)ionocyteacidosis
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環境污染及氣候變遷導致的水域酸化已嚴重威脅水生生物的生存。神經內分泌系統是感受體外環境變化並啟動體內生理反應的重要媒介,然而其在魚類酸鹼適應中的調節角色仍所知有限。過去研究證實內皮素藉由對於氫離子幫浦的轉譯後調控進行斑馬魚表皮排酸功能的調節,而本研究旨在探討內皮素在斑馬魚酸環境適應的短期調節中可能的角色。孵化後斑馬魚胚胎面臨酸性(pH4)環境時,表皮的排酸能力在6小時內即顯著增加,內皮素基因表現亦於酸處理後4小時內顯著上升,而此時氫離子幫浦表現量並無明顯變化,且表皮離子細胞分化基因表現量在酸處理12小時候才開始上升,而其他內分泌激素基因表現於酸處理較晚期才產生變化,暗示內皮激素可能參與短期排酸能力的調控,而長期的酸環境適應機制於面臨酸性壓力12小時候才開啟。又斑馬魚胚胎面臨較低強度的酸性刺激(pH 5.5)時氫離子幫浦和表皮離子細胞分化基因表現皆未產生變化,然而內皮素基因表現已顯著上升,暗示內皮素可能參與適應較低強度酸性刺激的調節功能。

Water area acidification caused by industrial pollution and climate change has threatened the survival of aquatic habitants. As a primary link between environmental changes and physiological responses, the role of neuroendocrine system in acid-base regulation under pH disturbance in fish remains unclear. Previous study indicated that Endothelin-1 (EDN1) was involved in regulation of skin acid secretion in zebrafish embryos. The aim of this study is to explore the role of EDN1 in acute regulation of acid secretion upon acidic challenge in fish. The results showed that skin acid secretion of zebrafish embryos was stimulated in 6hr after low pH (pH4) treatment, but no concomitant changes were observed on the expression of H+-ATPase (HA). Expression of EDN1was also increased in 4hr after treatment while the expression of other hormones increase at latter timepoints. Moreover, the expression of ionocyte differentiation factors increased in 12hr after treatment, implying the long-term regulatory mechanism to acidic stress may be turned on in a latter stage and the involvement of EDN1 in short-term regulation of acid secretion. During acclimation to moderate acid conditions (pH 5.5), expression of EDN1 was upregulated while no significant difference was observed in the transcription levels of H+-ATPase and ionocyte differentiation factor, implying that EDN1 may be involved in regulating moderate acid challenge.

致謝 3
摘要 6
Abstract 7
Introduction 9
Acid-base regulation in fish 9
Zebrafish as a model to study acid-base homeostasis mechanism 10
Endothelin-1 and transepithelial proton secretion 12
Purpose 14
Materials and methods 16
Experimental animals 16
Low pH transfer experiment 16
H+-selective electrode technique 17
Measurement of whole body H+ efflux 18
Total RNA extraction 19
Reverse-transcription (RT) 19
Real-time quantitative PCR analysis (Q-PCR) 20
Microinjection of antisense morpholino oligonucleotides 20
Immunocytochemistry 21
Western blot analysis 22
Statistical analysis 23
Results 24
Effects of ednraa knockdown on the expression of H+-ATPase 24
Time-course changes in proton secretion of zebrafish embryos after transfer to acidic (pH 4) water 24
Time-course changes in mRNA expression of H+-ATPase and HRC-differentiation-related gene in zebrafish embryos after transfer to acidic (pH 4) water 25
Time-course changes in mRNA expression of EDN1/EDNRAA in zebrafish embryos after transfer to acidic (pH 4) water 25
The effects of EDN1 knockdown on acute response of proton secretion in zebrafish embryos after transfer to acidic (pH4) water 26
Time-course changes in mRNA expression of acid acclimation-related hormones in zebrafish embryos after transfer to acidic (pH 4) water 27
HR cell density and atp6v1a mRNA expression of 3dpf zebrafish larva acclimated to different pH FW 28
Expression of endothelin system in 3dpf zebrafish larva acclimated to different pH FW 28
Discussion 30
Temporal response of proton secretion in zebrafish embryos after low pH transfer 30
EDN1 is important for acute regulation of proton secretion upon acid challenge 32
EDN1 is distinctively important for acute regulation of proton secretion among hormones 34
Endothelin may be involved in acid acclimation at moderate pH environment 36
Conclusion 37
References 38
Tables and figures 43


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