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研究生:王贊評
研究生(外文):Tsan-Ping Wang
論文名稱:黑鯛鹽度緊迫相關基因在鰓上的表現
論文名稱(外文):Salinity effects in the expression of stress-related genes in gill of the protandrons black porgy,Acanthopagrus schlegeli
指導教授:張清風張清風引用關係
指導教授(外文):Ching-Fong Chang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
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:2009
畢業學年度:97
語文別:中文
論文頁數:72
中文關鍵詞:黑鯛緊迫
外文關鍵詞:Black porgyGillStress
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在廣鹽性硬骨魚類中,魚鰓除了為一重要調節體內外滲透壓平衡的多功能器官外,仍扮演免疫與緊迫反應的角色。本實驗目的是探討腦及鰓組織中,受到鹽度緊迫下相關基因的表現量及其鰓上表現的位置。實驗設計分別以急速改變滲透壓之實驗、類固醇注射實驗以及鰓組織切片染色實驗。並利用即時定量PCR(real-time PCR)測定基因表現量以及免疫、原位雜交染色來偵測表現在鰓組織中的位置,來探討黑鯛受到緊迫下,腦及鰓組織中緊迫相關基因的表現。
在本研究中發現,急速改變滲透壓會導致腦中的hsp70以及Na+/K+ATPase α 在第七天的淡水適應中表現量顯著上升,在鰓組織中則發現crh在第一天淡水適應中略為上升,而crhr1、hsp70、il-1β、Na+/K+ATPase α以及prlr均有顯著的上升,但在淡水適應第七天後crh和crh1表現量卻下降許多。在給予注射類固醇的實驗中,注射CRH(10 μg/kg BW)可以促進前腦、下視丘及腦下垂體crh的表現量上升,而注射Dexamethasone(3.0 μg/g BW)會促進前腦和腦下垂體crh的表現量上升,而抑制下視丘crh的表現,亦會抑制前腦crhr1表現。注射CRH會促進鰓組織中crh表現量,並且與注射劑量成正比關係。
藉由免疫染色,可以發現Na+/K+ATPase α 分布於鰓組織上的MR細胞的位置,並利用及原位雜交染色可以發現鰓組織中crh及crhr1在淡水中的表現量高於海水,以及crh及crhr1分布在細胞的確切位置。可以藉由以上的結果發現,在鹽度緊迫環境刺激下,鰓對於crh、crhr1、hsp70、il-1β、Na+/K+ATPase α以及prlr在短時間以及長時間內的調控亦扮演著一個重要的調控角色,以維持正常於魚體內的恆定。
In euryhaline teleosts, gills play an important role in osmoregulation and ionoregulation, but also have an immunization and stress response function. The objectives of the present study were to investigate the expression and possible roles of CRH, CRHR1, HSP70, IL-1β, Na+/K+ATPase α and PRLR genes in the gill and brain of black porgy (Acanthopagrus schlegeli) in response to stress.
We conducted salinity stress experiments by transferring the fish to freshwater (FW) from seawater (SW) and also CRH and Dexamethasone and the changes in respective gene expressions were analyzed in the gill and brain by quantitative real-time PCR measurements. Transfer of fish from SW to FW resulted in an increased expression of HSP70 and Na+/K+ATPase α mRNA expression in brain on day 7. In contrast, CRHR1,HSP70, IL-1β, Na+/K+ATPase α and PRLR mRNA expressions in the gill exhibited a significant increase on both day1 and day 7.Injection of CRH resulted in increased CRH expression in both brain and gill. On the other hand, injection of Dexamethasone resulted in an increase in CRH gene in the forebrain and pituitary, while CRHR1 gene decreased in forebrain.
Immunohistochemical and in situ hybridization studies were also conducted to identify Na+/K+ATPase α, crh and crhr1 localization in gill. The results suggest important roles for gill in during stress.
目錄
. 頁次
謝辭.............................................................................................................i
中文摘要…………………………………………………………….…...ii
英文摘要…………………………………………………………………iv
目錄 ………………………………………………………………..…....v
圖表目錄………………………………………………………...……...viii
第一章 文獻整理
一、魚類的緊迫反應……………………………………………………1
二、促腎上腺皮質素釋放素(Corticotropin-Releasing Hormone, CRH).2
三、促腎上皮質釋放素受體(Corticotropin-Releasing Hormone
receptor,CRH-R)………………………………………………………….3
四、熱休克蛋白 70(Heat-Shock Protein 70, HSP70)…………………..4
五、魚類免疫系統及白細胞介白素一β(Interleukin-1β, IL-1β)……….5
六、鈉鉀幫浦(Na+/K+-ATPase)………………………………………….6
七、泌乳激素受體(Prolactin receptor, PRLR)…………………………..7
八、實驗動物…………………………………………………………….8
九、研究目的…………………………………………………………….9

第二章 鹽度緊迫相關基因在鰓上的表現………………...………….11
一、前言…………………….………………………………………….11
二、實驗材料及方法….……………………………………………….13
1. 實驗魚種.............................................................................................13
2. 實驗設計…………………………………………………………….13
3. 實驗方法…………………………………………………………….16
4. 實驗藥品………………………………………………………….…27
5. 酵素及生化分析試劑……………………………………………….28
6. 實驗溶液配方……………………………………………………….28
7. 統計分析…………………………………………………………….29
三、結果…………………………………………………………….….30
1. 探討黑鯛急速改變鹽度適應一天、七天及十四天對腦及
鰓組織中crh、crhr1、hsp70、il-1β以及Na+/K+ATPase α基
因表現量的影響…………………………………………………....30
2. 探討黑鯛在注射不同劑量之類固醇激素,對於前腦、
下視丘及腦下垂體的crh以及crhr1的基因表現量………….….31
3. 探討黑鯛在注射不同劑量之類固醇激素,對於鰓組織
的crh、crhr1、prlr以及Na+/K+ATPase α的基因表現量…….....32
4. 藉由不同的染色方式觀察鰓絲細胞上,crh、crhr1及
Na+/K+ATPase的表現位置………………………………………...33
四、討論………………………………………………………………..35
第三章 結論………………………………………………………...….39
參考文獻………………………………………………………………..55
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