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標題: 日本鰻食道對於海水去鹽機制之探討-鈉、鉀、氯共同運輸蛋白家族(SLC12A)之表現、內分泌調控與功能
Esophageal Desalination of Seawater in Eel: Expression, Hormonal Regulation, and Roles of Na+, K+, Cl-Cotransporter (SLC12A) Family
作者: 李宗翰
關鍵字: 基礎研究;euryhaline teleost;漁業類;廣鹽性硬骨魚;鰻魚;食道;鈉;鉀;氯共同運輸蛋白;可體松;去鹽作用;eel;esophagus;Na+;K+;Cl- cotransporter;cortisol;desalination
海水硬骨魚類在吞嚥海水過程中,食道會進行去鹽作用。先前的研究發現海水魚食道對於鈉、氯離子通透性較高,但是對水分通透性極低。然而對於食道上何種細胞參與此鈉、氯離子由管腔內進入血液中的運輸機轉仍缺乏明確的證據。從魚類鰓上離子調節機制的研究中發現鈉,鉀,氯共同運輸蛋白(SLC12A)家族,在氯離子進出鰓的運輸作用上扮演重要的角色。因此,本研究選用具有較長食道的廣鹽性日本鰻做為實驗魚種。第一年目標是以分子生物層面分析表現於食道黏膜層的SLC12A成員,並比較環境鹽度對其mRNA和蛋白質表現量的影響。第二年計畫以建立食道黏膜層細胞的初級細胞培養系統,藉由不同離子培養基的刺激,探討SLC12A成員受到不同鈉、氯離子的濃度刺激後的表現變化,並且建立活體螢光染色法,即時追蹤氯離子進出細胞的流動率,並搭配專一性抑制劑的處理,以偵測出黏膜層細胞SLC12A成員的活性。第三年研究探討魚類適應海水的重要荷爾蒙-可體松(cortisol)是否調控黏膜層細胞SLC12A成員的表現。首先證實其受體(receptors) -葡萄醣皮質素受體(glucocorticoid receptor, GR)和礦物性皮質素醛固酮受體(mineralocorticoid receptor, MR)的表現,接著以體內注射cortisol的實驗探討其對SLC12A蛋白成員的影響。最後,利用初級細胞培養的系統,搭配可cortisol receptor的抑制劑處理,釐清cortisol是經由GR或MR的路徑傳遞對SLC12A成員的影響。

Esophageal desalination is essential for marine teleosts drinking seawater to compensate thediffused water. Previous studies reported that the esophagus of marine fish is Na+ and Clpermeablebut water impermeable. However, the type of epithelial cells responsible for thetransport of Na+ and Cl- from lumen to the circulatory system in the esophagus is unclear.Members of Na+,K+,Cl- cotransporter (SLC12A) family were found to be crucial for Cltransportsystem of fish gills. This project will use Japanese eel with long esophagus as theexperimental animal. In the 1st year, gene sequences of the member of SLC12A family in theeel esophagus will be identified. The expressions and distributions of SLC12A familyproteins in the mucosa of esophagus will be compared between the freshwater- andseawater-acclimated eels. In the 2nd year, epithelial cells from the esophageal mucosa will beisolated and the primary culture system for treating the media with different Na+ and Clconcentrationwill be developed. The method of microfluorescent imaging will be applied totrace the changes of Cl- flux on the target cells exposed to the media with the inhibitors ofSLC12A family proteins to analyze the activity of the transporters. In the 3rd year, cortisoleffects on the expression of SLC12A family will be illustrated. Cortisol receptors -glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) will be identified from eelesophagus. Finally, the cultured cells will be treated with the inhibitors of cortisol receptors torealize the signal pathways of cortisol which affected the expressions of SLC12A family ofeel esophagus.
其他識別: NSC99-2313-B005-004-MY3
Appears in Collections:生命科學系所

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