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標題: 虱目魚(Chanos chanos)、墨綠凹鼻魨(Tetraodon nigroviridis)與星點東方魨(Takifugu niphobles)適應在不同鹽度環境中鰓上氯離子運輸蛋白之分布與數量
Differential distribution of chloride transporters in gills of three seawater- and fresh water-acclimated euryhaline teleosts, Chanos chanos, Tetraodon nigroviridis, and Takifugu niphobles
作者: 沈宜達
Shen, I-Dar
關鍵字: NKA;鰓;NKCC;CFTR;AE1;CLC-3;gill;salinity;Milkfish;spotted green pufferfish;grass pufferfish;immunofluorescent staining;鹽度;虱目魚;墨綠凹鼻魨;星點東方魨;免疫螢光染色;免疫化學染色;氯離子運輸蛋白
出版社: 生命科學系所
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虱目魚(Chanos chanos)、墨綠凹鼻魨(Tetraodon nigroviridis)以及星點東方魨(Takifugu niphobles)皆屬於廣鹽性硬骨魚,可適應於淡水與海水。因為先前的研究指出虱目魚與墨綠凹鼻魨生存在不同鹽度適應下有不同的Na+ /K+-ATPase (NKA)活性反應,所以藉由氯離子運輸蛋白的位置去了解和比較鰓上氯離子運輸機制在兩群間的差異。目前已知氯離子運輸蛋白包括:Na+ /K+ /2Cl- cotransporter (NKCC);cystic fibrosis transmembrane conductance regulator (CFTR);anion exchanger 1 (AE1)以及chloride channel 3 (CLC-3)。為了解氯離子運輸蛋白分佈於廣鹽性魚類鰓表皮細胞上的位置與數量,本論文利用雙重免疫螢光染色配合共軛焦顯微鏡比較其在淡水(0‰鹽度)或海水(35‰鹽度)馴化後之變化。實驗結果指出馴養於淡水或海水的虱目魚、墨綠凹鼻魨以及星點東方魨,其NKA、NKCC、AE1以及CLC-3均位於細胞基部,而CFTR在淡水中並未出現,在海水中則位於細胞頂端的位置。這說明在鰓表皮細胞上是由NKA耗能啟動氯離子的調節路徑,NKCC或AE1則負責帶動氯離子進入細胞,當氯離子過多時,由CFTR或CLC-3分別將細胞中的氯離子分別帶出至外界或血管中,保持細胞內氯離子的濃度。此外,馴養在淡水或海水中的墨綠凹鼻魨與星點東方魨,氯離子運輸蛋白僅分布在鰓絲表皮上;而馴養在淡水的虱目魚,氯離子運輸蛋白則會另增生於鰓薄板上。計算免疫反應細胞(immunoreactive cells)數量的結果則顯示馴養於淡水的虱目魚,NKA或NKCC免疫反應細胞在鰓薄板上數量顯著增加,且NKCC免疫反應細胞數量在鰓表皮上明顯少於NKA免疫反應細胞數量。而在馴養於海水的墨綠凹鼻魨中,NKA免疫反應細胞數量顯著高於其在馴養於淡水的個體;星點東方魨的NKA免疫反應細胞數量在淡海水則沒有顯著差異。综合上述結果顯示與鰓表皮NKA免疫反應相符之細胞,其細胞上所表現之四種氯離子運輸蛋白的差異性,說明三種不同棲地的廣鹽性魚類為配合生理上的需要,會有不同的調節機制。

Milkfish (Chanos chanos), spotted green pufferfish (Tetraodon nigroviridis) and grass pufferfish (Takifugu niphobles) are all euryhaline species of seawater or freshwater origins. Since previous studies revealed that Milkfish, and spotted green pufferfish exhibited different adaptive changes in Na+/K+-ATPase (NKA) activity following salinity challenge, it is thus intriguing to realize and compare Cl- transport mechanisms in gills between these two groups by immunolocalization of Cl- transporters, i.e., Na+/K+/2Cl- cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 1 (AE1), and chloride channel 3 (CLC-3). In order to illustrate the distributions and numbers of those Cl- transporters in gill epithelia, euryhaline fish from different habitats were acclimated to fresh water (FW; 0‰ salinity) or seawater (SW; 35‰ salinity) for more than one month, and cryosections of gills were observed and analyzed by the confocal laser scanning microscope after double-immunofluorescent staining. The antibody staining results showed that in gills of milkfish, spotted green pufferfish and grass pufferfish, NKA as well as NKCC, AE1, and CLC-3 were colocalized in the basolateral parts of immunoreactive epithelial cells of FW- or SW-acclimated individuals, while CFTR were localized only in the apical sides of NKA immunoreactive cells in SW fish. In SW milkfish as well as FW or SW spotted green pufferfish and grass pufferfish, immunoreactive cells were only distributed in filaments; in FW milkfish, however, immunoreactive cells were found in both filaments and lamellae. Moreover, in the FW-acclimated milkfish, the numbers of NKA and NKCC immunoreactive cells in gill lamellae increased significantly, whereas the number of NKCC immunoreactive cells was significantly lower than that of NKA immunoreactive cells in gill filaments and lamellae. On the other hand, the number of NKA immunoreactive cells was significantly higher in SW-acclimated spotted green pufferfish than that in FW-individuals, while the amounts of NKA immunoreactive cells were similar in SW- and FW-acclimated grass pufferfish. Taken together, differential expression of four Cl- transporters colocalized in the NKA immunoreactive cells of gill epithelia implicated different mechanisms of Cl- transport among three euryhaline species from different habitats to match the physiological requirements.
其他識別: U0005-0507200617393800
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