黃鵬鵬Hwang, Pung-Pung臺灣大學:漁業科學研究所李依純Lee, Yi-ChunYi-ChunLee2010-05-102018-07-062010-05-102018-07-062008U0001-2107200822051700http://ntur.lib.ntu.edu.tw//handle/246246/181667在哺乳動物中，SLC4蛋白質家族主要分為三類型碳酸氫根運輸蛋白 (anion exchanger, AE; Na+/HCO3- cotransporter, NBC; Na+-driven Cl-/HCO3- exchangers, NDCBE)、概括由十個基因所組成，主要負責維持生物體內pH値的恒定調節，此外，哺乳類腎臟更發現多類型的碳酸氫根運輸蛋白參與離子再吸收的機制。過去研究中發現，SLC4蛋白質家族成員中的AE1 (anion exchanger) 和NBCe1 (sodium bicarbonate cotransporter) 參與魚類鰓上離子細胞的酸鹼平衡以及鈉離子吸收機制，然而迄今仍缺乏有力的分子證據。因此本實驗目的以斑馬魚為模式動物探討SLC4蛋白質家族在鰓上離子調節吸收的機制中所扮演的角色。 藉由NCBI以及Ensembl基因體資料庫預測出斑馬魚中共有十三型SLC4基因家族成員，其中十型在本研究中已經成功的被選殖出。由原位雜交 (in situ hybridization)與免疫螢光化學分析實驗發現，zAE1b (zebrafish anion exchanger, zAE1b) 以及zNBCe1b (zebrafish Na+/HCO3- cotransporter)表現在富含氫離子幫浦(H+-ATPase-rich cells, HR) 的離子細胞中並且證實位於該類細胞的基側膜上。此外在初步功能分析實驗中，以基因專一的反股核酸 (morpholino-modified antisense oligonucleotide) 降低其表現，同時以定量PCR檢測其他參與鈉吸收和酸鹼平衡機制之基因表現情形，發現這些參予調控的基因均受影響。在斑馬魚環境適應的實驗中更進一步發現，低鈉環境下NBCe1b mRNA表現量減少而AE1b增加，酸環境下呈現相同地表現。綜合以上結果，本研究中首次提供在in vivo層次上的分子生理證據，證明兩型新的碳酸氫根運輸蛋白(AE1b和NBC1b)參與鰓上鈉離子吸收與酸鹼平衡的機制。In mammal, bicarbonate transporters play an important role in pH regulation, and ten isoforms are grouped into the solute carrier 4 (SLC4) family. Some SLC4 members have been proposed to be associated with ion regulation and acid-base balance in fish, but no convincing evidence is available to support so far. The present study used zebrafish as a model to examine the role of SLC4 members in fish ion regulation and acid/base balance mechanisms. Ten genes of the zebrafish SLC4 have been successfully cloned. Using triple in situ hybridization and/or immunochemistry, 2 novel isoforms, zslc4a1b (zebrafish anion exchanger, zAE1b) and zslc4a4b (zebrafish Na+/HCO3- cotransporter, zNBCe1b), were identified to specifically expressed in the basolateral membrane H+-ATPase rich (HR) cells of zebrafish skin/gills. The preliminary knockdown experiments of the zAE1b and zNBCe1b showed the two novel genes were associated with Na+ uptake and acid-base regulation by qRT-PCR analysis on relevant gene expresions. The qRT-PCR experiments revealed that acclimation to low-Na+ environment up-regulated the mRNA expression of zAE1b, but down-regulated that of zNBCe1b. On the other hand, exposure to acidic freshwater stimulated the zAE1b expression while declined that of zNBCe1b. Taken together, two novel isoforms of zNBC1b and zAE1b were found to specifically express in HR cells of zebrafish, and molecular physiologically demonstrated to be involved in the mechanisms of Na+ uptake/acid-base regulation.Table of Contents文摘要 1bstract 3ntroduction 4LC4 family as a group of HCO3- transporters 4he role of SLC4 in mammalian kidney 6odium uptake and acid/base balance in freshwater fish 7ims of this study 9aterial and Method 11xperimental animals 11cclimation experiments 11olecular cloning and sequences analysis 12reparation of total 12T-PCR and 5’ and 3’ RACE 12n situ hybridization 13hole-mount immunohistochemistry 14estern blot analysis 16eal-time polymerase chain reaction (qRT-PCR) 16ranslational knockdown with antisense morpholino oligonucleotides 17tatistical analysis 18esults 19dentification, molecular cloning and sequencing of slc4 gene family by database mining 19T-PCR and whole mount in situ hybridization analysis of mRNA expression patterns of slc4 gene family 20olocalization of slc4 members with H+-ATPase and Na+/K+ ATPase by in situ hybridization and immunohistochemistry in zebrafish HR cells 21mmunohistochemistry of AE1 and NBCe1 21ffect of environmental Na+ concentration on NBCe1 expression in zebrafish gills 22xpressions of relevant genes in zslc4a1b and zslc4a4b morphants 22ffect of environmental pH and Na+ concentration on zslc4a1b and zslc4a4b expression in zebrafish gills 23iscussion 24eferences 33ables 41igures 44application/pdf1483730 bytesapplication/pdfen-US離子調節碳酸氫根運輸蛋白ion regulationbicarbonate transporterhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181667/1/ntu-97-R95b45016-1.pdf