臺灣博碩士論文加值系統

廣鹽性的硬骨魚類主要藉調節其離子和滲透壓的平衡來面對環境中鹽度的改變。在現今硬骨魚海水適應的鰓細胞模型中，富含粒線體細胞(Mitochondira-rich cells, MR cells)的離子通道和運輸蛋白負責最主要的離子調節，維持體液的恆定。其中最重要也被研究最透徹離子通道包括:鈉鉀幫浦(Na+/K+-ATPase, NKA)、鈉鉀二氯共通運輸蛋白(Na+/K+/2Cl- cotransporter, NKCC)和纖維性囊腫穿膜傳導調節蛋白(Cystic fibrosis transmembrane conductance regulator, CFTR)。本研究利用日本種稻田魚(Oryzias latipes)作為研究對象，分析這些離子運輸蛋白的生理功能和基因表現，發現日本種稻田魚的存活率測試在適應過海水之後有顯著的增加。顯示出適應過海水的日本種稻田魚應有特別的轉換調控機制維持其存活，因此研究日本種稻田魚可以提供線索以了解鹽度適應性和基因調控的關係。
將日本種稻田魚鰓細胞中離子通道之胺基酸序列個別進行演化樹分析，再利用即時定量聚合酉每連鎖反應(qRT-PCR)分析適應不同環境鹽度的日本種稻田魚鳃組織，發現atp1a1a.1, atp1b1a, atp1b1b, slc12a2a和abcc7可能與海水適應的離子調節相關，因其mRNA會受到海水刺激而表現增加；其中atp1b1a和atp1b1b由海水轉移回淡水表現量會維持增高的情形，可能共同參與維持日本種稻田魚適應過後的海水調節能力。同時以atp1a1a.1, atp1b1b, slc12a2a和abcc7核酸探針標定其基因表現位置，首次提供分子證據直接證實這些基因皆表現在相同的細胞。
另一方面，偵測細胞在的不同環境鹽度適應下增生和死亡的表現，發現轉移到海水時細胞增生和死亡率皆高於淡水；利用olfoxi3做標定發現細胞分化有增加的現象。基於上述的結果，我們認為當日本種稻田魚鰓上的離子通道受到鹽度的刺激，其細胞週期變短，且一些未分化的細胞會加速分化成海水型富含粒線體細胞表現以適應鹽度的改變。綜合上述之結果，本實驗首次提供分子生理證據，證明由淡水轉移到海水時，日本種稻田魚的海水型富含粒線體細胞相關基因參與滲透壓離子調節機制以對抗外在鹽度的改變。

Euryhaline teleosts have to cope with the fluctuating salinities of the environments in which they inhabit during their life time. In current model of gill salt scecretion of seawater- (SW) adaptated teleosts, mitochondria-rich cells (MR cells) play the major roles in the active ion secretion mechanism. The ion secretion mechanism in SW gill MR cells is achieved by the balsolateral Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC), and an apical membrane located cystic fibrosis transmembrane conductance regulator (CFTR). However, this model still lacks sufficient molecular evidences and leaves some controversies in the previous studies.
The present study used Japanese medaka (Oryzias latipes, OL) as the model species to provide molecular and physiological evidence to support the roles of the relevant transporters in the current NaCl secretion model of SW type MR cells. The mortality experiment showed that OL enhanced their salinity adaptability after a pre-acclimation to SW and even after back to FW for 1 wk. This study examined the mRNA expression patterns of 8 transporter isoforms, and found that 3 NKA isoforms (atp1a1a.1, atp1b1a and atp1b1b), 1 NKCC (slc12a1a) and 1 CFTR (abcc7) mRNA levels were up-regulated during SW exposure, suggesting that these genes may play critical roles in salt secretion. Interestingly, the elevated mRNA levels of atp1b1b and slc12a2a in SW medaka were prolonged even after transfer back to FW for 1 wk, indicating that occurrence of a latent salt secretion mechanism of these 2 genes may be necessary for the subsequent salinity challenge as we mentioned above. Furthermore, atp1a1a.1, atp1b1b, slc12a2a and abcc7 were found to be co-expressed in the same MR cell, providing the first molecular evidence for their isoform-specific identities.
The cell number of proliferation (by PH3 stainning) and apoptosis (by TUNEL assay) in gill MR cells showed that the gill cell’s turnover rate is faster in SW than in FW. In addition, olfoxi3 expression indicated the differentiation of MR cells during SW acclimation. NKCC and CFTR mRNA expression levels highly related to the number of SW-type MR cells. According to these results, we proposed that the differentiation of immature cells into SW-type MR cells can be accelerated to enhance NaCl secretion pathway in SW.
In summary, several isoforms were isolated from gill MR cells of OL, and in vivo molecular evidences demonstrated their roles in iono/osmo-regulation and cell differentiation mechanisms in fish gills. This supports the notion that the expressions of these ion transporters correlate with the NaCl secretion function in MR cells, and also offer new insights into the studies on salinity tolerance in euryhaline teleosts.

Table of content
中文摘要…..........…………………………………………………………...1
Abstract………….………………………………………………………….3
Introducation…….…………………………………………………………...5
Salinity acclimation mechanism in euryhaline teleosts………………………….....5
Transporters related to salinity acclimation………………………………………6
Differentiation of MR cells………………….…………………………………8
Medaka as a model to explore salinity acclimation mechanism………………….....9
Aims of study………………………………………………………………..10
Materials and Methods……………………………………………………...12
Animals…………………………………………………….........................12
Acclimation experiments……………………………………………………..12
Preparation of total RNA……………………………………………………..13
Reverse transcription-PCR analysis……………………………………………13
Gene expressions in different tissues…………………………………………...15
Molecular cloning and sequencing analysis...…………………………………...15
Fluorescence double in situ hybridization………………………………………16
Quantitative real-time PCR …………………………………………………...17
Phospho-Histone H3 staining………………………………………………….18
Indirect TUNEL labeling assay………………………………………………..18
Statistical analysis……………………………………………………............19
Results……………………………………………………..........................20
Seawater acclimation and survival rate………………………............................20

Phylogenetic analysis of medaka genes………………………..........................20
RT-PCR analysis of gene expressions in different tissues………...........................21
Effects of salinity on mRNA expressions of ion transporters in medaka gills….…...22
Whole-mount in situ hybridization expression of NKA, NKCC and CFTR genes…..23
Cell proliferation and apoptosis……….............................................................23
Time course changes of foxi3, atp1a1a.1, atp1b1b and slc12a2a gene expressions rates in medaka gills after transfer to BW/SW.............................................................24
Discussion………………………………………………….........................25
References………………………………………………….........................33
Tables…………………………………………………................................41
Figures…………………………………………………..............................42

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