臺灣博碩士論文加值系統

離子細胞或富含粒腺體細胞為一群特化之表皮細胞，能主動吸收或排出離子維持體液滲透壓平衡。本研究以日本種稻田魚 (青鱂魚) (Japanese Medaka, Oryzias latipes) 為模式動物，闡明廣鹽性硬骨魚類短期適應海水細胞及分子生理機制。
本研究將青鱂魚離子細胞歸類為四型，淡水有三型：HA細胞、NCC細胞及NHE細胞，海水只有一型SW細胞。NHE細胞占所有NKA免疫反應性離子細胞絕大多數，頂膜表現鈉氫交換蛋白 (Na+/H+ exchanger-3, NHE3)、氯離子通道 (cystic fibrosis conductance regulator, CFTR)、基側膜表現鈉鉀幫浦 (Na+/K+-ATPase, NKA) 及鈉鉀氯共同運輸蛋白 (Na+/K+/2Cl- cotransporter-1a, NKCC1a)。NCC細胞較少數，頂膜表現 (Na+/Cl- cotransporter, NCC)。
海水轉移適應過程中，上述離子運輸蛋白表現量產生對應消長。在海水適應青鱂魚胚胎表皮NKCC1a顯著增加而NCC不表現。成魚轉移海水實驗當中，鰓上NHE3基因表現量下降但是CFTR基因表現量上升。我們以NKA標定青鱂魚胚胎離子細胞，發現轉移過程當中離子細胞密度不變，而細胞體積變大。
短期轉移海水過程中，青鱂魚胚胎表皮P63幹細胞標密度在1、7小時無顯著變化，至1天則有些許下降。TUNEL assay染色結果顯示表皮細胞凋亡作用無顯著變化。離子細胞分化相關轉錄因子foxi3與gcm2表現量在海水轉移24小時皆無顯著差異，這些實驗結果顯示青鱂魚胚胎表皮細胞新生、分化及死亡作用在短期轉移海水變化皆不顯著。
DMBT1為哺乳類Hensin同源蛋白，Hensin為促進腎臟閏細胞產生轉型作用的重要因子。我們發現青鱂魚DMBT1表現在離子細胞上，海水轉移過程中，DMBT1免疫染色反應性變低，然而基因層級表現變化不顯著。抑制dmbt1後再轉移海水則導致胚胎孵化延遲，我們推測dmbt1可能參與海水轉移適應過程中細胞轉型作用。
綜言之，本研究推測廣鹽性硬骨魚類短期適應海水時，表皮細胞更新作用無顯著變化，離子細胞的數目亦不變，但是離子細胞型態及運輸蛋白表現因應環境鹽度變化受到調控，淡水型離子細胞可能直接轉型為海水型離子細胞，而DMBT1可能參與短期海水轉移細胞轉型作用。

Ionocytes, mitochondrion-rich cells (MRCs), are specialized epidermal cells which maintain ionic homeostasis of body fluid by actively taking up or excreting ions. The purpose of this study is to examine the regulatory mechanism of cell turnover and transporters expression of ionocytes in Japanese Medaka (Oryzias latipes) during seawater (SW) acclimation.
Four types of ionocyte were identified in medaka embryo, the freshwater (FW) type were NHE cell, NCC cell and HA cell and only one SW type ionocyte. The NHE cells expressing apical Na+/H+ exchanger-3 (NHE3), cystic fibrosis conductance regulator (CFTR), and basolateral Na+/K+-ATPase (NKA) and constituted the majority of FW ionocytes. We also found that most of ionocytes were basolateral expressing NKCC1a and co-localized with NKA suggesting that they are belonged to NHE cell. The cell number of NCC cell expressing apical Na+/Cl- cotransporter (NCC) was in minority.
Expressions of above-mentioned transporters were regulated in response to SW stimulation. NKCC1a signal was up-regulated and no apical NCC signal was found in SW-acclimated embryo. The gene expression of NHE3 (slc9a3) was down-regulated but CFTR (abcc7) was up-regulated in medaka gill during 24 hours SW transfer. In 2-days SW transfer medaka embryo, the density of NKA-immunoreactive ionocytes was not changed, but the cell size became larger.
Ionocyte turnover was examined on medaka yolk sac membrane during short-term SW adaptation. P63 is an epidermal stem cell marker, immunocytochemistry of P63 showed no significant difference in the density of P63-cells after 1 hour and 7 hours SW transfer, but a little decrease showed after 1 day SW transfer. The result of TUNEL assay indicated that cell apoptosis was not significant at 12, 24 and 48 hours after SW transfer. By qRT-PCR, The expression of ionocyte differentiation marker foxi3 and gcm2 did not differ between FW and 24-hour SW transfer medaka gill. These results suggest that proliferation, differentiation and apoptosis of epidermal cells were not changed in short-term SW adaptation response.
In mammalian, deleted in malignant brain tumor 1 (dmbt1) encodes Hensin which transform b-intercalated cell into a-intercalated cell in cortical collecting duct during systemic metabolic acidosis. In medaka, dmbt1 was also expressed in ionocytes. The protein expression of DMBT1 was down-regulated in 36 hours SW transfer embryo by immunocytochemistry. However, there was no difference in the gene expression of dmbt1 during 24 hours SW tranfer. Loss-of-function experiment showed higher mortality in dmbt1 morpholino-injected group than in control. Morpholino knock down accompany with SW transfer treatment showed obvious delay in the development of medaka morphant. We suggested dmbt1 might involve in transformation of ionocytes during SW acclimation.
Taken together, we demonstrated a model of short-term SW adaptation in medaka which suggests that FW ionocytes directly transform to SW ionocytes and dmbt1 might involve in this process.

口試委員會審定書...i
誌謝..............ii
摘要.............iii
Abstract...........v
目錄.............vii
圖目錄..........viii
表目錄............ix
前言...............1
材料與方法.........9
結果..............13
討論..............16
參考文獻..........22
附圖..............33
附表..............47
附錄..............50

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