黃鵬鵬臺灣大學:漁業科學研究所丁楓峻Ting, Feng-ChunFeng-ChunTing2010-05-102018-07-062010-05-102018-07-062009U0001-0308200911405100http://ntur.lib.ntu.edu.tw//handle/246246/181695鈣離子參與許多重要的生理反應，因此動物如何調控鈣離子恆定是相當重要的議題。淡水環境中的鈣離子濃度變化劇烈，當淡水魚成魚面對環境鈣離子變化時，位於鰓上皮組織的富含粒線體細胞(mitochondria-rich cell, MR cell)上的離子通道或運輸蛋白會受到調控並改變表現，進而維持體內的離子變動不會過於激烈。然而在淡水魚的胚胎發育時期，鰓並未發育完全，離子調控主要透過一些位在表皮上的富含粒線體細胞。對於內分泌調控魚類鈣離子吸收機制的研究目前非常少，已知在哺乳動物的實驗模式中，vitamin D可以調控腸道和腎臟的表皮鈣離子通道 (Epithelium calcium channel, ECaC)表現量，但在魚類仍不清楚，是否在原始的魚類中就已存在維他命D調控鈣離子吸收機制值得進一步探討。實驗探討維他命D對斑馬魚鈣離子吸收機制的影響，結果發現維他命D受器基因(vitamin D receptor, VDR)在胚胎受精後三小時內就開始表現；另一方面，並發現VDR基因廣泛分佈在成魚的各組織中。將斑馬魚胚胎浸泡維他命D以後分析魚體總鈣含量、鈣離子流進胚胎體內的速率及與鈣離子相關的離子通道或運輸蛋白的基因表現量，結果發現皆有上昇的趨勢。利用Morpholino默化 (knockdown) 斑馬魚維他命D-a型受器(zVDRa)的表現，發現到胚胎體內的鈣離子含量、鈣離子流進體內的速率和 zECaC mRNA 的表現量，都有顯著的下降，但是卻不影響 zNCX1b和 zPMCA2的表現量。藉由上述結果推論斑馬魚胚胎在適應鈣環境改變過程中，維他命D是扮演調控鈣離子吸收機制的重要因素之一 。How animals maintain Ca2+ balance is an important issue because Ca2+ serves a variety of physiological roles. Ca2+ concentration usually fluctuates in freshwater environment. Facing Ca2+ changes, aquatic vertebrates must adjust Ca2+ absorption ability by regulating the expression of apical epithelial calcium channel (ECaC) on skin epithelial mitochondria-rich cells (MR-cells) to compensate intracellular Ca2+ level. During embryo stage, however, gills of freshwater teleosts are not well developed. At this stage, MR-cells on skin epithelium are main roles for ion regulation. To date, there are fewer studies on how endocrines regulate Ca2+absorption in teleosts, while a number of studies have shown that vitamin D affects the expression of ECaC in intestine and kidney in mammals. To see if this mechanism proposed in mammals has already existed in fish, which are more primitive than mammals in the aspect of evolution, is valuable for further research.he present study focused on effects of vitamin D on Ca2+ absorption in zebrafish. The mRNA expression of zebrafish vitamin D receptor-a (zVDRa) was detected as early as 3 hours postfertilization (hpf) and ubiquitously expressed in all tissues of zebrafish. Moreover, Ca2+ content, Ca2+ influx, and mRNA expression of zECaC were all increased, following vitamin D treatment. Finally, we found that Ca2+ content, Ca2+influx and the mRNA expression of zECaC decreased after zVDRa morpholino injection, but the expression of zebrafish Na+-Ca2+ exchanger 1b (zNCX1b) and zebrafish plasma membrane Ca2+-ATPase 2 (zPMCA2) were not affected. This study suggests that during the adaptation to fluctuating Ca2+ environment, vitamin D is one of the important factors regulating Ca2+ absorption in zebrafish embryos.Acknowledgement 1要 2bstract 3ntroduction 5he importance of Ca2+ to vertebrates 5a2+channels and transporters for Ca2+ absorption 6ndocrine control of Ca2+ regulation in teleost fish 7itamin D and its receptor in fish 8urpose of this study 11aterials and Methods 12xperimental animals 12est chemicals and concentrations 12rolonged zebrafish embryo test (96 h) 12easurement of the whole-body Ca2+ content 13easurement of whole-body Ca2+ influx 14NA extraction 15everse transcriptase PCR (RT-PCR) analysis 15uantitative real-time RT-PCR 16icroinjection of antisense morpholino oligonucleotides (MOs) 16tatistical analysis 17tructural Analysis 17esults 18xpression patterns of zVDRa in various tissues of zebrafish 18ffects of environmental Ca2+ levels on zVDRa and CYP27A1-like mRNA expressions 18ffects of 3-day exposure to vitamin D on growth rate, Ca2+ content, Ca2+ influx and Ca2+ absorption related genes mRNA expressions in zebrafish embryos 19ffects of the zVDRa morpholino on Ca2+ content, Ca2+influx and Ca2+ absorption related genes mRNA expressions in zebrafish embryos 19tructural characterization of zECaC 20iscussion 21erspectives and Significance 24eferences 26ables and Figures 30application/pdf1226905 bytesapplication/pdfen-US鈣維他命D硬骨魚離子調節受器Calciumvitamin Dteleostsionoregulationreceptorshttp://ntur.lib.ntu.edu.tw/bitstream/246246/181695/1/ntu-98-R95b45005-1.pdf