斑馬魚胚胎時期鈣離子吸收機制之研究

Abstract

對脊椎動物而言，骨骼能支撐整個身體並且提供動物運動的能力，而鈣離子則為骨骼形成時，最重要的元素。由於魚類生活在水中，因此水成為魚類吸收鈣離子最直接且重要的來源。鰓在過去的研究中被證實為鈣吸收最主要的器官（佔 80 ％以上）。在鰓上，鈣離子需經由主動且耗能的吸收，主要由富含粒線體細胞 ( MR cell ）負責。在鈣離子吸收的分子機制方面，Ca2+-ATPase 以及 Na+-Ca2+ exchanger 兩種離子運輸蛋白質曾在富含粒線體細胞的基底膜被發現，負責將鈣離子由細胞質運送到血液中。至於在頂端膜中，鈣離子通道（Ca2+ channel ）被推測為負責將鈣離子由水中運送進細胞內的媒介，但缺乏進一步生理以及分子生物學的證據。本研究的目的即希望確認此一鈣離子通道的種類，以及其在魚類鈣離子吸收所扮演的角色。斑馬魚的胚胎，這種近來被廣泛應用於基因表現以及發育生物學研究的材料，被選為本研究中所使用的模式動物。我們在斑馬魚的鰓中選殖出上皮細胞型鈣離子通道之基因。同時我們觀察此基因在成體各組織，和胚胎時期各個發育階段的表現，以及其在細胞層次的表現情形。此外，在斑馬魚胚胎發育時期的魚體鈣離子含量、鈣離子流入變化，以及富含粒線體細胞之分化趨勢皆被檢測。 由斑馬魚選殖出的鈣離子通道，基因全長 2578bp ，轉譯出的蛋白質則為 709 個胺基酸。根據各物種鈣離子通道之親緣分析，斑馬魚、鱒魚和河魨的鈣離子通道基因被歸為一群，和哺乳類與兩生類的基因分開。顯示出此一上皮細胞型鈣離子通道基因，很可能在哺乳類和魚類分歧之後有被複製的情形。在基因表現方面，此一基因於本研究中，所檢測的所有組織中皆有表現，另外在胚胎中，則是從受精後 24 小時聞始表現。斑馬魚胚胎之鈣離子流入，開始於受精後第 36 小時，鈣離子含量則在孵化復大量累積。富含粒線體細胞最早在受精後 24 小時的胚胎上出現，而其對外的開口則於受精36小時後出現。由上述實驗，富含粒線體細胞的分化和胚胎鈣離子開始流入的時間相符，此外，這也顯示對仔魚而言，由外界獲取充足的鈣離子是非常重要的。而鈣離子通道基因在胚胎發育過程中，開始表現的趨勢則和先前富含粒線體細胞的分化，以及胚胎鈣離子開始流入的時間相吻合。而更進一步的，此基因在鰓上表現位置，和富含粒線體細胞共同出現在斑馬魚鰓絲的周圍。 於本次研究中，斑馬魚的上皮細胞型鈣離子通道基因首次被選殖，並表現在檢測的所有組織中，也包含了富含粒線體細胞。顯示此一蛋白質對仔魚以及成魚的鈣離子吸收和平衡，很可能扮演相當重要的角色。

Vertebrates use Ca2+ as the major element for the formation of skeleton, which provides support and movement ability for individuals. In fish, water serves as a main source of Ca2+ than food, and gill has been proved to be the major Ca2+ uptake site that is responsible for more than 80% Ca2+ absorption. Ca2+ uptake occurs actively and also transcellularly through mitochondria-rich (MR) cells. The activities of plasma membrane Ca2+-ATPase (PMCA) and Na+-Ca2+exchanger (NCX) have been identified in basolateral membrane of MR cells. PMCA and NCX have also been shown to be responsible for extruding Ca2+ into plasma. In the apical membrane, a Ca2+ channel is presumed to mediate Ca2+ entry, but there are no substantial molecular or physiological evidences for such channel in fish. The purpose of the present study is to identify the Ca2+ channel and elucidate its role in Ca2+ absorption. Zebrafish has been used extensively for the research of gene expression and developmental biology was selected as the model animal. In the present study, zebrafish ECaC (zECaC) from gill was cloned, sequenced, and the tissue distribution, developmental expression, and cellular localization of the zECaC were also studied. In addition, Ca2+influx, Ca2+content, and MR cell differentiation in different developmental stages of embryos were examined. The cloned zECaC is 2578bp in legnth and encodes a protein of 709 amino acids. According to the phylogenetic analysis, trout and zebrafish ECaC were clustered together and formed a distinct group from amphibian and mammalian ones. It indicates that the duplication of ECaC may occur after the divergence of fish and mammals. zECaC was found to express ubiquitously in all the tissues examined, and started to express in embryos at 24 hours post fertilization (hpf). Ca2+ influx started to increase at 36hpf while Ca2+ content accumulated after hatching. MR cells appeared on the embryos at 24hpf, but first opening was observed at 36hpf. According to the results, the timing of MR cell differentiation corresponded with the data of Ca2+ influx, and it implies a definite need for Ca2+ uptake from ambient environment during larval development. The zECaC expression pattern during development correlated with the first appearance of MR cell and also Ca2+ influx. Moreover, zECaC expression in gill was localized along the gill filaments where MR cells were concentrated. In conclusion, zECaC is first cloned in the present study, and it is expressed in all the tissues examined, including MR cells in gill. The wide tissue distributions suggest that zECaC may play a key role in Ca2+ absorption in developing embryo and also in adult fish.