2017-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656629摘要：本計劃擬研究斑馬魚 lrrc8a 基因之表現特性、其與體積感應陰離子通道 (VSOAC) 組成之關係以及於胚胎發育中所扮演之角色。過去的研究指出，細胞對低滲透壓環境之調節反應中，至少有一半的效應來自有機滲透壓調節物質，如：牛磺酸等胺基酸；惟將有機滲透壓調節物質送出細胞外的 VSOAC，其分子組成一直不明，直到最近的研究顯示，LRRC8A 為VSOAC 之必要元件。過去對遺傳疾病第五型丙球蛋白缺乏貧血症之研究顯示，lrrc8a 的缺失乃是肇因，會使循環系統缺少B 細胞，而Lrrc8a 基因剔除小鼠則造成T 細胞之發育異常。另一方面，有其他研究指出血流之剪力會影響血球分化，而我們先前研究也發現牛磺酸缺乏會使心臟發育異常。由於牛磺酸是調控血液動力持衡的重要因子，且 VSOAC 為目前已知唯一將牛磺酸運至細胞外之通道。因此本計畫擬研究斑馬魚lrrc8a，及其對循環系統及血流動力形成之影響。目前我們已在斑馬魚基因中找到兩個 lrrc8a 同源基因，預計將探討其於胚胎發育時表現情形。為釐清斑馬魚lrrc8a 與VSOAC 之關係，將以HEK293 細胞確認斑馬魚 Lrrc8a 與其他 LRRC8 家族蛋白之表現位置，並測試 lrrc8a 基因是否能恢復由於 LRRC8A 缺失造成 293 細胞無法應對低滲透壓環境之反應。此外，將利用本實驗室已建立之CRIPSPR/Cas9 系統產製lrrc8a 基因剔除魚，以比較其與反義核&#33527;酸降低lrrc8a 表現及正常斑馬魚之胚胎發育情形，並觀察VSOAC 在胚胎及循環系統發育中之功能。本計劃之目標為：一、確認lrrc8a 之分子特性；二、產製lrrc8a 基因剔除斑馬魚；三、研究VSOAC 於胚胎發育之生理功能。<br> Abstract: The goal of this project is to characterize zebrafish lrrc8a genes to determine their role in constituting volume sensitive organic osmolyte and anion channel (VSOAC) and their potential roles during embryogenesis. The regulation and homeostasis on osmolality is critical for all active cells, while previous study indicated that organic osmolytes such as taurine are accountable for at least half of the total regulatory volume decrease of a cell in hypo-osmotic environment. Over two decades, the properties and VSOAC were widely studied and described, while its true molecular identity remained elusive until 2014. Two independent research groups adapting a similar strategy and identified that leucine-rich repeat containing 8A (LRRC8A) is an essential component in VSOAC in HEK293 cells. Interestingly, previous findings indicated that loss of this gene lead to type 5 agammaglobulinemia, an autosomal dominant genetic disorder featuring the absence of circulating B cells, while Lrrc8a knockout mouse only showed abnormality in T cell development with normal B cell function. Considering the importance of osmoregulation in living cells, it is surprising that the loss of VSOAC shows so little abnormalities. Previous studies done by other groups indicated that hematopoiesis is influenced by shear stress triggered by hemodynamics, while our previous study suggested that taurine deficiency led to cardiac abnormalities. Since taurine has been considered an important player in hemodynamic homeostasis and VSOAC is the only known mechanism to efflux taurine from within a cell to extracellular environment, in this project, we will identify and characterize zebrafish lrrc8a gene for its role as an essential component of VSOAC and for its role during embryogenesis, especially during the development of circulatory system and the formation of hemodynamics. To this end, we have identified two candidate zebrafish lrrc8a homologs in the zebrafish genome. We will investigate their temporal and spatial expression profiles during zebrafish embryogenesis. Furthermore, to validate their participation of VSOAC, we will confirm their co-localization with other LRRC8 family in HEK293 cells and will test whether these lrrc8a candidates can rescue the VSOAC activity in LRRC8A deficient 293 cells. To study their physiological role during embryogenesis, we will generate gene knock-out fish by CRISPR/Cas9 technology, which we have already established a working system in our lab. Finally, we will compare the embryogenesis in normal, morpholino knockdown and knockout embryos to find out the potential roles of VSOAC during embryogenesis, especially in the development of circulatory system. Aim 1: To molecularly characterize the zebrafish lrrc8a genes; Aim 2: To generate lrrc8a knockout zebrafish; Aim 3: To determine the physiological role of VSOAC during embryogenesis.心血管系統血流動力學lrrc8a滲透壓調節牛磺酸斑馬魚cardiovascular systemhemodynamicslrrc8aosmoregulationtaurinezebrafish