請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51508完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃銓珍(Chang-Jen Huang) | |
| dc.contributor.author | Shan Su | en |
| dc.contributor.author | 蘇珊 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:36:50Z | - |
| dc.date.available | 2018-02-24 | |
| dc.date.copyright | 2016-02-24 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-01-26 | |
| dc.identifier.citation | Reference
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51508 | - |
| dc.description.abstract | 特定離子細胞在狹鹽性硬骨魚類取代了哺乳類腎臟在滲透壓、水份以及酸鹼值等等的調節功能。透過表現在細胞膜上的特定離子通道可以將斑馬魚離子細胞區分為富鈉鉀幫浦細胞, 鈉氯離子運輸蛋白細胞, 富氫幫浦細胞, SLC26細胞 以及泌鉀細胞，在功能上分別負責：鈣離子運輸、氯離子/HCO3運輸, 氯離子運輸, 氫離子/鈉離子運輸, 以及鉀離子運輸。過去的研究中，已經對前四種離子細胞的功能有一定的了解，然而，泌鉀細胞對於鉀離子的調節模式還是未知的。近來學者指出，斑馬魚帶有一個與哺乳類Kir1.1(ROMK)同源的蛋白，Kcnj1a.1。其表現無論在原前腎或是皮膚細胞都跟鈉鉀幫浦次單元異構物 atp1a1a.4都具有共位現象。現在，斑馬魚除了kcnj1a.1之外還預測及比對出另外五個重覆複製的基因，命名為Kcnj1a.2到Kcnj1a.6。由於序列的相似，且具有高度保守的內向整流鉀離子功能domain，本研究的目的即在定義Kcnj1a亞家族成員在斑馬魚的表現以及調控鉀離子的功能。透過全體雙螢光雜合實驗發現，只有kcnj1a.1在腎細胞有表現，且與atp1a1a.4有共位現象。另外五個基因的表現則廣泛分布在卵黃囊、軀幹及少部分頭等區域的皮膚細胞，並且跟atp1a1a.4有部分共位現象。由於kcnj1a.2,3以及4的mRNA表現訊號較明顯，我們選殖出這三個基因的全長並且在HEK293細胞中過量表現，經由電生理實驗發現只有Kcnj1a.2在調控鉀離子具有非電壓依賴性，且其反轉電流最接近於鉀離子通道。在檢測斑馬魚體內鉀離子含量的實驗中發現，降低飼養斑馬魚水體環境中的鉀含量，會降低其體內鉀含量。然而提高水中鉀的濃度，相對於對照組而言，高鉀水飼養的斑馬魚體內鉀含量並沒有顯著差異。利用MO 技術來降低Kcnj1a在斑馬魚發育過程蛋白質表現，發現受精後三天的斑馬魚體內鉀含量如同低鉀水飼養的情況類似都會降低。因此，由上述結果證實Kcnj1a在斑馬魚胚胎發育階段扮演著調控鉀離子進出的角色。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:36:50Z (GMT). No. of bitstreams: 1 ntu-105-D97b46011-1.pdf: 2210267 bytes, checksum: 3cad5d9be286855f5022c9f8cfc544ee (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | Content…………………………………………………………………………………………… I
中文摘要…………………………………………………………………………………………III Abstrate……………………………………………………………………………………………IV Abbreviations…………………………………………………………………………………… VI Introduction…………………………………………………………………………………………1 Kcnj1 family in mammals………………………………………………………………… 1 Kcnj1a genes and proteins in fishes……………………………………………………………2 Ionocytes……………………………………………………………………………………… 4 Na+-K+-ATPase plays an important role of osmoregulation in zebrafish development………4 Zebrafish as an experimental model for ionocytes development………………………………5 Specific aims…………………………………………………………………………………………8 Materials and methods………………………………………………………………………………9 Zebrafish care……………………………………………………………………………… 9 Generation of a transgenic zebrafish line carrying atp1a1a.4:GFP …………………………9 RNA Probes synthesis and whole-mount insitu hybridization of Kcnj1a family genes………10 Construction of expression plasmids…………………………………………………………10 Cell cultures……………………………………………………………………………………11 Transactivation assay…………………………………………………………………………12 Western blot and immunocytochemistry assay………………………………………………12 Electrophysiology………………………………………………………………………13 Acclimation experiments………………………………………………………………………13 Injection of specific Kcnj1a morpholino………………………………………………………14 Zebrafish embryos whole-body Potassium content detection…………………………………14 Whole-mount immunostaining……………………………………………………………… 14 Results…………………………………………………………………………………………… 15 Genomic organization of zebrafish Kcnj1a family……………………………………………15 Spatial and temporal expression pattern of six Kcnj1a genes at 3 dpf zebrafish embryo……15 II Assessment the promoter activity of the zatp1a1a.4 in cultured cells and zatp1a1a.4 stable transgenic fish generation……………………………………………………………………16 Colocalization of endogenous kcnj1a.1~6 and gfp in Tg-(ATP1a1a.4: GFP) ………………17 Cellular localization of Kcnj1a.2, Kcnj1a.3 and Kcnj1a.4 in HEK293T cells……………………………………………………………………………………………18 Functional assay of potassium channels of zebrafish Kcnj1a.2, Kcnj1a.3 and Kcnj1a.4 by whole-cell patch clamp in vitro………………………………………………………………19 Whole body potassium content in various potassium contained environment in zebrafish…20 Kcnj1a loss-of-function results in decreased whole body potassium content…………………21 Discussion…………………………………………………………………………………………22 References…………………………………………………………………………………………26 Figures………………………………………………………………………………………………32 Fig. 1. Genomic organization of the zebrafish Kcnj1a genes and alignment of amino acid sequences of six zebrafish Kcnj1a genes………………………………………………………32 Fig. 2. Expressing patterns of zKcnj1a family mRNAs in zebrafish embryos at 72 hpf. …34 Fig. 3. Analysis the promoter activity of atp1a1a.4 in vitro and investigate the expression patterns of transient GFP driven by 5'upstream region of 1a1a.4 in vivo. …………………36 Fig. 4. The expressing pattern of green fluorescent protein in transgenic zebrafish Tg-(ATP1a1a.4:GFP) line. …………………………………………………………………37 Fig. 5. Double in situ hybridization of the genes of zKcnj1a family and GFP in Tg-(ATP1a1a.4:GFP) fish. …………………………………………………………………39 Fig. 6. Expression of Kcnj1a.2, Kcnj1a.3 and Kcnj1a.4 proteins in HEK-293T cells.. ………………………………………………………………………………41 Fig. 7. Expression and cellular localization of Kcnj1a.2, Kcnj1a.3 and Kcnj1a.4 proteins in HKE293T cells ………………………………………………………………………………42 Fig. 8.Whole-cell patch clamp of Kcnj1a.2, Kcn1a.3 and Kcnj1a.4 transient expressed HEK293 cells. ………………………………………………………………………………44 Fig. 9. Effect of whole body potassium content and phenotypes of zebrafish embryos by environment ion concentration changing and Kcnj1 morpholino knockdown………………45 Table………………………………………………………………………………………………47 | |
| dc.language.iso | en | |
| dc.title | 內向整流鉀離子通道亞家族成員 (Kcnj1a) 調控斑馬魚表皮離子細胞的鉀離子平衡 | zh_TW |
| dc.title | Inwardly rectifying potassium channels (Kcnj1a) are involved in potassium homeostasis of zebrafish ionocytes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 黃鵬鵬,黃聲蘋,陳建璋,張茂山(Mau-Sun Chang) | |
| dc.subject.keyword | 內向整流鉀離子通道,鈉鉀幫浦,離子細胞,斑馬魚, | zh_TW |
| dc.subject.keyword | Kcnj1a,Na+-K+-atpase,ionocyte,zebrafish, | en |
| dc.relation.page | 47 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-01-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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