斑馬魚之新型鈉氯離子運輸蛋白表現與功能之研究

Expression and Function of a Novel Na-Cl Cotransporter Isoform in Zebrafish (Danio rerio)

王怡方(Yi-Fang Wang)

指導教授 ： 黃鵬鵬

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00645

摘要

鈉氯離子運輸蛋白(thiazide-sensitive Na-Cl cotransporter, NCC)主要表現於哺乳類腎臟腎小管的遠端彎曲小管表皮細胞頂端，鈉氯離子運輸蛋白的功能為將腎小管管腔中的鈉離子和氯離子共同運輸進細胞中，其所扮演的角色是負責遠曲小管氯化鈉的再吸收，而一種常用於治療高血壓的醫療用利尿劑thiazide是對鈉氯離子運輸蛋白有專一性的抑制劑。然而，目前在鈉氯離子運輸蛋白方面的研究大部分僅限於in vitro實驗，因此，本實驗的目的在於提供鈉氯離子運輸蛋白對於鈉氯離子運輸所扮演的角色和調控機制in vivo層次之證據。
在本實驗中發現，斑馬魚不同於哺乳類，擁有兩型鈉氯離子運輸蛋白，其中之一(zNCCk)大量表現於腎臟。而另一型則是專一的表現在斑馬魚成魚的鰓上(zNCCg, NCBI accessory number EF591989)。在定量PCR實驗中，進一步發現，將斑馬魚生活適應在高鈉離子和低氯離子濃度環境的人工水中，斑馬魚鰓上的zNCCg mRNA會被刺激而表現增加。此外，本實驗首次提供完整的in vivo證據證明鈉氯離子運輸蛋白專一性的抑制劑metolazone對於鈉及氯離子的吸收均有影響。而當zNCCg被基因專一反股核酸(morpholino-modified antisense oligonucleotide)抑制其蛋白表現後，斑馬魚胚胎的氯離子吸收會因此減少；同時，當zNCCg的capped mRNA和morpholino一同顯微注射到1-4細胞期的斑馬魚胚胎後，幾乎可以完全地將因為morpholino抑制而造成的氯離子運輸異常補救回接近胚胎正常表現量。綜合以上結果，本實驗首次提供在in vivo層次上的分子生理證據，證明斑馬魚皮膚及鰓上的新型鈉氯離子運輸蛋白(zNCCg)參與氯離子的吸收機制。

關鍵字

斑馬魚 ； 鈉氯離子 ； 運輸蛋白 ； 離子調控

並列摘要

Thiazide-sensitive Na-Cl cotransporter (NCC), SLC12A3, is a kidney-specific transporter in mammalian and is mainly expressed in the apical membrane of the distal convoluted tubule (DCT) cells. In mammalian DCT, NCC is responsible for co-transporting Na+ and Cl- from lumen into DCT cells, playing a major role in NaCl reabsorption. NCC also acts as the target for the thiazide diuretics, which is used to treat hypertension. However, only a few in vivo data were available to elucidate the transport mechanism of NCC. In the present study, zebrafish was used as an in vivo model to examine the role of NCC in Na+/Cl- uptake mechanisms. Two NCCs were cloned and sequenced. One (zNCCk) was mainly expressed in kidney and another (zNCCg, NCBI accessory number EF591989) was in gill. Real-time RT-PCR analysis indicated that mRNA expression of the zNCCg was induced by a high-Na+-low-Cl- environment. Incubating with metolazone, a specific inhibitor of NCC, was found to impair both Na+ and Cl- influx in 5-dpf zebrafish embryos. Knockdown of the zNCCg translation with specific morpholino caused a significant decrease in Cl- uptake of zebrafish embryos, while co-injection with the zNCCg capped mRNA completely rescued the defect in morphants. These results for the 1st time provide in vivo molecular physiological evidence for the involvement of the zNCCg in Cl- uptake mechanisms in zebrafish skin/gill.

並列關鍵字

thiazide ； zebrafish ； Na-Cl cotransporter ； isoform ； NCC ； osmoregulation

參考文獻

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