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標題: | 抗利尿激素誘導第二型水通道蛋白透過Mal2調節的胞轉機制運送到腎臟集尿管細胞的頂膜 Vasopressin Induces Apical Aquaporin-2 Trafficking via Mal2-Mediated Transcytosis in Renal Collecting Duct Cells |
作者: | Pei-Yu Chen 陳珮毓 |
指導教授: | 余明俊(Ming-Jiun Yu) |
關鍵字: | 抗利尿激素,第二型水通道蛋白,腎臟集尿管細胞,蛋白Mal2, Vasopressin,Aquaporin-2,Renal collecting duct cells,Mal2, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 當腎臟集尿管細胞受到抗利尿激素(antidiuretic hormone vasopressin)的刺激後,會促使細胞中的第二型水通道蛋白(aquaporin-2, AQP2)由細胞內液胞被運送到細胞頂膜(apical plasma membrane),藉此調節腎臟集尿管對水分的排除。在先前的蛋白質體學研究中鑑定到Mal2,一個已知與細胞頂膜蛋白質之胞轉作用(transcytosis)有關的脂筏(lipid raft)蛋白質,因此在這個研究中,我們想探討Mal2是否會參與並調解受抗利尿激素刺激後,AQP2運送至細胞頂膜的胞轉作用。在抗利尿激素類似物(dDAVP)刺激小鼠集尿管細胞株(mpkCCD)後的不同時間點,以免疫螢光染色(immunofluorescence staining)的方法觀察到,在短時間dDAVP的處理過後,AQP2會出現在細胞的側膜。而隨著dDAVP處理時間的增加,位於細胞側膜的AQP2逐漸減少,伴隨著在細胞頂膜的AQP2逐漸增加,這意味著當細胞受到dDAVP的刺激後,AQP2被運送到頂膜的過程會透過胞轉機制。當mpkCCD細胞的側膜被單寧酸(tannic acid)固定,再經dDAVP或forskolin刺激後,可以觀察到一部分的AQP2聚集在細胞的側邊區域,而有另一部分的AQP2則是聚集在細胞頂膜,表示當細胞受到dDAVP或forskolin刺激後,AQP2從細胞內液胞被送至頂膜的路徑分為兩條:其一為直接運送,其二則透過胞轉機制。然而在不表現Mal2的mpkCCD 細胞中,當此細胞的側膜被單寧酸(tannic acid)固定後,此時forskolin的刺激無法誘導AQP2聚集在細胞的側邊區域,此結果說明了Mal2會參與並調解集尿管細胞受抗利尿激素刺激後,AQP2運送至細胞頂膜的胞轉作用。 In kidney collecting duct cells, vasopressin (AVP) triggers redistribution of aquaporin-2 (AQP2) water channel protein from intracellular vesicles to the apical plasma membrane to regulate renal water excretion. In our previous proteomic study, we identified Mal2, a lipid-raft membrane protein known to mediate apical membrane protein trafficking via transcytosis. This study examined roles of Mal2 in AVP-induced apical AQP2 trafficking via transcytosis. Time course immunofluorescence showed staining of AQP2 in the lateral plasma membrane of the mpkCCD cells after short-term AVP analog dDAVP stimulation. The lateral AQP2 staining disappeared gradually with concomitant appearance of apical AQP2 staining, suggesting indirect transcytotic mechanism for apical AQP2 targeting upon dDAVP stimulation. When the lateral membrane was fixed with tannic acid, a portion of AQP2 accumulated in the lateral space of the cells in response to dDAVP or forskolin. Note that another portion of AQP2 was detected in the apical space of the cells. The observations indicated that dDAVP or forskolin induced apical AQP2 targeting via both direct and indirect transcytotic pathway. The forskolin-induced lateral AQP2 staining in the presence of tannic acid was completely absent in the cells lacking Mal2. Our data are compatible with a role of Mal2 in vasopressin-induced indirect apical AQP2 targeting via the transcytotic mechanism. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18479 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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