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標題: | 轉錄因子Elf3參與抗利尿激素所誘導的水通道蛋白2號基因之表現 Transcription Factor Elf3 Mediates Vasopressin-Regulated Aquaporin-2 Expression in mpkCCD Cells |
作者: | Chia-Ching Ma 馬家慶 |
指導教授: | 余明俊(Ming-Jiun Yu) |
關鍵字: | 抗利尿激素,水通道蛋白2號,腎臟集尿管細胞,轉錄因子Elf1,Elf3, Vasopressin,aquaporin 2,renal collecting duct cells,Elf1,Elf3, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 抗利尿激素(AVP)是一種多胜肽賀爾蒙,當其從腦下垂體釋放到血液中時, AVP會經由腎臟集尿管來調控尿液中水分的排出,藉此穩定體內水分的平衡。AVP其主要是透過增加集尿管細胞內水通道蛋白2號(aquaporin 2, AQP2)基因的轉錄及轉譯,來增加集尿管細胞對於水的通透性,藉此減少尿液中水分的排出。當AVP無法正常地誘導AQP2基因表現時,會造成一些和身體水分平衡相關的疾病;然而AVP是透過怎樣的分子機制去調控AQP2基因的轉錄,仍然是不清楚的。最近microarray和proteomic的研究指出,轉錄因子Elf1以及Elf3可能參與在AVP所誘導的AQP2基因轉錄的機制當中,因此在我的研究中,想利用小鼠集尿管細胞的細胞株(mpkCCD),來研究轉錄因子Elf1和Elf3是否參與在AVP所誘導的AQP2基因表現的機制當中。由即時聚合酶鏈鎖反應的實驗結果知道當mpkCCD細胞受到抗利尿激素類似物dDAVP的刺激之下, AQP2 mRNA的表現量,會隨著dDAVP刺激的時間增長,而有逐漸增加的趨勢。利用shRNA將mpkCCD細胞中的Elf1以及Elf3進行knockdown,由即時聚合酶鏈鎖反應的分析結果得知,不管是在Elf1或是Elf3 knockdown的細胞當中,其dDAVP所誘導AQP2 mRNA的表現量,都較控制組細胞來的低,尤其在Elf3 knockdown的細胞中, AQP2 mRNA的下降是更為明顯。進一步利用西方點墨法的分析,觀察到mpkCCD細胞Elf3的knockdown,會顯著性降低細胞內dDAVP所誘導的AQP2蛋白質的表現量。由反轉錄聚合酶連鎖反應的分析,看到在mpkCCD細胞當中, Elf3有兩種isoform的表現,分別在mpkCCD細胞中表現這兩種Elf3的isoform,都會造成細胞內dDAVP所誘導的AQP2 mRNA的表現量增加,以及增強AQP2基因啟動子的活性。總結上述的結果,在我的研究中發現,轉錄因子Elf3參與在抗利尿激素所誘導AQP2基因表現的機制當中,並且扮演著正向調控者的角色。 Released from the pituitary, vasopressin (AVP) is a peptide hormone that regulates renal water excretion by the collecting ducts. AVP increases transcription and translation levels of the molecular water channel protein aquaporin-2 (AQP2) gene to increase water permeability of the collecting ducts thereby decreasing water excretion. Dysregulation of AQP2 gene expression induced by AVP is associated with many water balance diseases; however the molecular mechanism by which vasopressin regulates AQP2 gene transcription is poorly understood. Several recent microarray and proteomic studies have alluded to potential roles of the ETS-like transcription factor Elf1 and Elf3 in vasopressin-mediated AQP2 transcription. The goal of my study was to investigate whether the transcription factor Elf1 and Elf3 are involved in the regulation of vasopressin-mediated AQP2 gene expression in a mouse collecting duct cell model (mpkCCD). Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the vasopressin analog dDAVP induced AQP2 mRNA expression in a time-dependent manner in mpkCCD cells. Either stable Elf1 knockdown or Elf3 knockdown significantly reduced dDAVP-induced and basic AQP2 mRNA expression, but Elf3 knockdown reduced more AQP2 mRNA production than Elf1 knockdown. Immunoblotting analysis showed that Elf3 knockdown also decreased dDAVP-induced AQP2 protein expression. Furthermore, qRT-PCR and promoter reporter assay revealed that overexpression of either Elf3 isoform 1 or isoform 2 increased AQP2 mRNA levels and promoter activity in the presence and the absence of dDAVP. Collectively, my study shows that transcription factor Elf3 mediates vasopressin-induced and basal AQP2 expression in the mpkCCD cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60879 |
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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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