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標題: | 探討deptor基因表現對斑馬魚肝和腸發育的影響 Study the role of zebrafish deptor gene expression in the development of liver and gut |
作者: | Yong-Sin Jhang 張詠馨 |
指導教授: | 黃銓珍(Chang-Jen Huang) |
關鍵字: | deptor,mTOR,斑馬魚,肝臟,腸道,發育, deptor,mTOR,zebrafish,liver,gut,development, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 目前已知mammalian target of rapamycin (mTOR) 會調控許多生物機轉,包含細胞生長、存活、以及細胞自噬。先前研究指出 mTOR抑制劑-rapamycin會抑制斑馬魚腸道上皮細胞生長、形態發生與分化。DEP-domain-containing mTOR-interacting protein (DEPTOR) 是mTORC1與 mTORC2的抑制劑,與其他蛋白相互作用調節了細胞增殖與發育。然而,目前對DEPTOR的了解以及發育中的角色還不清楚。因此,在本研究中,我們希望透過斑馬魚動物模式來探討DEPTOR在肝腸早期發育中的功能。
首先,我們選殖了斑馬魚的deptor基因,並將其蛋白序列與人類、老鼠的DEPTOR蛋白序列做比對分析,結果顯示三者蛋白序列之間的保守性相當高。接著我們利用全覆式原位雜合實驗來探討斑馬魚deptor基因內生性mRNA的表現位置,發現於腦部、腸道及肝臟等器官上都有表現。此外,我們以注射deptor mRNA的方式來增加DEPTOR蛋白的表現,結果顯示外觀沒有明顯變化,但會在72 hpf時降低肝臟標記lfabp的表現,120 hpf時降低腸標記ifabp的表達以及減少腸道杯狀細胞數目。 注射deptor mRNA也會降低了raptor和ztor mRNA的表現,從免疫墨點法結果也可得知注射deptor mRNA會抑制mTORC1 下游蛋白S6K 的磷酸化。綜合上述結果顯示,deptor基因會參與了斑馬魚早期肝臟腸道發育。 The mammalian target of rapamycin (mTOR) signaling pathway can regulate cell growth, survival, and autophagy. Previous studies indicated that rapamycin is the mTOR inhibitor. It inhibits zebrafish intestinal epithelial growth, morphogenesis, and differentiation. DEP-domain-containing mTOR-interacting protein (DEPTOR) is the inhibitor of mTORC1 and mTORC2 complex, which is compromised with other proteins to regulate genes expression in cell proliferation and development. However, the role of DEPTOR in the development of liver and intestine is still unknown. Therefore, in this study, we used zebrafish as an animal model to investigate the role of DEPTOR during early development. We cloned zebrafish deptor gene and performing amino acids sequence alignment between human, mice, and zebrafish and it was highly conserved among these three species. We then performed whole-mount in situ hybridization experiment to analyze the endogenous deptor mRNA expression pattern. The zdeptor mRNA was expressed in the brain from 24 to 72 hpf and in the gut between 48 and 72 hpf. Overexpression of zerbrafish deptor mRNA decreased the expression of liver markers lfabp and intestinal marker ifabp in 72 and 120 hpf. Likewise, the injection of deptor mRNA in 120 hpf decreased alcian blue–stained goblet cell numbers compared to control embryos. In addition, overexpression of deptor mRNA also decreased the expression of raptor and ztor mRNA. The results from western blot indicated that injection of zdeptor mRNA would inhibit mTORC1 downstream protein S6K phosphorylation. In summary, our data indicated that zebrafish deptor gene was involved in liver and gut development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20200 |
DOI: | 10.6342/NTU201800285 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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