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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃銓珍(Chang-Jen Huang) | |
| dc.contributor.author | Yong-Sin Jhang | en |
| dc.contributor.author | 張詠馨 | zh_TW |
| dc.date.accessioned | 2021-06-08T02:42:05Z | - |
| dc.date.copyright | 2018-02-23 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-02-06 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20200 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T02:42:05Z (GMT). No. of bitstreams: 1 ntu-107-D01b46007-1.pdf: 2621165 bytes, checksum: ab23f32dd9950609eadd1b9ae9c35128 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 論文口試委員審定書 i
誌謝 ii 中文摘要 iii Abstract iv Content v Introduction 1 mTOR protein 1 The mTORC1 and mTORC2 1 mTOR plays an important role in the development of early embryos. 3 Rapamycin 3 DEPTOR 4 DEP domain and PDZ domain 4 Materials and methods 7 Zebrafish care and maintenance 7 Zebrafish strains 7 Cloning of full-length of zebrafish and mouse deptor 7 Microinjection of zdeptor and mdeptor mRNA in zebrafish embryos 7 Scoring microinjection phenotypes and statistics 8 RNA Probes synthesis and whole-mount in situ hybridization 8 Cell cultures 9 Cloning of full-length of zebrafish and mouse deptor protein expression vectors 9 The mouse and zebrafish deptor proteins expressed in 293T cells 9 Alcian blue staining 10 Whole-mount immunostaining 10 Western blot 11 RT-PCR 12 Results 13 Genomic organization of zebrafish ortholog of DEPTOR (zdeptor). 13 Expression profiles of deptor mRNA at different developmental stages in zebrafish embryos. 13 Injection of zdeptor mRNA caused abnormal formation of intestine and liver. 14 The deptor antibody labeled cells were found in zebrafish developing gut and liver. 14 Injection of deptor mRNA affects the development of intestine and liver and related marker genes expression. 15 The zdeptor mRNA injection inhibited raptor expression which is a zTORC1 component gene in gut. 16 Injection of deptor mRNA has decreased the amount of S6K protein and increased the p-AKT phosphorylation. 17 The injection of deptor mRNA inhibited the liver cells proliferation. 18 Discussions 19 References 22 Figures 28 Figure 1. Alignment of amino acid sequences of zebrafish, mouse, and human DEPTOR proteins. 28 Figure 2. Expression patterns of zebrafish deptor in various tissue of different developmental stages 29 Figure 3. Injection of zdeptor mRNA caused abnormal formation of intestine and liver 31 Figure 4. Injection of deptor mRNA led to abnormal intestinal development at 120 hpf. 32 Figure 5. Injection of deptor mRNA led to abnormal liver and intestine development. 34 Figure 6. Injection of deptor mRNA decreased the liver and intestine developmental related genes expression 35 Figure 7. Injection of deptor mRNA downregulated ztorc1 expression in developing intestine 37 Figure 8. Detection of p-AKT, AKT, p-S6K, and S6K protein amounts in zebrafish embryos. 38 Figure 9. Whole-mount immunostaining analysis of pHH3+ cells (red) in lfabp:EGFP (green) liver reporter zebrafish at 72 hpf. 39 Table 40 Table 1. primer sequences 40 Table 2. qPCR primer sequences 41 APPENDIX 42 Figure.1. ztor expression during embryonic and larval development. 43 Figure 2. Ventral view of liver development in zebrafish. 44 | |
| dc.language.iso | en | |
| dc.title | 探討deptor基因表現對斑馬魚肝和腸發育的影響 | zh_TW |
| dc.title | Study the role of zebrafish deptor gene expression in the development of liver and gut | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 黃鵬鵬,張茂山,周志銘,鄭嘉雄 | |
| dc.subject.keyword | deptor,mTOR,斑馬魚,肝臟,腸道,發育, | zh_TW |
| dc.subject.keyword | deptor,mTOR,zebrafish,liver,gut,development, | en |
| dc.relation.page | 44 | |
| dc.identifier.doi | 10.6342/NTU201800285 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-02-07 | |
| 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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