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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱家瑩(Chia-Ying Chu) | |
| dc.contributor.author | Jing-Jie Syu | en |
| dc.contributor.author | 許靖婕 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:38:39Z | - |
| dc.date.available | 2021-07-10T21:38:39Z | - |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76853 | - |
| dc.description.abstract | 生物藉由再生的過程以長回失去的組織，或更新體內老化細胞來維持個體原 本正常功能。扁形動物中的渦蟲具有強大的再生能力，利用體內廣布的多功能成體 幹細胞(neoblasts)，在受傷時增生並分化成所有類型的細胞來修復失去的組織。 許多基因參與成體幹細胞的增生與分化，這些基因都需要在轉錄或後轉錄層級精 確地被調控，以維持成體幹細胞正常功能。在 DEAD-box RNA 解旋酶家族的成員 中，DDX6 的同源蛋白於不同真核生物中都具有高度保守性。過去的研究指出， DDX6 透過降解 mRNA 和抑制轉譯的方式調控哺乳類上皮幹細胞及神經幹細胞。 先前研究顯示，渦蟲的 DDX6 同源基因 DjCBC-1 高度表現在成體幹細胞及神經系 統，以 RNAi 方式減弱 DjCBC-1 的表現會降低渦蟲再生時的存活率、改變渦蟲腦 部發育與功能。本研究論文以高度專一性的抗 DjCBC-1 抗體驗證 DjCBC-1 蛋白的 渦蟲全身性分佈，尤其高度表現於腦及再生組織中，與先前以原位雜合偵測 mRNA 的結果相似。此外 DjCBC-1 在細胞內分布於細胞核與細胞質聚集點。利用此抗體 進行免疫沈澱，成功純化分離 DjCBC-1 的 RNA-蛋白複合體，分析其中的 RNA 組 成，顯示 DjCBC-1 結合並調控專一表現於 neoblast 的 mRNA，藉此影響成體幹細 胞的數量與分化。 | zh_TW |
| dc.description.abstract | Regeneration is a process of regrowing the missing body parts or renewing aging cells to preserve the normal functions of an organism. Regeneration in planarians requires the somatic stem cells, named neoblasts, which proliferate and differentiate into all types of cells. Many genes involved in regeneration are regulated transcriptionally or post- transcriptionally. DEAD-box proteins are one of the post-transcriptional regulators. DDX6 is a highly conserved DEAD-box protein in eukaryotes. Studies showed that DDX6 regulates differentiation of epidermal and neural stem cells in mammals, by regulating mRNA degradation and/or translational repression. In planarian, the DDX6 ortholog, DjCBC-1, is highly expressed in neoblasts and neurons. Depletion of DjCBC- 1 decreased the survival rate, perturbed the brain structure, and delayed the formation of eyespots during regeneration. Long-term depletion of DjCBC-1 by RNAi also abolished the negative phototaxis of planarian. Here, I examined the expression of DjCBC-1 at the protein level. DjCBC-1 protein is expressed ubiquitously and is highly expressed in the brain and regenerating tissue. This finding is consistent with previous results, which were observed using whole-mount in situ hybridization (WISH). DjCBC-1 is localized to the nucleus and cytoplasm foci at the subcellular level. The depletion of DjCBC-1 increased the number of neoblasts. Finally, using an antibody specifically recognizing DjCBC-1, I immuno-precipitated DjCBC-1 RNA-protein complex and pulled down the neoblast- specific RNAs. My results suggested the function of DjCBC-1 in regulating target mRNA levels and participating in the maintenance of neoblasts. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:38:39Z (GMT). No. of bitstreams: 1 U0001-1308202015483800.pdf: 3005609 bytes, checksum: eeb5b13b5a560fd799218b051a7c7d7b (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 摘要 iii Abstract iv Contents v 1. Introduction 1 1.1 Regeneration in planarian 1 1.2 Neoblasts in planarian regeneration 3 1.3 Planarian nervous system and behavior 5 1.4 DDX6 protein and homolog in planarian 7 2. Materials and Methods 10 2.1 Animals 10 2.2 Immunofluorescence (IF) staining 10 2.3 Western Blot 11 2.4 RNA isolation and cDNA preparation 11 2.5 Quantitative reverse-transcription PCR (RT-qPCR) 12 2.6 Fluorescence-activated cell sorting (FACS) 12 2.7 Synthesis and purification of digoxigenin (DIG)-labeled RNA probes and double-strand RNA 13 2.8 dsRNA-mediated RNA interference (RNAi) experiment 14 2.9 Whole-mount in situ hybridization (WISH) 14 2.10 Immunoprecipitation 16 3. Results 17 3.1 Generation and functional characterization of a highly specific polyclonal anti-DjCBC-1 antibody. 17 3.2 DjCBC-1 was highly expressed in the brain and assembled around the nucleus. 18 3.3 DjCBC-1 was highly expressed in regenerating tissue of planarian. 18 3.4 DjCBC-1 was highly expressed in isolated mitotic neoblasts (X1) and neoblasts / progenitor cell populations (X2) of planarian. 19 3.5 Depletion of Djcbc-1 increased the number of mitotic cells. 20 3.6 Depletion of Djcbc-1 increased the expression level of cNeoblasts maker genes. 21 3.7 DjCBC-1 associated the mRNA of tgs-1, a neoblast-specific transcription factor. 22 3.8 Depletion of Djcbc-1 increased the average level of tgs-1 in each neoblast, without changing the expression pattern of tgs-1+ cells. 23 3.9 DjCBC-1 and Tgs-1 co-regulate the cell cycle of neoblasts. 23 3.10 Depletion of Djcbc-1 delayed the proliferating response of neoblasts during regeneration. 24 3.11 The DjCBC-1 protein binds to mex3-1 mRNA. 25 3.12 Depletion of Djcbc-1 perturbed the tissue homeostasis of eyespots and optic chiasm. 26 3.13 Depletion of Djcbc-1 increased the expression level of the differentiated neuron marker genes after Djcbc-1 RNAi. 27 4. Discussion 27 4.1 Summary and significance 28 4.2 The polyclonal anti-DjCBC-1 antibody can specifically pull down DjCBC-1 protein. 28 4.3 DjCBC-1 is required for cell differentiation and regulate cell cycle. 29 4.4 DjCBC-1 associated and regulated mex3-1 which is required for mediating cell fated to different lineages. 31 4.5 Depletion of Djcbc-1 affect the second mitotic proliferating response of neoblasts after missing tissue and during regeneration. 31 4.6 The DjCBC-1 protein is highly expressed in the brain, neoblasts, and blastema. 32 4.7 DjCBC-1 is required for the brain and the optic nervous system homeostasis. 33 4.8 Perspectives 34 5. Figures 36 Figure 1. The customized anti-DjCBC-1 antibody specifically recognized DjCBC-1 protein. 37 Figure 2. DjCBC-1 was expressed in intact planarian and highly expressed in the brain. 39 Figure 3. The DjCBC-1 was expressed in the cytoplasm surrounding the nucleus of the epidermal cells. 40 Figure 4. DjCBC-1 was highly expressed in regenerating tissue of planarian. 42 Figure 5. DjCBC-1 was highly expressed in FACS-isolated X1 (mitotic neoblasts) and X2 (non-mitotic neoblast and progenitor cell populations) population from the body of planarian. 44 Figure 6. Depletion of Djcbc-1 increased the mitotic cell number. 47 Figure 7. Depletion of Djcbc-1 increased the expression level of cNeoblasts marker genes. 49 Figure 8. The mRNA of cNeoblast genes were associated with and regulated by DjCBC-1. 51 Figure 9. DjCBC-1 and Tgs-1 might co-regulate cell cycle. 53 Figure 10. Depletion of Djcbc-1 affected the bimodal mitotic response at wound sites during regeneration. 56 Figure 11. The mRNA of RNA binding protein (mex3-1) was associated and regulated with DjCBC-1. 57 Figure 12. Depletion of Djcbc-1 perturbed the structure of optic never and impaired the maintenance of head homeostasis. 59 Figure 13. The expression levels of differentiated neuron marker genes was affected after Djcbc-1 depletion. 60 Figure 14. Schematic showing function of DjCBC-1 in homeostasis of planarian neoblasts and regeneration. 61 6. Appendix 62 7. References 63 | |
| dc.language.iso | en | |
| dc.subject | 再生 | zh_TW |
| dc.subject | 渦蟲 | zh_TW |
| dc.subject | 組織恆定性 | zh_TW |
| dc.subject | DjCBC-1 | zh_TW |
| dc.subject | DDX6 | zh_TW |
| dc.subject | DEAD-box核糖核酸解旋酶 | zh_TW |
| dc.subject | DjCBC-1 | en |
| dc.subject | DEAD-box helicase 6 | en |
| dc.subject | DDX6 | en |
| dc.subject | planarian | en |
| dc.subject | homeostasis | en |
| dc.subject | regeneration | en |
| dc.title | 探討 DjCBC-1 蛋白於渦蟲神經系統與成體幹細胞恆定性之調控功能 | zh_TW |
| dc.title | Deciphering the regulatory function of DjCBC-1 in homeostasis of planarian neoblasts and nervous system | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 郭典翰(Dian-Han Kuo),周銘翊(Ming-Yi Chou) | |
| dc.subject.keyword | DEAD-box核糖核酸解旋酶,DDX6,DjCBC-1,組織恆定性,再生,渦蟲, | zh_TW |
| dc.subject.keyword | DEAD-box helicase 6,DDX6,DjCBC-1,homeostasis,regeneration,planarian, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU202003280 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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