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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊西苑(Hsi-yuan Yang) | |
| dc.contributor.author | Peng-han Su | en |
| dc.contributor.author | 蘇芃翰 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:21:16Z | - |
| dc.date.available | 2013-07-30 | |
| dc.date.copyright | 2013-07-30 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2013-07-25 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61974 | - |
| dc.description.abstract | 巢蛋白 nestin 是一種第六類中間絲蛋白，專一性地表現於神經及肌肉幹細胞中。Nestin 幾乎從未在成熟神經元中被發現過，此外，它的異構物也從未被報導過。在本研究中，我們利用單神經元 RT-PCR 技術，自成熟大鼠背根神經元中，鑑定第一個 nestin 異構物— Nes-S。在此異構物中，原本 nestin 蛋白絕大部份的C端區域，包括整個重覆片段區域 ( repeat region )，皆因選擇性剪切 ( alternative splicing ) 而移除。該剪切亦同時造成了下游序列的框移突變 ( frameshift mutation )，導致切點下游產生了一段長32胺基酸 (aa)的全新片段，以及讓轉譯提前結束的停止密碼子 ( premature stop codon )。Nes-S 蛋白總長度為 408-aa，其中包含 7-aa 長的 N 端區域、一個完整、307-aa 長的 Rod domain，以及一段 94-aa 長的 C 端區域，其預測的分子量為 45.9kD。在不表現胞質中間絲蛋白的 SW13 細胞株中，Nes-S 無法自我組裝形成中間絲纖維。取而代之的是，Nes-S 在表現 vimentin 的 SW13 細胞中，能夠與 vimentin 共同組裝形成中間絲纖維。在 N2a 神經母細胞瘤細胞株、以及初代培養的背根神經元中，Nes-S 亦可和 peripherin ，或是 neurofilament heavy chain 共同形成中間絲纖維。為了研究 Nes-S 的表現分佈狀況，我們從其尾端的變異序列區域，挑選並合成出適合的多胜片段，並製造專一辨識該片段的兔多株抗體。利用此抗體進行免疫轉漬法實驗，我們發現，直至大鼠出生後第五天，Nes-S 始於背根神經節中表現，且該表現會一直持續存在至成體。此外，利用同一支抗體進行免疫轉漬實驗和免疫標記實驗發現，在我們檢視的組織當中，Nes-S 僅會表現於會將其神經延伸至身體周邊或四肢的神經元中，其中包括三叉神經節和 superior cervical ganglion 之神經元，以及胸椎脊髓中的交感神經元和運動神經元。最後，透過 MTT assay，我們發現當 Nes-S 被轉殖於 N2a 細胞中時，將減緩遭受氧化壓力下的 N2a 細胞自戕反應。此外，若將初代培養之成鼠背根神經元中既有的 Nes-S 以 RNAi 敲減，將使該細胞喪失於長期培養環境中存活的能力。過去十年以來，越來越多證據指出基因選擇性剪接是另一種調控中間絲蛋白功能的方式。而這些中間絲蛋白異構物亦可能進一步參與細胞壓力反應路徑，甚至參與神經退化性疾病的進程。Nes-S 是一種新的、能在成熟神經元中行使保護作用的中間絲蛋白，未來專注于 Nes-S 上的研究，將可能有機會為神經退化性疾病研究領域，產生新的貢獻。 | zh_TW |
| dc.description.abstract | Nestin is a type VI intermediate filament (IF) protein predominantly expressed in neurogenic and myogenic stem cells. Nestin has hardly been discovered in mature neurons, and its isoforms have never been reported. In this study, the first nestin isoform, Nes-S, was identified by RT-PCR on RNA obtained from single neurons of dorsal root ganglia (DRG) of adult rats. In this isoform, the majority of the nestin C-terminal domain, including the whole repeat region, is removed. This splicing also changes the open reading frame, and results in a novel 32-aa tail sequence and a premature stop codon. The Nes-S protein is 408-amino acid (aa) in length, containing a complete 7-aa N-terminal domain, a complete 307-aa rod domain, and a 94-aa C-terminal domain, with a predicted molecular weight of 45.9 kD. Nes-S cannot form filaments by itself in cytoplasmic IF-free SW13 cells. Instead, it co-assembles into filaments with vimentin in vimentin+ SW13 cells, and with peripherin and neurofilament heavy chain in both N2a neuroblastoma cells and primarily cultivated DRG neurons. To study its distribution pattern, an isoform specific antibody was generated against a peptide derived from the novel tail sequence of Nes-S. Immunoblotting assay with this antibody showed that its expression first appears in DRG at postnatal day 5 and persist to adulthood. In addition, immunoblotting and immunofluorescence microscopy with the same antibody showed that, among the adult neural tissues we examined, expression of Nes-S was only observed in neurons whose processes extend towards the limbs or the periphery of body, including neurons of DRG, trigeminal ganglia, superior cervical ganglia, as well as motor and sympathetic neurons of the thoracic spinal cord. Finally, we showed by MTT assay that Nes-S exerts a cytoprotective function when transfected into N2a neuroblastoma cells, and knockdown of endogenous Nes-S impaired the survival of primary DRG neurons during prolonged cultures. For the last decade, emerging evidences have revealed that alternative splicing serves as an additional way to regulate IF functions, and the IF isoforms may be involved in cellular stress responses as well as the pathogenesis of neural degenerative diseases. Nes-S is a new neuronal IF protein that exerts a cytoprotective function in mature neurons, and future studies on this protein may shed new lights on the research of neural degenerative diseases. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:21:16Z (GMT). No. of bitstreams: 1 ntu-101-D95b43002-1.pdf: 64808283 bytes, checksum: b261d0e5bc403d32f78e8b63b3e79eef (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | Contents
口試委員審定書 謝詞 中文摘要 Abstract Chapter 1. Introduction 1 1.1 Introduction to intermediate filament protein 1 1.1.1 Structure and classification 1 1.1.2 Mechanical and non-mechanical functions of intermediate filament proteins 1 1.1.3 Intermediate filament protein isoforms 2 1.2 Introduction to nestin, a type VI intermediate filament protein 4 1.2.1 Structure and classification 4 1.2.2 Expression regulation of nestin 5 1.2.3 The temporal expression pattern of nestin 6 1.2.4 Mechanical and non-mechanical functions of nestin 6 Chapter 2. Objectives 8 Chapter 3. Materials and Methods 9 3.1 Reagents and antibodies 9 3.2 Sequences 9 3.3 Animals 9 3.4 Primary cultures of adult rat dorsal root ganglia neurons 9 3.5 Probe synthesis for Northern blotting and in situ hybridization 10 3.6 Northern blotting 11 3.7 In situ hybridization 11 3.8 Generation of isoform-specific antibody 12 3.9 Immunofluorescence microscopy 12 3.10 Single neuron RT-PCR 14 3.11 Plasmid and cloning 15 3.12 Site-directed mutagenesis 16 3.13 Cell culture and transfection 16 3.14 Intermediate filament-enriched preparations 17 3.15 MTT assay 18 3.16 RNAi knockdown of nestin isoform in primary dorsal root ganglia neurons 18 3.17 Identification of the transfected primary DRG neurons by live-cell imaging 19 Chapter 4. Results 20 Part I- Identification of Nes-S, a novel nestin isoform, in dorsal root ganglia neurons 20 4.1 In situ hybridization evidence for a nestin transcript variant in adult DRG neurons 20 4.2 Epitope mapping of the Rat-401 antibody 20 4.3 Amplification of the 5’ region of Nes-S 21 4.4 Amplification of the 3’ region of Nes-S 22 4.5 Amplification of the whole coding region of Nes-S 22 4.6 Confirmation of the existence of Nes-S mRNA by Northern blotting and in situ hybridization 23 4.7 Identification of endogenous Nes-S protein by an isoform-specific antibody 24 Part II-Characterization of the Nes-S protein 25 4.8 Expression of Nes-S in dorsal root ganglia neurons of adult rats 25 4.9 Filament assembly property of Nes-S in SW13 cells 25 4.10 Filament assembly property of Nes-S in N2a neuroblastoma 26 4.11 Nes-S expression during postnatal DRG development 26 4.12 Tissue distribution of Nes-S 27 Part III-Functional analysis of the Nes-S protein 28 4.13 Effect of Nes-S in cell proliferation of N2a neuroblastoma cells 28 4.14 The cytoprotective effect of Nes-S in N2a neuroblastoma cells 28 4.15 The threonine-316 residue of nestin is indispensable for its cytoprotective function 29 4.16 The role of Nes-S in survival of primarily cultivated adult DRG neurons 30 Chapter 5. Discussions 31 5.1 Nes-S shares similar splicing patter with SynL, the low molecular weight isoform of type VI IF protein synemin 31 5.2 Nes-S inherit the same filament assembly property from nestin 31 5.3 The role of Cdk5 signaling in the cytoprotective function of Nes-S 32 5.4 Controversial results reported from recent studies on nestin knock mice 34 5.5 The neural protective role of Nes-S in vivo 34 Chapter 6. Conclusions 36 References 37 Tables 48 Figures 51 List of Figures Figure 1. The nestin transcript variant in DRG neurons of adult rats 51 Figure 2. Collecting single DRG neurons 53 Figure 3. Epitope mapping of the mouse monoclonal antibody Rat-401 55 Figure 4. Identification of the Nes-S transcript variant in DRG neurons by RT-PCR 57 Figure 5. Cloning and sequencing of the complete Nes-S transcript 59 Figure 6. Nucleotide and predicted amino acid sequence of nestin-FL and Nes-S 61 Figure 7. Northern blotting and in situ hybridization of Nes-S mRNA in DRG 63 Figure 8. Identification of Nes-S protein by immunoblotting of IF-enriched preparations of DRG with anti-AY14 65 Figure 9. Immunolabeling of cultured cells with anti-AY14 67 Figure 10. Expression of Nes-S in DRG neurons of adult rats 69 Figure 11. Immunofluorescence localization of Nes-S protein in DRG neurons of adult rats 71 Figure 12. High magnification confocal microscopy of Nes-S IFs in DRG neurons of adult rats 73 Figure 13. Filament assembly properties of Nes-S 75 Figure 14. Filament assembly property of Nes-S in transiently transfected N2a neuroblastoma cells 77 Figure 15. Postnatal expression and tissue distribution of Nes-S 79 Figure 16. Confocal microscopy of neuroepithelium of the embryonic rats 83 Figure 17. Expression of Nes-S in intestinal parasympathetic neurons 85 Figure 18. The cytoprotective effect of transiently expressed Nes-S in N2a neuroblastoma cells 87 Figure 19. The cytoprotective effect of Nes-S in N2a neuroblastoma cells 89 Figure 20. The cytoprotective effects of Nes-S, Nes-S-T316A, and Nes-S-T316D in stable clones of N2a neuroblastoma cells 92 Figure 21. Impaired cell survival in primary DRG neurons of adult rats upon RNAi knockdown of Nes-S 94 Figure 22. Multiple alignment analysis of nestin cDNA sequences of rat, mouse and human 96 List of Appendixes Appendix 1. The interaction between Cdk5 and mutated NestS proteins 98 | |
| dc.language.iso | en | |
| dc.subject | 背根神經節 | zh_TW |
| dc.subject | 感覺及運動神經元 | zh_TW |
| dc.subject | 中間絲 | zh_TW |
| dc.subject | 巢蛋白異構物 | zh_TW |
| dc.subject | 選擇性剪接 | zh_TW |
| dc.subject | alternative splicing | en |
| dc.subject | dorsal root ganglia | en |
| dc.subject | intermediate filament | en |
| dc.subject | nestin isoform | en |
| dc.subject | sensory and motor neurons | en |
| dc.title | 背根神經元巢蛋白異構物Nes-S：辨識、鑑定與細胞保護功能 | zh_TW |
| dc.title | Identification, Characterization and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 黃步敏(Bu-Miin Huang),李心予(Hsinyu Lee),溫進德(Jin-Der Wen),黃元勵(Yuan-Li Huang) | |
| dc.subject.keyword | 選擇性剪接,背根神經節,中間絲,巢蛋白異構物,感覺及運動神經元, | zh_TW |
| dc.subject.keyword | alternative splicing,dorsal root ganglia,intermediate filament,nestin isoform,sensory and motor neurons, | en |
| dc.relation.page | 99 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-07-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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