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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 涂熊林(Hsiung-Lin Tu) | |
dc.contributor.author | Chieh-Teng Cheng | en |
dc.contributor.author | 鄭捷登 | zh_TW |
dc.date.accessioned | 2021-06-08T03:31:03Z | - |
dc.date.copyright | 2021-02-22 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-01-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21321 | - |
dc.description.abstract | 細胞如何反應細胞激素的刺激並傳遞發炎信號至下游對於先天免疫反應的維持與細胞命運的決定至關重要。轉錄因子 NF-κB 是免疫信號路徑中的重要調控者,當受到細胞激素的刺激後會表現細胞質到細胞核之轉位活性;然而造成 NF-κB 在單細胞之間所展現的數位性活化狀態,即全有全無的潛在原因仍然是未知的。在此研究中我們假設單細胞間 NF-κB 的活性是透過細胞激素受體差異表現的程度所調控,從而表現出細胞間 NF-κB 活化的異質性。利用 CRISPR/Cas9 介導的螢光蛋白標記與延時顯微技術,我們建立了一個多通道的活細胞影像平台得以在腫瘤壞死因子α (TNF) 輸入期間追蹤內生性的腫瘤壞死因子受體1 (TNFRI)、NF-κB 以及細胞核之信號。我們的結果顯示即便在相同的細胞激素刺激下,差異化的 TNFR1 表現水平可同時以數位化與梯度化的訊號傳遞模式介導單一細胞間 NF-κB 的活化狀態。進一步我們利用全內角反射螢光顯微鏡於活細胞中觀察 TNFRI 受 TNF 刺激前後的動態,發現當 TNF 刺激後有高程度的 TNFR1 發生寡聚化並形成更高階的群聚,我們假設這些高階的 TNFR1 群聚狀態可能作為 NF-κB 活化的定量標記。本研究涵蓋了單細胞至單分子層級的定量生物學分析,可探討細胞激素受體在是否其表現程度上所受到的調控,如膜上的蛋白表現量或聚集狀態為造成單細胞間 NF-κB 活化高度異質性的來源之一。 | zh_TW |
dc.description.abstract | How cells respond to cytokine stimulation and consequently transmit inflammatory signaling downstream is critical for proper immune function maintenance and cell fate determination. Transcription factor NF-κB, a central regulator in immune signaling, exhibits cytoplasmic-to-nuclear translocation upon cytokine stimulation. However, possible source underlying digital pattern, i.e. all-or-none, NF-κB activation at single-cell level remains unclear. In this study, we hypothesize that NF-κB activation in single cells is regulated by fundamental differences at the receptor level, and this in turn manifests into the observed heterogeneous cell-to-cell NF-κB activation. Using CRISPR/Cas9-mediated fluorescent tagging and time-lapse microscopy, we developed a multi-channel live-cell imaging platform to track endogenous tumor necrosis factor receptor 1 (TNFR1), NF-κB, and nucleus signals during TNF stimulation. Our data indicates that both digital and graded information processing are involved in single-cell NF-κB activation states across differential TNFR1 abundance levels even under the same TNF input. Moreover, we applied total internal reflection fluorescence (TIRF) microscopy to observe differential TNFR1 dynamics before and after TNF stimulation in live cells. It was found that high levels of TNFR1 undergoes oligomerization and forms high-order clusters after TNF input. We inferred that high-order TNFR1 clustering might serve as a quantifiable marker for downstream NF-κB activation. This study offers the quantitative analysis of TNFR1 and NF-κB dynamics in live cells at high resolution, i.e. single-molecule and single-cell resolution, allowing us to probe the possible mechanism underlying heterogeneous NF-κB activation at single-cell level. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:31:03Z (GMT). No. of bitstreams: 1 U0001-2701202111412300.pdf: 21878075 bytes, checksum: 91c304fb4de53c1fb2885dfb0509499b (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 摘要 iii ABSTRACT iv CONTENTS v LIST OF FIGURES vii Chapter 1 Introduction 1 1.1 NF-κB Signaling in Inflammation 1 1.2 Tumor Necrosis Factor Receptor 1 2 1.3 Live Single-cell Imaging and Endogenous Fluorescence Tagging 3 1.4 Total Internal Reflection Fluorescence Microscope 4 1.5 Specific Aim 4 Chapter 2 Materials and Methods 6 2.1 Cell Line and Cell Culture 6 2.2 High-content Imaging Platform 6 2.3 Cytokine Stimulation 7 2.4 Plasmid Construction of CRISPR-Cas9-mediated Gene Tagging 8 2.4.1 Polymerase chain reaction (PCR) 8 2.4.2 Construction of sgRNA/Cas9 vector 8 2.4.3 Construction of KI donor vector 9 2.5 Establishment and Characterization of the KI Cell Line 10 2.5.1 Transfection 10 2.5.2 Cell sorting 10 2.5.3 Western blotting 11 2.6 TIRF Microscopy 12 2.6.1 Sample preparation for TIRF microscopy 12 2.6.2 Time-laspe TIRF microscopy 12 2.7 Quantitative Image Analysis 13 2.7.1 Cell tracking and NF-κB dynamics 13 2.7.2 Single-particle tracking of TNFR1 13 Chapter 3 Results 14 3.1 Single-cell NF-κB Activation under Varied TNF Dose Stimulation 14 3.2 Establish Tnfr1-mCherry KI MEFs via CRISPR Mediated Fluorescent Protein Tagging 15 3.3 A Combination of Digital and Graded Patterns in NF-κB Activity Across Differential TNFR1 Abundance Levels 17 3.4 TIRF Imaging Reveals Endogenous TNFR1 Forming Ligand-independent Clustering Status on Cell Membrane 20 3.5 TNFR1 Undergoes Higher-order Clustering upon TNF Stimulation. 21 Chapter 4 Discussion 23 Chapter 5 Figures 25 REFERENCE 50 PLASMID MAP 58 | |
dc.language.iso | en | |
dc.title | 以活體單細胞研究細胞激素受體於 NF-κB 活化過程之動態 | zh_TW |
dc.title | Live single-cell study of cytokine receptor dynamics in NF-κB activation | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊維元(Wei-Yuan Yang),黃筱鈞(Hsiao-Chun Huang) | |
dc.subject.keyword | 核因子活化B細胞κ輕鏈增強子,腫瘤壞死因子受體1,單細胞分析,內生性螢光蛋白標記,定量生物學, | zh_TW |
dc.subject.keyword | NF-κB,TNFR1,Single-cell analysis,CRISPR/Cas9-mediated fluorescent tagging,Quantitative biology, | en |
dc.relation.page | 59 | |
dc.identifier.doi | 10.6342/NTU202100202 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-01-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 基因體與系統生物學學位學程 | zh_TW |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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