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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 安形高志(Takashi Angata) | |
| dc.contributor.author | Po-Chun Huang | en |
| dc.contributor.author | 黃柏鈞 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:28:32Z | - |
| dc.date.available | 2023-08-16 | |
| dc.date.copyright | 2018-08-16 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-13 | |
| dc.identifier.citation | 1. Crocker, P.R., J.C. Paulson, and A. Varki, Siglecs and their roles in the immune system. Nat Rev Immunol, 2007. 7(4): p. 255-66.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70454 | - |
| dc.description.abstract | 人類唾液酸結合蛋白14 (Siglec-14)為唾液酸結合蛋白家族成員之一，其具有增強細菌誘導的髓細胞促發炎反應。在臨床研究病例中，Siglec-14蛋白已被證實具有影響某些細菌誘導的反應。雖然Siglec-14蛋白已知屬於第一型膜蛋白，但是研究發現人類血液中存在著分泌型Siglec-14蛋白。然而，對於分泌型Siglec-14蛋白產生的機制與其存在人體內所扮演的角色，其研究所知甚少。本研究的目的著重於探索分泌型Siglec-14蛋白如何產生與其在人體內的功能。
我發現Siglec-14蛋白具有兩種傳訊RNA剪接變異體，分別為具備或是缺少內含子5的變異體。由於內含子5帶有終止密碼子，造成蛋白轉譯提前終止。缺乏內含子5的Siglec-14，其外顯子6可以被轉譯形成跨膜部位並組成第一型膜蛋白。藉由專一性辨認分泌型Siglec-14之C端胜肽片段的抗體，實驗證實由分泌型Siglec-14傳訊RNA剪接異變體所轉譯的蛋白是存在於人類血清中的。 在膜型Siglec-14蛋白表現的髓細胞實驗中，我分析重組分泌型Siglec-14蛋白對於促發炎細胞激素的影響。結果發現分泌型Siglec-14蛋白抑制無莢膜流感嗜血桿菌引起的髓細胞發炎反應並呈現劑量依賴性的狀況。此結果可能係由分泌型Siglec-14蛋白阻礙膜型Siglec-14與類鐸受體2 (Toll-like receptor 2)之間在細胞表面上的交互作用。內含子5的一部分序列會形成一種RNA二級結構名為G-四聯體，它可能涉及內含子5剪接的效率。綜上所述，分泌型Siglec-14蛋白在膜型Siglec-14蛋白引發的促發炎反應中扮演著負向調控的角色。 | zh_TW |
| dc.description.abstract | Human Siglec-14 is a member of the Siglec family of sialic acid-binding proteins. Siglec-14 enhances anti-bacterial myeloid cell pro-inflammatory responses. In some clinical conditions, Siglec-14 was shown to influence some bacterially induced responses in patients. Although Siglec-14 is a type 1 transmembrane protein, a soluble form of Siglec-14 was also found in human serum. However, the mechanism how soluble Siglec-14 is generated and what role it plays remain unknown. In this study, I aimed to understand how the soluble Siglec-14 is generated and what the function of soluble Siglec-14 is.
I found two major mRNA splice variants that either contain or lack intron 5, which contains an in-frame stop codon. The variant containing intron 5 yields soluble form by premature termination (as the exon 6 encodes transmembrane domain of Siglec-14), while the one without intron 5 yields the transmembrane form. I prepared antibodies against the C-terminal peptide segment unique to the soluble form of Siglec-14, and confirmed that the soluble form in human serum is indeed generated by the splice variant I found. I analyzed the influence of recombinant soluble Siglec-14 on pro-inflammatory cytokine production by membrane-bound Siglec-14 (mSiglec-14)-expressing myeloid cells. Soluble Siglec-14 suppressed non-typeable Haemophilus influenzae (NTHi)-stimulated response in a dose-dependent manner, which may be the result of interrupting the interaction between mSiglec-14 and toll-like receptor 2 on cell surface. A part of the intron 5 assumes an RNA secondary structure called G-quadruplex, which may be involved in the regulation of intron 5 splicing. In summary, I propose that soluble Siglec-14 is a negative regulator for the pro-inflammatory responses triggered by transmembrane Siglec-14. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:28:32Z (GMT). No. of bitstreams: 1 ntu-107-D00b46016-1.pdf: 2325286 bytes, checksum: 7dfa28c6fa7857c94d385f62208f3316 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii Table of Contents v List of Figures viii List of Tables xi CHAPTER I: Introduction 1 1.1 Siglec family and their distributions in human tissues 1 1.2 Function of CD33-rSiglecs in immune system 2 1.3 Human Siglec-14 in immune system 3 1.4 Fine-tuning of immune responses by soluble receptors 4 1.5 Aim of this study 6 CHAPTER II: Materials and Methods 7 2.1 Cell lines and culture 7 2.2 3' Rapid amplification of cDNA end (3'RACE) for Siglec-14 mRNA 8 2.3 Determination of the relative abundance of mRNA variants by quantitative PCR 8 2.4 Production of rabbit polyclonal antibody that recognizes C-terminal segment of sSiglec-14 9 2.5 Preparation of N-terminal His6-tagged sSiglec-14 expression construct 10 2.6 Mutation of N-terminal His6-tagged sSiglec-14 expression constructs 11 2.7 Production of recombinant N-terminal His6-tagged sSiglec-14 protein variants with Expi293F cell 12 2.8 Gravity purification of recombinant N-terminal His6-tagged sSiglec-14 protein variants from culture supernatant 13 2.9 Non-typeable Haemophilus influenzae (NTHi) culture 14 2.10 Analysis of THP-1 pro-inflammatory responses elicited with NTHi or Pam3CSK4 by ELISA 15 2.11 Analysis of multiple chemokines and cytokines by Magnetic Luminex Assay 16 2.12 Analysis of differentially expressed genes in sSiglec-14-treated human monocytes 17 2.13 Production of N-terminal FLAG-tagged mSiglec-14 expression constructs 18 2.14 Preparation of THP-1 cells expressing N-terminal FLAG-tagged Siglec-14 (FLAG-Siglec-14/THP-1) with retroviral expression system 19 2.15 Proximity biotin labeling and identification of Siglec-14-interacting proteins 20 2.16 Co-immunoprecipitation of mSiglec-14 with TLR2 22 2.17 Far-UV circular dichroism (CD) spectroscopy of synthetic 27-nucleotide RNA 23 2.18 Nuclear magnetic resonance (NMR) spectroscopy of synthetic 27-nucleotide RNA 24 2.19 Preparation of mini-gene constructs for Sigec-14 containing intron-5 25 CHAPTER III: Results 26 3.1 Alternative mRNA splicing generates sSiglec-14 26 3.2 sSiglec-14 splicing variant mRNA is abundant 27 3.3 Presence of sSiglec-14 protein corresponding to the mRNA splicing variant 28 3.4 sSiglec-14 suppresses pro-inflammatory responses of the cells that express mSiglec-14 30 3.5 Analysis of differentially expressed genes in sSiglec-14-treated human monocytes. 32 3.6 sSiglec-14 interferes with the interaction between mSiglec-14 and TLR2 34 3.7 Intron 5 of mRNA splice variant encoding sSiglec-14 contains a G-quadruplex structure 36 3.8 G-rich segment in the intron 5 of Siglec-14 pre-mRNA suppresses the splicing of the intron 39 CHAPTER IV: Discussion 40 CHAPTER V: Figures 47 CHAPTER VI: Tables 80 CHAPTER VII: References 85 | |
| dc.language.iso | en | |
| dc.subject | 類鐸受體2 | zh_TW |
| dc.subject | 無莢膜流感嗜血桿菌 | zh_TW |
| dc.subject | 選擇性剪接 | zh_TW |
| dc.subject | G-四聯體 | zh_TW |
| dc.subject | 唾液酸結合蛋白14 | zh_TW |
| dc.subject | 分泌型唾液酸結合蛋白 | zh_TW |
| dc.subject | alternative splicing | en |
| dc.subject | G-quadruplex | en |
| dc.subject | non-typeable Haemophilus influenzae | en |
| dc.subject | Toll-like receptor 2 | en |
| dc.subject | soluble Siglec | en |
| dc.subject | Siglec-14 | en |
| dc.title | 分泌型唾液酸受體其生成機制與抑制骨髓細胞發炎反應的功能探討 | zh_TW |
| dc.title | Soluble Siglec-14 is generated by alternative splicing and suppresses myeloid inflammatory responses | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),邱繼輝(Kay-Hooi Khoo),林俊宏(Chun-Hung Lin),徐尚德(Shang-Te Danny Hsu) | |
| dc.subject.keyword | 唾液酸結合蛋白14,分泌型唾液酸結合蛋白,選擇性剪接,無莢膜流感嗜血桿菌,類鐸受體2,G-四聯體, | zh_TW |
| dc.subject.keyword | Siglec-14,soluble Siglec,alternative splicing,non-typeable Haemophilus influenzae,Toll-like receptor 2,G-quadruplex, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU201803214 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-13 | |
| 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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