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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 | 江伯倫(Bor-Luen Chiang) | |
dc.contributor.author | Tien-Hsuan Chen | en |
dc.contributor.author | 陳恬萱 | zh_TW |
dc.date.accessioned | 2021-06-16T02:31:06Z | - |
dc.date.available | 2020-09-24 | |
dc.date.copyright | 2015-09-24 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-30 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53841 | - |
dc.description.abstract | 近來研究指出間葉幹細胞(mesenchymal stem cells; MSCs)在發炎環境中扮演著調節者的腳色,針對不同種類的發炎刺激,會引發細胞內完全相反的反應。本研究針對小鼠骨髓間葉幹細胞中介白素-1beta(IL-1beta)的基因表現及細胞內製造途徑進行調查。
在BALB/c小鼠模式中,取其腹腔巨噬細胞(peritoneal macrophage; pM)、骨髓衍生巨噬細胞(bone marrow derived macrophage; BMDM)作為對照組,以LPS (lipopolysaccharide)與第二刺激物 (Alum或ATP)的組合模擬NLRP3發炎複合體活化及IL-1beta製造途徑。實驗結果發現,間葉幹細胞在不受刺激的情況下即製造少量IL-1beta,製造途徑仍然透過NLRP3發炎複合體活化。但是,製造出來的IL-1beta並不如預期在經後轉譯修飾後隨即被釋放出細胞外,而是堆積在間葉幹細胞的細胞質內,表達未知的生理意義。經文獻回顧,我們假設一種抗微生物胜肽(antimicrobial peptide) alpha型防禦素 (alpha-defensin)能在不影響製造途徑的前提下,調控IL-1bera分泌的能力。比諸作為對照組的巨噬細胞,alpha型防禦素的製造在間葉幹細胞內旺盛許多。並且,在使用免疫沉澱(immunoprecipitation)的成果中,我們也看到alpha型防禦素與IL-1beta間具直接相互結合的能力。然而,關於兩者之間關係的直接證據,尚待繼續研究。 總結而言,本篇研究在間葉幹細胞中發現了自發性活化的NLRP3發炎複合體及在該細胞中IL-1beta具有獨特的分布現象。我們進一步假設了alpha型防禦素在發炎環境中的腳色與IL-1beta的分泌相關。本研究的主要貢獻在於報導一個全新且獨特存在於間葉幹細胞中的反應途徑,使我們對於該細胞在發炎環境中的反應有更深一層的認識。 | zh_TW |
dc.description.abstract | In recent studies, the role of mesenchymal stem/stromal cells (MSCs) has become a modulator of inflammation in response to different inflammatory signals. Among all, we focus on the profile of NLRP3 inflammasome and its main product cytokine IL-1beta.
Secretion and processing pathway of IL-1beta in BALB/c mice thioglycollate-elicited peritoneal macrophages (pMs), bone marrow derived macrophages (BMDMs) and BM-MSCs were investigated at both protein and RNA level after lipopolysaccharide (LPS) and a secondary stimulator (Alum or ATP) treatment. Interestingly, we found the NLRP3-caspase 1-IL-1beta pathway autonomously launched without LPS stimulation in MSCs. Moreover, MSCs maintain this small amount of IL-1beta in their cytosol for unknown physiological purpose. Since this discovery is apart from our knowledge of secretion right after production, we further postulated that mouse alpha-defensins, cryptdin 1 and cryptdin 4, might play a modulatory role in blocking the release of IL-1beta based on the previous reports and our observation of their larger amount in MSCs compared to pMs. Furthermore, the immunopreicipitation data also supported alpha-defensins would bind with IL-1beta in the cell. In conclusion, we demonstrated autonomous NLRP3 inflammasome activation in MSCs and a possible new role of alpha-defensins involved in inflammation. We believed that this investigation here might shed light on further understanding the mechanism of IL-1beta release and also therapeutic strategies of high-efficacy MSC-based therapy in autoimmune diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:31:06Z (GMT). No. of bitstreams: 1 ntu-104-R02449001-1.pdf: 5455886 bytes, checksum: a18910cb459f4f957814cd091d4016de (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝/Acknowledgement ii 中文摘要 iii Abstract iv Contents vi Figure Contents xii Table Contents xiii Chapter I Introduction 1 1. Overview of Mesenchymal Stem/Stromal Cells 2 1.1 Characterization of MSCs 2 1.2 Immunoregulatory Activities of MSCs 3 1.3 MSCs in inflammation 4 1.4 MSC-based cellular therapy 4 2. IL-1beta production in inflammation 5 2.1 Characterization of IL-1beta in inflammation 5 2.2 Regulation of IL-1beta production 6 2.3 Caspase-1 independent processing pathway 6 3. Overview of alpha-defensin 7 3.1 Characterization of alpha-defensin 7 3.2 Mechanisms of alpha-defensin bactericidal activity 8 3.3 The role between alpha-defensin and IL-1beta release 9 4. Hypothesis and Specific aims 10 Chapter II Materials and Methods 11 Part1 Materials 12 1. Cell Culture 12 1.1 Animals 12 1.2 Cell culture medium and buffer 12 1.3 MACS cell purification 13 2. Flow cytometry 14 3. Characterization of MSC 14 3.1 MSC differentiation medium 14 3.2 Imobilzer and indicator 15 3.3 T cell proliferation assay 15 4. IL-1beta inducing model 16 5. Immunoblot 16 5.1 Protein extraction and quantification 16 5.2 Gels, buffers, and reagents 16 5.3 Antibodies 17 6. Immunoprecipitation 18 7. Enzyme-linked immunosorbent assay (ELISA) 18 8. Detection of RNA expression 19 8.1 RNA extraction 19 8.2 Reverse transcription-polymerase chain reaction (RT-PCR) 19 8.3 Quantitative real-time polymerase chain reaction (qPCR) 20 9. shRNA knockdown system 21 Part2 Methods 23 1. Cell Culture 23 1.1 Preparation of bone marrow derived MSCs and macrophages (BMDM) 23 1.2 Preparation of peritoneal macrophages (pMs) 23 1.3 CD4+ T cell isolation 24 1.4 General cell culture process 25 1.5 MTT cytotoxicity assay 25 2. Flow cytometry 25 3. Characterization of MSC 26 3.1 MSC differentiation assay 26 3.2 T cell suppression assay 26 4. IL-1beta inducing model 27 5. Immunoblot 27 5.1 Protein extraction 27 5.2 BCA assay 28 5.3 Gel electrophoresis 28 5.4 Transfer and blocking 28 5.5 Detection and stripping 28 6. Immunoprecipitation 29 7. Enzyme-linked immunosorbent assay (ELISA) 29 8. Detection of RNA expression 30 8.1 RNA extraction 30 8.2 Reverse transcription-polymerase chain reaction (RT-PCR) 31 8.3 Quantitative real-time polymerase chain reaction (qPCR) 31 9. shRNA knockdown system 32 9.1 Amplification and isolation of plasmids 32 9.2 293 T transfection 32 9.3 Lentivirus titration 32 9.4 MSC infection 33 10. Statistical analysis 33 Chapter III Results 34 1. Experimental design 35 2. MSCs produced both precursor and mature IL-1beta without LPS stimulation. 36 3. NLRP3 inflammasome expressed and activated in post-translational level without LPS stimulation in MSCs. 37 4. Cryptdins serve as a potential regulator of IL-1beta release in MSCs 38 5. Knockdown of cryptdins in MSCs 39 Chapter IV Discussion 41 1. Regulation of NLRP3 inflammasome in MSCs 42 2. Physiological purpose of IL-1beta accumulation in MSCs 44 3. Regulation of IL-1beta release 46 4. Macrophages from difference source in LPS inflammation model 47 5. Conclusions 48 References 50 Figures 61 Tables 74 | |
dc.language.iso | en | |
dc.title | 小鼠骨髓間葉幹細胞介白素-1beta的分泌機制與alpha型防禦素間相關性之探討 | zh_TW |
dc.title | Study on the role of alpha-defensin in NLRP3 inflammasome dependent IL-1beta secretion of mesenchymal stem cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐立中,伍安怡 | |
dc.subject.keyword | 間葉幹細胞,NLRP3發炎複合體,IL-1beta,alpha型防禦素,LPS發炎模式, | zh_TW |
dc.subject.keyword | Mesenchymal stem/stromal cell,NLRP3 inflammasome,IL-1beta,alpha-defensin,LPS inflammation model, | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-30 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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