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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 許輔(Fuu Sheu) | |
dc.contributor.author | Chia-Hung Pan | en |
dc.contributor.author | 潘家弘 | zh_TW |
dc.date.accessioned | 2021-06-07T18:08:17Z | - |
dc.date.copyright | 2012-07-20 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-17 | |
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Journal of Agricultural and Food Chemistry 60: 4914-4922. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16288 | - |
dc.description.abstract | 金針菇免疫調節蛋白 (FIP-fve) 為蕈類免疫調節蛋白家族之一,其具有抑制系統性過敏反應,增加人類周邊血單核細胞之細胞激素介白素-2 (IL-2) 及 干擾素-gamma (IFN-gamma)之能力。本研究室先前研究中發現FIP-fve 不同於裂殖素直接活化T淋巴細胞之特性,其需要抗原呈獻細胞 (APC, antigen-presenting cells) 之存在才可發揮刺激T淋巴細胞之功能,並可提升 T 細胞接受體 (TCR, T cell receptor) Valpha 19 之基因表現量,因此推測其作用特性可能類似於超抗原。
超抗原為一系列微生物蛋白質,其可透過直接與T細胞以及APC之作用而造成過度的免疫反應。為了證明FIP-fve具有類超抗原之特性,可TCR以及MHC結合,利用共同免疫沉澱方法研究蛋白質之間的交互作用。結果發現FIP-fve與TCR Vbeta, TCR Valpha, 及第二型主要組織相容性複合物 (MHC, major histocompatibility complex) 之間有直接連接之證據。 第二部分,前人研究中顯示FIP-fve也具有刺激小鼠週邊巨噬細胞累積Th1趨向性細胞激素介白素-12以及介白素-18mRNA之累積。本研究利用骨髓衍生型樹突細胞 (bone marrow derived dendritic cells, BMDCs) 作為抗原呈獻細胞,探討FIP-fve是否也能刺激此類抗原呈獻細胞之成熟與活化。介白素-6樹突細胞活化所需細胞激素之檢測中,發現FIP-fve不存在直接活化此類抗原呈獻細胞之能力 另外,欲探討在BMDCs之存在下,FIP-fve仍能表現超抗原之活性與否,故利用流式細胞儀檢測FIP-fve是否可刺激DO11.10 小鼠 (OVA323-339 特異性) 體內 CD4 輔助型T細胞。結果顯示,在6小時之共培養下FIP-fve 可使細胞早期活化之表面分子CD69提升7%,而分泌IFN-gamma之CD4+ T 細胞在經過12小時之共培養後有9.81% 之比例,較控制組約高出3.5%。此結果證實FIP-fve具有T細胞早期活化之功能,與抗原特異性呈現在72 h有最多分泌IFN-gamma之T 細胞有所不同。 最後,考慮到T 細胞產生之 IFN-gamma也可能進一步輔助回饋活化樹突細胞,故研究經過共培養後,在OVA323-339 特異性T細胞被FIP-fve刺激下,骨髓衍生性樹突細胞是否進而成熟活化。結果顯示,與樹突細胞成熟相關之細胞激素介白素-6、及干擾素-gamma之產生量在三天共培養下均顯著提升。另外,樹突細胞表面分子CD40以及MHC class II之表現也有所提升,顯示FIP-fve除了透過超抗原之特性刺激T細胞之外,更能使T細胞活化樹突細胞,達成免疫調節之效。 關鍵字:金針菇免疫調節蛋白,超抗原,共同免疫沉澱,骨髓衍生型樹突細胞,OVA323-339 特異性T 細胞 | zh_TW |
dc.description.abstract | FIP-fve is a member of the fungal immunoregulatory protein family. It was demonstrated to suppress systematic hypersensitive reaction and enhance cytokines IL-2 and IFN-gamma secretion by human peripheral blood mononuclear cells. We previously found the characteristic of FIP-fve induced lymphocytes activation was different from mitogens and its T cell activating function of FIP-fve was antigen presenting cells (APC)-dependent. In addition, FIP-fve enhanced the gene expression of TCR Valpha19. These observations inferred that FIP-fve could perform superantigens resembling function.
Superantigens are a series of microbial proteins that bridge the APC and T cells, causing non-specific T cell activation. To prove if FIP-fve was superantigens-like and has the characteristic in binding with both TCR and MHC, co-immunoprecipitation approach was used to study the protein-protein interaction. Results indicated the direct binding of FIP-fve with TCR Vbeta, TCR Valpha, and MHC class II. Second, we found that FIP-fve could induce murine peritoneal macrophages to express the mRNA of Th1 cytokine IL-12 and IL-18. In addition, murine bone marrow derived dendritic cells (BMDCs) were used as APCs to investigate whether FIP-fve could activate dendritic cells (DCs) to mature. We found that FIP-fve had no ability to directly activate DCs to secrete maturation cytokines IL-6. Third, to confirm if FIP-fve could perform the superantigen resembling ability in the presence of BMDCs, FACS analysis were used to detect whether FIP-fve could stimulate CD4+ helper T cells from OVA323-339 specific DO11.10 mice. We found that in the OVA-specific co-culture system, FIP-fve treatment enhanced the CD69+ population by 7%, and increased the population of IFN-gamma secreting CD4+ T γcells by 3.5% within 6 h of incubation. The result demonstrated the T cell activation of FIP-fve was earlier than OVA323-339. Finally, we used the co-culture system to confirm whether DCs could be activated by OVA323-339 specific T cells stimulated by FIP-fve. Results showed that maturation cytokines secretion of dendritic cells such as IL-6 and IFN-gamma were enhanced during 3 days of co-culture. Furthermore, DC surface markers CD40 and MHC class II expression was also up-regulated, suggesting that the FIP-fve-activated T cells could reciprocally induce the maturation of BMDCs. In conclusion, four features were found in the superantigen-resembling protein FIP-fve. First, FIP-fve could bind to MHC class II, TCR Vbeta, and TCR Valpha molecules. Second, FIP-fve alone could not activate BMDCs. Third, FIP-fve-activated T cells were antigen independent in the presence of BMDCs. Last, FIP-fve-activated T cells reciprocally caused the maturation of BMDCs. Keywords: FIP-fve, superantigen, co-immunoprecipitation, bone marrow derived dendritic cells, OVA323-339 specific T cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T18:08:17Z (GMT). No. of bitstreams: 1 ntu-101-R99628212-1.pdf: 2561332 bytes, checksum: 30245be725c3baa49680dac1cfcc936c (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Content
口試委員審定書 I 誌謝 II 摘要 IV Abstract VI Content VIII Figure caption X 1. Introduction 1 1.1. PREFACE 1 1.2. OVERVIEW OF FLAMMULINA VELUTIPES 2 1.3. HISTORICAL REVIEW IN FIP-FVE RESEACH 3 1.4. CHARACTERISTICS OF SUPERANTIGENS 4 1.5. PROTEIN-PROTEIN INTERACTIONS APPROACH 6 1.6. THE EVIDENCE OF FIP-FVE IS A SUPERANTIGEN-LIKE PROTEIN 7 1.7. PURPOSE OF THE PRESENT STUDY 8 2. Materials and Methods 11 2.1. MATERIALS 11 2.1.1. Mice 11 2.1.2. Reagents and chemicals 11 2.1.3. Antibodies 12 2.1.4. Commercial kits 13 2.1.5. Disposable parts and Instruments 13 2.1.6. Disposable Materials 14 2.1.7. Buffers 14 2.1.7.1. 10X PBS 14 2.1.7.2. 10X TBS 14 2.1.7.3. Complete RPMI medium 14 2.1.7.4. RBC lysis buffer 15 2.1.7.5. MACS buffer 15 2.1.7.6. FACS buffer 15 2.1.7.7. Blocking buffer 15 2.1.7.8. Non-denaturing lysis buffer 15 2.2 METHODS 15 2.2.1. Purification and biochemical characteristics of FIP-fve 15 2.2.2. BCA protein assay 16 2.2.3. Preparation of splenocytes 17 2.2.4. Co-immunoprecipitation (Co-IP) 17 2.2.5. Western blot analysis 18 2.2.5. Preparation of bone marrow derived dendritic cells 19 2.2.6. T lymphocytes purification 20 2.2.7. Total tyrosine-phosphorylated protein detection 21 2.2.8. Flow cytometry 21 2.2.9. Statistical analysis 23 3. Results 24 3.1 THE BIOCHEMICAL CHARACTERISTICS OF FIP-FVE 24 3.2 THE EVIDENCE OF PROTEIN-PROTEIN INTERACTION BETWEEN FIP-FVE, MHC AND TCR 25 3.3 FIP-FVE COULD NOT STIMULATE BMDCS OR T CELLS RESPECTIVELY. 26 3.4 FIP-FVE STIMULATES DO11.10 T CELLS IN AN OVA323-339 INDEPENDENT PATHWAY 27 3.5 MATURATION OF BMDCS IN MIXED LYMPHOCYTE REACTION 28 4. Discussion 30 4.1. THE HOMODIMER FORMATION OF FIP-FVE WAS RELATED TO SUPERANTIGENS 30 4.1.1. The FIP-fve used in this study is a homodimer 30 4.1.2. Structural perspectives of FIP-fve related to superantigens 30 4.2. FIP-FVE INTERACTS WITH T CELLS AND ANTIGEN-PRESENTING CELLS SURFACE MARKER 31 4.2.1 MHC class II, TCR Vbeta, and TCR Valpha interacted with FIP-fve 32 4.2.2. No interaction evidence between MHC class I and FIP-fve 32 4.3. THE IFN-gamma SECRETION BY FIP-FVE IS ANTIGEN INDEPENDENT 33 4.3.1. FIP-fve could not activate BMDCs 33 4.3.2. FIP-fve was not a mitogen that directly stimulated T cells 34 4.3.3. FIP-fve stimulated DO11.10 CD4+ T cells to secret IFN-gamma 35 4.3.4. FIP-fve stimulated DO11.10 CD4+ T cells might activate BMDCs to mature in IFN-gamma dependent pathway 36 4.4. CONCLUSION 37 4.5. FUTURE PERSPECTIVES 38 5. References 41 | |
dc.language.iso | en | |
dc.title | FIP-fve類超抗原特性之探討 | zh_TW |
dc.title | Study on the Superantigen-resembling Properties of FIP-fve | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周志輝(Chi-Fai Chau),蔣恩沛(En-Pei Chiang),繆希椿(Shi-Chuen Miaw),莊雅惠(Ya-Hui Chuang) | |
dc.subject.keyword | 金針菇免疫調節蛋白,超抗原,共同免疫沉澱,骨髓衍生型樹突細胞,OVA323-339 特異性T 細胞, | zh_TW |
dc.subject.keyword | FIP-fve,superantigen,co-immunoprecipitation,bone marrow derived dendritic cells,OVA323-339 specific T cells, | en |
dc.relation.page | 62 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-07-17 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
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