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標題: | FIP-fve類超抗原特性之探討 Study on the Superantigen-resembling Properties of FIP-fve |
作者: | Chia-Hung Pan 潘家弘 |
指導教授: | 許輔(Fuu Sheu) |
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, FIP-fve,superantigen,co-immunoprecipitation,bone marrow derived dendritic cells,OVA323-339 specific T cells, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 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 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16288 |
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顯示於系所單位: | 園藝暨景觀學系 |
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