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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊雅雯(Ya-Wun Yang) | |
dc.contributor.author | Wen-Hui Luo | en |
dc.contributor.author | 羅文蕙 | zh_TW |
dc.date.accessioned | 2021-06-16T08:06:09Z | - |
dc.date.available | 2019-10-20 | |
dc.date.copyright | 2014-10-20 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-06-23 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58110 | - |
dc.description.abstract | 本論文的研究目的為探討poly(lactic-co-glycolic acid)(PLGA)奈米粒子透過小鼠骨髓中的Lin2-CD11b+Gr-1highLy-6Clow細胞(Gr-1high)與Lin2-CD11b+Gr-1low -Ly-6Chigh細胞(Gr-1low),交叉呈現PLGA奈米粒子之抗原所造成OT-1 CD8+ T細胞的影響,以及使用500 nm polystyrene Yellow-Green Fluoresbrite microspheres (YG-MP)來研究的生體分佈動力學。
我們發現Gr-1high與Gr-1low細胞可在in vitro環境將PLGA奈米粒子內包覆之ovalbumin (OVA)抗原,經由交叉呈現使OT-1 CD8+ T細胞產生增生反應、表現CD25與CD69、分泌IL-2、TNF-α、IFN-γ等活化反應,並且可在in vitro環境使OT-1 CD8+ T細胞活化為殺手型T細胞,產生perforin與granzyme B,並進行專一性細胞毒殺反應。此外我們也發現Gr-1low細胞的交叉呈現能力比Gr-1high細胞更好。將PLGA/OVA NPs注射到小鼠體內後,也會產生抗原專一性免疫反應,包括對OVA專一性IgG1、IgG2a、IgG2b抗體的產生、以及OT-1 CD8+ T細胞的增生反應。YG-MP在小鼠體內生體分佈的結果顯示在血液與骨髓中最主要吞噬YG-MP的細胞為B220-CD11b+Gr-1highLy-6Clow顆粒球,在脾臟則為B220+CD11b- B細胞。YG-MP主要分佈在marginal zone以及紅髓區(red pulp)。 綜合以上結果,我們確認了Gr-1+細胞,包括Gr-1high與Gr-1low細胞,具有交叉呈現PLGA/OVA NPs的抗原的作用。 | zh_TW |
dc.description.abstract | We attempt in this study to investigate the effect of ovalbumin (OVA) encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on cross-presentation of soluble antigens, mediated by Gr-1+ cells from mouse bone marrow, including Lin2‾CD11b+Gr-1highLy-6Clow (Gr-1high) and the Lin2‾CD11b+Gr-1lowLy-6Chigh (Gr-1low) subpopulations, to OT-1 CD8+ T cells. We also examined the biodistribution kinetics of 500 mm polystyrene nanoparticles, Yellow-Green Fluoresbrite microspheres (YG-MPs), in the mouse immune system by flow cytometry.
Our results showed that PLGA/OVA NPs-primed Gr-1+ cells stimulated the proliferation of OT-1 CD8+ T cells in vitro, whereas Gr-1low cells exhibited higher capability of antigen presentation than that of Gr-1high cells. Treatment of Gr-1+ cells with PLGA/OVA NPs upregulated the expression of activation markers CD25 and CD69, as well as the pro-inflammatory mediators, such as IL-2, TNF-α, and IFN-γ. PLGA/OVA NPs-primed Gr-1+ cells also induced the intracellular expression of perforin and granzyme B, and promoted antigen-specific cytotoxic lymphocyte (CTL) effect in vitro. Injection of mice with PLGA/OVA NPs induced the production of antigen-specific antibodies and stimulated the proliferation of OT-1 CD8+ T cells in vivo. Flow cytometric analysis showed that the major phagocytes of YG-MPs are B220‾CD11b+Gr-1highLy-6Clow granulocytes in the blood and bone marrow, and B220+CD11b‾ B cells in the spleen. Immunostaining and microscopic examination illustrated that YG-MPs are mainly distributed in the marginal zone and red pulp of the spleen. In summary, this study demonstrated that treatment of Gr-1+ cells with PLGA/OVA NPs induced cross-presentation of soluble antigens both in vitro and in vivo. | en |
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dc.description.tableofcontents | 目錄
口試委員審定書……………………………………………………………………………………..# 誌謝…………………………………………………………………………………………………..# 中文摘要 i Abstract ii 第一章 文獻回顧 1 1.1. 免疫系統 1 1.2. 顆粒球 3 1.3. PLGA奈米粒子 4 第二章 研究動機目的與實驗設計 6 第三章 材料 8 3.1. 藥品 8 3.2. 抗體 9 第四章 方法 15 4.1. PLGA奈米粒子的製作方法(43) 15 4.2. 測定PLGA奈米粒子的外觀(morphology)、表面電位與粒子大小(43) 15 4.3. 測定PLGA奈米粒子的OVA包覆量 16 4.4. PLGA奈米粒子所包覆OVA之釋放動力學 17 4.5. 老鼠品系 17 4.6. 細胞培養 18 4.7. 初代細胞(primary cells)的處理 18 4.8. 小鼠骨髓樹突狀細胞(Bone marrow-derived dendritic cells,BMDCs)的培養 19 4.9. 細胞enrichment與純化(purification)的過程 19 4.10. 5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester (CFSE)的標記方法 20 4.11. 以Giemsa stain觀察細胞型態 20 4.12. 以MTT測試PLGA奈米粒子對於小鼠骨髓細胞的毒性 20 4.13. PLGA奈米粒子影響Gr-1high與Gr-1low兩群細胞表面抗原表現的測定 21 4.14. PLGA奈米粒子對於Gr-1high、Gr-1low生長影響的觀察 22 4.15. OT-1 CD8+T細胞在in vitro的增生(proliferation)實驗以及增生後細胞產生之變化的觀察 22 4.16. OT-1 CD8+ T細胞細胞週期(cell cycle)的測定 23 4.17. OT-1 CD8+T細胞胞內產生cytokines、perforin、以及granzyme B的測定(Intracellular cytokine staining, ICS)(54, 55) 24 4.18. 專一性殺手型T細胞的細胞毒殺能力試驗[Cytotoxic T Lymphocyte assay (CTL) assay in vitro] 24 4.19. 偵測小鼠體內產生對OVA專一性抗體的酵素連接免疫吸附法(Enzyme-linked immunosorbent assay,ELISA) 26 4.20. OT-1 CD8+T細胞在小鼠體內的增生實驗 27 4.21. BMDCs吞噬奈米粒子實驗in vitro 27 4.22. 奈米粒子在小鼠體內之分佈實驗 27 4.23. 以顯微鏡觀察細胞或組織之免疫螢光染色法(Immunofluorescence, IF) 28 4.24. 實驗中所使用的流式細胞儀的規格 30 4.25. 分析數據所使用的統計方法 31 第五章 結果 32 第一部分 PLGA-OVA對於Gr-1high與Gr-1low細胞的影響 32 5.1. PLGA奈米粒子之外型、大小、表面電位、OVA包覆率與包覆量 32 5.2. PLGA奈米粒子內包覆之OVA的釋放動力學於前12小時為突釋(initial burst)時期,並於120小時後釋放完畢 32 5.3. PLGA奈米粒子對於小鼠骨髓細胞的毒性耐受度為100 μg/ml 33 5.4. 經流式細胞分選儀分選出來的Lin2-CD11b+Gr-1highLy-6Clow 細胞呈現顆粒球的外觀;Lin2-CD11b+Gr-1lowLy-6Chigh細胞呈現單核球的外觀,並且部分細胞有CD115表現 33 5.5. PLGA-OVA會造成Gr-1(high)細胞的CD115與Gr-1(low)細胞的CD11c、F4/80、CD115、I-A/I-E表現下降.以及Gr-1(low)細胞的外型改變 34 第二部分 PLGA-OVA對於Gr-1high與Gr-1low細胞抗原呈現所造成OT-1 CD8+ T細胞的影響 35 5.6. PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原後使OT-1 CD8+ T細胞在in vitro產生增生反應,並表現T細胞活化表面抗原CD25、CD69 35 5.7. 在in vitro增生的OT-1 CD8+ T細胞會變得比未增生的T細胞更大 37 5.8. PLGA-OVA可經由Gr-1(high)細胞交叉呈現,使OT-1 CD8+ T細胞進入S phase以及G2/M phase的比例增加 37 5.9. PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原,使OT-1 CD8+ T細胞產生IL-2、TNF-α、IFN-γ這些細胞激素(cytokines) 38 5.10. PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原後,使OT-1 CD8+ T細胞產生perforin以及granzyme B 39 5.11. PLGA-OVA可經由Gr-1(high)與Gr-1(low)細胞在in vitro交叉呈現抗原後,使OT-1 CD8+ T細胞轉變為殺手型T細胞(CTL),並進行專一性細胞毒殺反應 40 5.12. PLGA-OVA可增加小鼠體內對OVA專一性IgG1、IgG2a抗體的產生 41 5.13. PLGA-OVA可在in vivo環境中使OT-1 CD8+ T細胞產生增生反應 41 第三部分 利用帶螢光之奈米粒子研究在小鼠體內主要吞噬奈米粒子的細胞種類 42 5.14. BMDCs於in vitro環境會吞噬YG-MP,並且於4小時後達最大吞噬比例 42 5.15. 小鼠血液與骨髓內主要吞噬奈米粒子的細胞為B220-CD11b+Gr-1highLy-6Clow 顆粒球,小鼠脾臟內主要吞噬奈米粒子的細胞為B220+CD11b- B細胞,而淋巴結並不是奈米粒子主要分布之處 43 5.16. 奈米粒子主要分佈在小鼠脾臟的marginal zone以及紅髓區 48 第六章 討論 50 第一部分 PLGA-OVA對於Gr-1high與Gr-1low細胞的影響 50 第二部分 PLGA-OVA對於Gr-1high與Gr-1low細胞抗原呈現所造成OT-1 CD8+ T細胞的影響 52 第三部分 利用帶螢光之奈米粒子研究在小鼠體內主要吞噬奈米粒子的細胞種類 55 第七章 結論 58 參考文獻 60 附錄 129 圖目錄 圖 一、本研究所使用的PLGA奈米粒子為表面平滑的圓球形粒子。 67 圖 二、PLGA奈米粒子內包覆之OVA的釋放動力學於前12小時為突釋(initial burst)時期,並於120小時後釋放完畢。 72 圖 三、PLGA奈米粒子對於小鼠骨髓細胞的毒性耐受度為100 μg/ml。 74 圖 四、經流式細胞分選儀分選出來的Lin2-CD11b+Gr-1highLy-6Clow 細胞呈現顆粒球的外觀;Lin2-CD11b+Gr-1lowLy-6Chigh細胞呈現單核球的外觀,並且部分細胞有CD115表現。 76 圖 五、PLGA-OVA會造成Gr-1(high)細胞的CD115與Gr-1(low)細胞的CD11c、F4/80、CD115、I-A/I-E表現下降.以及Gr-1(low)細胞的外型改變。 78 圖 六、 PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原後使OT-1 CD8+ T細胞在in vitro產生增生反應,並表現T細胞活化表面抗原CD25、CD69。 82 圖 七、在in vitro增生的OT-1 CD8+ T細胞會變得比未增生的T細胞更大。 86 圖 八、在in vitro增生的OT-1 CD8+ T細胞的細胞質與細胞核會變得比未增生的T細胞大。 88 圖 九、 PLGA-OVA可經由Gr-1(high)細胞交叉呈現,使OT-1 CD8+ T細胞進入S phase以及G2/M phase的比例增加。 90 圖 十、PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原,使OT-1 CD8+ T細胞產生IL-2、TNF-α、IFN-γ這些cytokines。 92 圖 十一、 PLGA-OVA可經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原後,使OT-1 CD8+ T細胞產生perforin以及granzyme B。 96 圖 十二、 PLGA-OVA經由Gr-1(high)以及Gr-1(low)細胞交叉呈現抗原後,可在螢光顯微鏡觀察到OT-1 CD8+ T細胞產生perforin以及granzyme B。 99 圖 十三、PLGA-OVA可經由Gr-1(high)與Gr-1(low)細胞在in vitro交叉呈現抗原後,使OT-1 CD8+ T細胞轉變為殺手型T 細胞(CTL),並進行專一性細胞毒殺反應。 104 圖 十四、PLGA-OVA奈米粒子可增加小鼠體內對OVA專一性IgG1、IgG2a抗體的產生。 106 圖 十五、PLGA-OVA可在in vivo環境中使OT-1 CD8+ T細胞產生增生反應。 109 圖 十六、BMDCs於in vitro環境會吞噬YG-MP,並且於4小時後達最大吞噬比例。 111 圖 十七、小鼠血液內主要吞噬奈米粒子的細胞為B220-CD11b+Gr-1highLy-6Clow 顆粒球。 113 圖 十八、小鼠骨髓內主要吞噬奈米粒子的細胞為B220-CD11b+Gr-1highLy-6Clow 顆粒球。 117 圖 十九、小鼠脾臟內主要吞噬奈米粒子的細胞為B220+CD11b- B細胞,而淋巴結並不是奈米粒子主要分布之處。 121 圖 二十、奈米粒子主要分佈在小鼠脾臟的marginal zone以及紅髓區。 126 表目錄 表 一、本研究所使用的PLGA奈米粒子之大小、表面電位、以及OVA包覆量的測定。 128 | |
dc.language.iso | zh-TW | |
dc.title | 以奈米粒子作為抗原遞輸載體之研究 | zh_TW |
dc.title | Studies of nanoparticles-mediated antigen delivery | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 伍安怡,李建國,繆希椿 | |
dc.subject.keyword | 交叉呈現,殺手型T細胞,顆粒球,奈米粒子,poly(lactic-co-glycolic acid), | zh_TW |
dc.subject.keyword | cross-presentation,cytotoxic T lymphocytes,granulocytes,nanoparticle,poly(lactic-co-glycolic acid, | en |
dc.relation.page | 133 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-06-23 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
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