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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許輔 | |
| dc.contributor.author | Po-Yu Chi | en |
| dc.contributor.author | 紀柏羽 | zh_TW |
| dc.date.accessioned | 2021-05-15T17:54:10Z | - |
| dc.date.available | 2014-08-08 | |
| dc.date.available | 2021-05-15T17:54:10Z | - |
| dc.date.copyright | 2014-08-08 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-07-28 | |
| dc.identifier.citation | 王紅利. 2011. 茯苓的松蔸栽培技術. 中國林副特產. 4:52-55.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5240 | - |
| dc.description.abstract | 茯苓蛋白 PCP (Poria cocos protein) 為由茯苓菌核萃取出的蛋白二聚體，分子量為 35.6 kDa，包含 14.3 kDa 單元及 21.3 kDa 含醣基單元。PCP 具有活化 RAW264.7 巨噬細胞及小鼠腹腔巨噬細胞的能力，在 anti-CD3/CD28 mAbs 存在的環境與 T 細胞共同培養可活化 T 細胞並分泌 TH1 細胞素 IFN-γ (interferon-gamma)，且於異位性皮膚炎之貼膚試驗中可抑制 TH2 免疫反應。因為 PCP 無法直接活化 CD90.2+ T 細胞，故本研究推測 PCP 必須利用脾細胞中的抗原呈現細胞 (antigen-presenting cells, APCs)，如樹突細胞 (dendritic cells, DCs) 使輔助型 T 細胞分化成 TH1 型，並設計試驗驗證之。首先，在探討 PCP 是否能活化 DCs 的研究中，PCP (100 μg/mL) 可增加小鼠 DCs 之 CD40、CD80 及 CD86 表現及 IL-6 及 IL-12p70 分泌量，證明 PCP 可活化 DCs。由 TLR (toll-like receptor) 2-/- 及 TLR4-/- 基因剔除鼠之 DCs 表面分子表現及 IL-6 細胞素分泌試驗結果推測 PCP 不完全透過 TLR2 或 TLR4 路徑活化 DCs。接著，在探討樹突細胞調節 TH 免疫反應之試驗中，預先與 PCP 及 OVA 抗原培養過的 DCs 可增加對 OVA 有特異性之 DO11.10 CD90.2+ T 細胞增生及分泌 IFN-γ，並減少 IL-4 分泌量，顯示 PCP 有助於 DCs 呈現 OVA 並使免疫反應導向 TH1。最後，於 OVA 誘導之小鼠氣喘試驗中，餵食 PCP (100 μg/day) 可增加支氣管肺泡灌洗液 (bronchoalveolar lavage fluid, BALF) IFN-γ 濃度及血清 OVA-specific IgG2a 含量，並降低 BALF 嗜酸性球 (eosinophils) 數、TH2 細胞素 (IL-4、IL-5、IL-13) 濃度、血清 OVA-specific IgE 含量及肺部組織的發炎細胞浸潤情形。由以上試驗結果證明 PCP 可透過活化樹突細胞使 TH1 型細胞分化，並抑制 TH2 型細胞分化，減少動物氣喘模式之小鼠 TH2 免疫反應。 | zh_TW |
| dc.description.abstract | Poria cocos protein (PCP), which is purified from dried sclerotium of Poria cocos, is a 35.6 kDa heterodimer protein including 14.3 and 21.3 kDa glycosyl subunits. In our previous results, PCP could active RAW264.7 cells and mouse peritoneal macrophages. This protein could also up-regulate murine T cells to secrete IFN-γ in the presence of anti-CD3/CD28 mAbs and inhibit TH2 immune-response in patch test for atopic dermatitis. However, PCP could not directly activate the differentiation of CD90.2+ T cells to TH1 cells, suggesting antigen-presenting cells as dendritic cells (DCs) could be responsible for the activation of PCP on TH1 cells.
In this study, we first found that PCP could up-regulate the expression of CD40, CD80, and CD86 and induce secretion of IL-6 and IL-12p70 on murine DCs. In the results of PCP incubated with DCs of TLR2-/- and TLR4-/- mice, we found that PCP might induce DCs activation through neither TLR2 nor TLR4 pathway. Second, PCP significantly increased the antigen-presentimg ability of murine DCs to promote cell proliferation and IFN-γ secretion of OVA-specific DO11.10 CD90.2+ T cells. Meanwhile, these DCs could inhibit the OVA-specific IL-4 secretion by DO11.10 CD90.2+ T cells. According to the results, we suggested that PCP can help DCs presenting antigen and skewing immune-response to TH1. Finally, in OVA-induced asthma mouse model, oral administration of PCP (100 μg/day) could increase the production of IFN-γ in bronchoalveolar lavage fluid (BALF) and OVA-specific IgG2a in serum. Oral administration of PCP could also down-regulate the amount of eosinophils and TH2 cytokines (IL-4, IL-5, and IL-13) in BALF, OVA-specific IgE in serum, and cell infiltration in mouse lung sections. In conclusion, PCP can activate DCs to promote differentiation of TH1 cells and suppress TH2 immune-response in mouse model of asthma. | en |
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| dc.description.tableofcontents | 總目錄
口試委員會審定書……………………………………………………………………..I 致謝…………………………………………………………………………………….II 中文摘要……………………………………………………………………………....III Abstract...…………………………………………………………………………...…IV 總目錄.…………………………………………………………………………...…...VI 圖目錄…………………………………………………………………………...……..X 縮寫對照表…………………………………………………………………………...XI 第一章 緒論………………………………………………………………………...1 1.1 茯苓之前人研究……………………………………………………………1 1.1.1 簡介…………………………………………………….……………1 1.1.2 茯苓之生長與栽種………………………………………….………1 1.1.3 茯苓之成分………………………………………………………….2 1.1.3.1 多醣類……………………………………………………...2 1.1.3.2 三萜類………………………………………….…………..3 1.1.4 茯苓之生物活性……………………………………….……………3 1.1.4.1 抗腫瘤活性………………………………………………...3 1.1.4.2 抗發炎……………………………………………………...4 1.1.4.3 抗氧化……………………………………………………...5 1.1.4.4 抗菌………………………………………………………...5 1.1.4.5 降血糖……………………………………………………...5 1.1.4.6 腎保護作用………………………………………………...6 1.2 茯苓蛋白之研究………………………………………………………………7 1.2.1 茯苓蛋白 PCP 之生化特性………………………….……………..7 1.2.2 茯苓蛋白 PCP 之生物活性…………………………….…………..7 1.2.2.1 PCP 於 APCs 之影響…………………………….………..7 1.2.2.2 PCP 於 T 細胞之影響…………………………….……….8 1.3 樹突細胞………………………………………………………………………9 1.3.1 樹突細胞簡介…………………………………………….………….9 1.3.2 樹突細胞之活化……………………………………………………10 1.3.3 樹突細胞之表面分子………………………………………………10 1.3.4 樹突細胞活化路徑…………………………………………………10 1.3.5 樹突細胞分泌之細胞素及功能……………………………………11 1.4 輔助型 T 細胞……………………………………………………………...11 1.4.1 輔助型 T 細胞之活化…………………………………………….11 1.4.2 TH1 型及TH2 型輔助 T 細胞簡介……………………..……….12 1.5 過敏性氣喘與 TH 免疫反應之相關性…………………………………….13 1.5.1 過敏反應介紹………………………………………………….......13 1.5.2 過敏反應之免疫發生步驟…………………………………………13 1.5.3 過敏性氣喘簡介…………………………………………………....14 1.5.4 如何透過調控 TH 免疫反應抑制過敏反應……………………...15 1.6 研究動機與目的…………………………………………………..................15 第二章 材料與方法……………………………………………………….………17 2.1 茯苓蛋白之萃取與分子量檢測……………………………………………..17 2.1.1 茯苓蛋白粗萃取…………………………………………………...17 2.1.2 DE-52 管柱層析…………………………………………………...18 2.1.3 蛋白去醣試驗……………………………………………………...20 2.1.4 SDS 電泳…………………………………………………………..20 2.1.5 PAS 醣蛋白染色…………………………………………………..22 2.2 茯苓蛋白與脾細胞、CD90.2+ T 細胞之培養與免疫活性試驗……………23 2.2.1 脾細胞之取得……………………………………………………...25 2.2.2 由脾細胞中分離 CD90.2+ T 細胞………………………………..25 2.2.3 茯苓蛋白與脾細胞、CD90.2+ T 細胞之培養……………………26 2.2.4 MTT 細胞活性試驗……………………………………………….26 2.2.5 ELISA 酵素免疫分析法測定 IFN-γ……………………………..26 2.3 茯苓蛋白與樹突細胞之培養與樹突細胞活化 T 細胞能力試驗…..…….27 2.3.1 骨髓衍生樹突細胞之取得方式…………………………………...29 2.3.2 茯苓蛋白與樹突細胞之培養……………………………………...30 2.3.3 樹突細胞與 DO11.10 CD90.2+ T 細胞之培養…………………..30 2.3.4 樹突細胞之表面分子檢測…………………………………….…..30 2.3.5 CFSE 細胞增生試驗………………………………………………31 2.3.6 IL-4、IL-6 及 IFN-γ 細胞素之ELISA 酵素免疫分析法測定...31 2.4 動物氣喘模式試驗…………………………………………………………..32 2.4.1 小鼠致敏步驟……………………………………….……………..33 2.4.2 小鼠餵食方式……………………………………………………...34 2.4.3 支氣管肺泡灌洗液 (BALF) 取得方式……………………….….34 2.4.4 劉氏細胞染色………………………………………………….…..35 2.4.5 IL-4、IL-5、IL-13 及 IFN-γ 細胞素之ELISA 測定…………..35 2.4.6 血液之取得及 OVA 特異性 IgE、OVA 特異性 IgG2a 之測定..35 2.4.7 肺部切片之取得及染色…………………………………………...36 2.5 統計方式…………………………………………………………………..…36 第三章 結果………………………………….……………………………………37 3.1 PCP 無法直接活化 T 細胞…………...…………………………….……37 3.1.1 PCP 之製備……………….……………………………………..…37 3.1.2 PCP 無法直接活化 CD90.2+ T 細胞……………….………...…37 3.2 PCP 可活化樹突細胞……………………………………………………...38 3.2.1 PCP 可促進 DC 表面分子之表現…………………………..…...39 3.2.2 PCP 提高樹突細胞分泌 IL-6 及 IL-12p70………………….….39 3.3 PCP-DC 抑制 DO11.10 T細胞之 TH2 免疫反應………………….…….40 3.3.1 T 細胞之 CFSE 細胞增生試驗結果……………………………..40 3.3.2 細胞上清液 IFN-γ 及 IL-4 濃度測定結果…………………….40 3.4 PCP 活化 DC 之路徑研究…………………………………………..…….41 3.4.1.1 PCP 刺激 TLR2-/- 小鼠樹突細胞…………………….……42 3.4.1.2 PCP 刺激 TLR4-/- 小鼠樹突細胞…………………….……42 3.5 餵食 PCP 於動物氣喘模式試驗………………………………………….43 3.5.1 BALF 嗜酸性球及 TH2 細胞素………………………………….43 3.5.2 BALF TH1 細胞素……………...……………………………….....44 3.5.3 血清 OVA-specific-IgE 及 OVA-specific-IgG2a………………...44 3.5.4 肺部切片 H&E 染色結果………………....………………...........45 第四章 討論……………………………………………….....................................46 4.1 PCP 活化樹突細胞之討論及其與 TLR4路徑之相關性.........................46 4.2 PCP 透過活化樹突細胞誘導 TH1 反應...................................................47 4.3 餵食 PCP 於小鼠氣喘動物模式之討論....................................................48 4.4 結論……………………………………………...........................................50 第五章 參考文獻………………………………………………………………….51 附圖……………………………………………………………………………………60 圖目錄 圖一、PCP 之 DE-52 層析、SDS-PAGE 及 PAS 醣染分析結果。………………..60 圖二、PCP 與脾細胞及 CD90.2+ T 細胞培養後之細胞活性。……………………61 圖三、PCP 刺激 BALB/c 脾細胞及 CD90.2+ T 細胞 IFN-γ 分泌之活性。……62 圖四、PCP 可增加 BALB/c BMDCs 表面分子 CD40、CD80 及 CD86 表現量。……………………………………………………………………………………..63 圖五、PCP 活化之 BMDCs 可提升 IL-6及 IL-12p70 分泌結果。………….......64 圖六、PCP 預培養之 BMDCs 可促 DO11.10 T 細胞增生。…………………….65 圖七、DO11.10 T 細胞與PCP 預培養之 BMDCs 培養後 IFN-γ 及 IL-4 分泌量。 ………………………………………….......................................……………….66 圖八、與 PCP 共培養之 TLR2-/- BMDCs 表面分子 CD40、CD80 及 CD86 表現。 …………………….……..….……………………………………………………67 圖九、與 PCP 共培養之 TLR4-/- BMDCs 表面分子 CD40、CD80 及 CD86 表現。 …………………….……………………………………………………………...68 圖十、與 PCP 共培養之 TLR2-/- 及 TLR4-/- 樹突細胞 IL-6 分泌量。…………...69 圖十一、動物氣喘模式之試驗步驟。…………………………………………............70 圖十二、餵食 PCP 可降低 BALB/c小鼠 BALF 之嗜酸性球數。…….................71 圖十三、口服 PCP 之氣喘小鼠 BALF TH2 細胞素 IL-4、IL-5 及 IL-13 分泌。………………………….....………………………………………….................................72 圖十四、口服 PCP 氣喘小鼠 BALF 之 TH1 細胞素 IFN-γ 分泌。…………..…73 圖十五、口服 PCP氣喘小鼠血清之 OVA 特異性 IgE 及 IgG2a 分泌。…………...74 圖十六、H&E 染色之小鼠肺部切片。……………………………………………….75 | |
| dc.language.iso | zh-TW | |
| dc.subject | 免疫調節蛋白 | zh_TW |
| dc.subject | 樹突細胞 | zh_TW |
| dc.subject | 輔助型 T 細胞 | zh_TW |
| dc.subject | 茯苓 | zh_TW |
| dc.subject | 呼吸道過敏 | zh_TW |
| dc.subject | bone marrow-derived dendritic cell | en |
| dc.subject | Poria cocos | en |
| dc.subject | allergic airway inflammation | en |
| dc.subject | T helper cell | en |
| dc.subject | immunomodulatory protein | en |
| dc.title | 茯苓免疫調節蛋白活化樹突細胞及調節 TH1 免疫反應 | zh_TW |
| dc.title | Poria cocos Immunomodulatory Protein Induces TH1 Immune Response by Activated Dendritic Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 周志輝,潘敏雄,繆希椿 | |
| dc.subject.keyword | 茯苓,免疫調節蛋白,樹突細胞,輔助型 T 細胞,呼吸道過敏, | zh_TW |
| dc.subject.keyword | Poria cocos,immunomodulatory protein,bone marrow-derived dendritic cell,T helper cell,allergic airway inflammation, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2014-07-28 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝暨景觀學系 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-103-1.pdf | 6.67 MB | Adobe PDF | 檢視/開啟 |
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