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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 許輔 | |
dc.contributor.author | Hui-Hsin Chang | en |
dc.contributor.author | 張慧欣 | zh_TW |
dc.date.accessioned | 2021-06-08T04:36:39Z | - |
dc.date.copyright | 2009-08-19 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-17 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22989 | - |
dc.description.abstract | 茯苓 (Poria cocos) 為重要的藥用真菌,具有多種藥理療效,惟其活性物質的結構及作用機轉尚未釐清,故本論文之研究目的為闡明茯苓活性成分,並針對其活化巨噬細胞及淋巴細胞之分子機制做進一步的研究。本研究自茯苓乾燥菌核中經由 DE-52 陰離子交換樹脂與膠體過濾層析純化所得之新免疫調節蛋白 PCP,層析與電泳結果顯示天然之 PCP 為異質雙體的醣蛋白,其分子量為 35.6 kDa,分別由 21.3 kDa 與 14.3 kDa 兩次單元蛋白以雙硫鍵鍵結所構成,其中 21.3 kDa 之次單元蛋白具有 N 型與 O 型之醣化修飾。
第一部份試驗主要偵測茯苓蛋白 PCP 對小鼠巨噬細胞之影響,於體外試驗顯示茯苓蛋白 PCP 能刺激 RAW 264.7 巨噬細胞產生 TNF-α 與 IL-1β,同時亦能調控 NF-κB 相關基因的表現量。於初代小鼠腹腔巨噬細胞共同培養發現,茯苓蛋白 PCP 能使其細胞增加 MHC class II 與 CD86 的表現,同時經由 TLR4 受器的辨識,透過 MyD88 依賴型信號傳導途徑,以達到活化巨噬細胞的效果。試驗中證明茯苓蛋白 PCP 能與 TLR4 受器結合,並使其細胞產生酪氨酸磷酸化;進一步利用 TLR4 免疫缺陷鼠 (C57BL/10ScN) 進行試驗,結果發現茯苓蛋白 PCP 活化巨噬細胞及與細胞結合的能力顯著降低。茯苓蛋白 PCP 經由酵素去醣基後會降低其引發的巨噬細胞反應,因此茯苓蛋白 PCP 上的醣基為 TLR4 調控腹腔巨噬細胞活化作用的主要因子。 第二部份探討茯苓蛋白 PCP 活化淋巴細胞的作用與機制,結果指出茯苓蛋白 PCP 能直接活化小鼠脾細胞,增加其細胞增生作用及細胞激素 IFN-γ 的產量,然而在 IL-5 分泌量則無提升之效果;定量即時聚合酶連鎖反應分析結果顯示茯苓蛋白 PCP 亦能有效提高 IL-2 與 IFN-γ 基因表現;經由流式細胞儀分析結果顯示,茯苓蛋白 PCP 雖無法增加小鼠脾細胞中 CD4+ 與 CD8+ T 細胞的細胞族群比例,卻能顯著增加 T 細胞活化標記 CD69 的表現量。進一步利用磁珠分離系統分選特定細胞,結果指出於 anti-CD3/CD28 抗體存在之下,茯苓蛋白 PCP 能增加純化後 CD90+ 細胞的增生能力,並呈劑量效應。藉由偵測細胞增生作用、細胞激素分泌量與 CD44 及 CD69 活化標記的表現,證實茯苓蛋白 PCP 具有活化 CD4+ 與 CD8+ T 細胞的效果。同時茯苓蛋白 PCP 能增加 CD4+ T 細胞 T-bet 轉錄因子的基因表現,促進其 STAT4 轉錄因子酪氨酸磷酸化,及 IL-2 與 IFN-γ 產量的增加。動物試驗結果顯示,口服茯苓蛋白 PCP 能抑制小鼠血清中 IgG1 的含量,並增加其血清 IgG2a 含量與脾細胞 IFN-γ 的分泌,同時茯苓蛋白 PCP 亦能提升 OVA 致敏小鼠體內 OVA 特異性 IgG2a 含量,推測茯苓蛋白 PCP 能促進第一型 T 輔助細胞 (Th1) 反應。此外,茯苓蛋白 PCP 經由酵素水解蛋白後,則顯著降低其原有活化 T 細胞的能力,此結果說明茯苓蛋白 PCP 主要藉由其蛋白分子誘發 T 細胞的活化。 綜合上述研究結果顯示,茯苓蛋白 PCP 為具開發潛力的免疫活化物質,可經由 TLR4 活化巨噬細胞,同時促進 Th1 的免疫反應。本研究結果有助於瞭解茯苓及其活性物質的功效,亦能促進其醫藥潛力與產業應用。 | zh_TW |
dc.description.abstract | Poria cocos (Schw.) Wolf is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and mechanisms involved have not been fully characterized. The objective of the present study was to investigate the bioactive substance from P. cocos and its molecular mechanism involved in immune modulation focused on macrophage and lymphocyte activation. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) as well as the regulation of nuclear factor-kappa B (NF-κB)-related gene expression. In primary mouse macrophages, we observed an increase in the expression of major histocompatibility complex (MHC) class II and CD86 molecules on peritoneal cavity macrophages. PCP directly activated macrophages to induce Toll-like receptor (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. We demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages.
Further investigation on lymphocyte activation indicated that PCP directly activated mouse splenocytes, markedly increased cell proliferation and the levels of interferon-gamma (IFN-γ) secretion but not IL-5 production. Similarly, the selectively enhanced transcriptional expression of IL-2 and IFN-γ by PCP was demonstrated using quantitative real-time PCR. Although there were slight increases in the total cell population of CD4+ and CD8+ T cells in PCP-stimulated splenocytes, PCP significantly increased expression of the activation marker CD69 on both splenic CD4+ and CD8+ T cells. The potent CD4+ and CD8+ T cell-activating capability of PCP was demonstrated by the enhancement of cell proliferation, cytokine secretion, activation marker CD44 and CD69 expression upon anti-CD3/CD28 costimulation. The expression of T-bet, tyrosine phosphorylation of STAT4, IFN-γ and IL-2 secretion during PCP-induced CD4+ T cell activation were upregulated. In contrast to the functional deficiency of deglycosylated PCP on macrophage activation, the core protein of PCP was shown to be involved strongly in induction of T cell activation, as demonstrated by inhibition of T cell response using deproteinized PCP. In vivo experiments indicated that oral administration of PCP (50 mg/kg body weight) suppressed the level of serum IgG1, and enhanced amounts of serum IgG2a and T helper 1 (Th1)-associated cytokine secretion in BALB/c mouse spleen cell cultures. Oral administration of PCP upon immunization with ovalbumin (OVA) exhibited that OVA specific IgG2a levels were also significantly increased compared with those of PBS-treated mice, suggesting that PCP could suppressed OVA-induced Th2 response to drive Th1 development. Taken together, these studies characterize a new potential immune stimulator, PCP, which induces TLR4-dependent activation within murine macrophages and triggers a Th1-dominant immune response. These observations provide strong support for further studies of PCP and P. cocos to explore their overall modulatory nature toward mammalian cells and reveal their pharmaceutical potential and industrial value. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:36:39Z (GMT). No. of bitstreams: 1 ntu-98-D94628008-1.pdf: 4044309 bytes, checksum: 33e630d6605c885f4a8ae811acc1d846 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 摘要 III Abstract V 1. Introduction 1 1.1. Preface 1 1.2. Overview of Poria cocos 3 1.2.1. Classification, morphology and chemical component of Poria cocos 3 1.2.2. Bioactivity of Poria cocos 4 1.3. Activation of innate and adaptive immune responses 9 1.3.1. General properties of immune responses 9 1.3.2. Involvement of Toll-like receptors in activation of innate immunity 10 1.3.3. Activation of adaptive immunity and T cell responses 13 2. Purpose of the present study 17 3. Materials and Methods 19 3.1. Materials 19 3.1.1. Mice 19 3.1.2. Reagents and chemicals 19 3.1.3. Antibodies 21 3.1.4. Specific primers for quantitative real-time PCR 21 3.1.5. Commercial kits 24 3.1.6. Instruments 25 3.2. Methods 25 3.2.1. Purification and biochemical characteristics of PCP 25 3.2.1.1. Purification and preparation of PCP 25 3.2.1.2. Molecular weight determination 27 3.2.1.3. Enzymatic protein deglycosylation and various treatments 28 3.2.1.4. Monoclonal antibodies 31 3.2.2. Macrophage activation induced by PCP 31 3.2.2.1. Cell cultures 31 3.2.2.2. Determination of TNF-α and IL-1β production 32 3.2.2.3. RNA isolation and RT-PCR analysis 33 3.2.2.4. Quantitative real-time PCR 34 3.2.2.5. Microarray analysis 35 3.2.2.6. Immunoprecipitation of TLR4 and Western blotting 36 3.2.2.7. Electrophoretic mobility shift assay (EMSA) 38 3.2.2.8. Fluorescence-activated cell sorting (FACS) 39 3.2.2.9. Macrophage binding study 39 3.2.2.10. Statistical analysis 41 3.2.3. Lymphocyte activation induced by PCP 41 3.2.3.1. Preparation of splenocytes 41 3.2.3.2. Purification of T cells 42 3.2.3.3. BrdU incorporation assay 43 3.2.3.4. Determination of cytokine production 44 3.2.3.5. Fluorescence-activated cell sorting (FACS) 45 3.2.3.6. Quantitative real-time PCR 46 3.2.3.7. Immunoprecipitation of STAT4 and STAT6 47 3.2.3.8. Oral administration of PCP and animal experiments 48 3.2.3.9. Quantification of total and OVA-specific antibodies 49 3.2.3.10. Statistical analysis 50 4. Results 51 4.1. Purification of PCP 51 4.2. Biochemical characteristics of PCP 52 4.3. PCP induces activation of NF-κB signaling pathway in mouse macrophage RAW 264.7 cells 54 4.4. TLR4 is involved in PCP-induced mouse peritoneal macrophage activation 56 4.5. PCP interacts with TLR4 in mouse peritoneal macrophages 57 4.6. Glycosylation of PCP is the key factor in PCP-signaling through TLR4 within murine macrophages 59 4.7. PCP directly activates mouse splenocytes 61 4.8. PCP activates purified T lymphocytes in the presence of anti-CD3/CD28 antibodies 62 4.9. PCP enhances effector functions of CD8+ T cells 64 4.10. PCP induces Th1-associated cytokines and transcription factors in CD4+ T cells 65 4.11. Oral administration of PCP enhances Th1-related immunoglobulin production and cytokine secretion by splenocytes 66 4.12. The protein core of PCP is essential for T cell activation 68 5. Discussion 70 5.1. PCP is a novel immunomodulatory protein which is not identical to other known functional proteins in nature 70 5.2. TLR4 is a limiting factor in PCP signal transduction with murine macrophages 71 5.3. PCP is a potent T cell activator mediating its effects via cytokine regulation 73 5.4. PCP promotes Th1 immune response in BALB/c mice 75 5.5. Polysaccharide and protein moieties of PCP represent distinct effects on macrophage and lymphocyte activation, respectively 77 5.6. The potential regulatory effects and functional applications of PCP 79 5.7. Conclusion 82 6. References 83 7. Figures 100 8. Tables 128 | |
dc.language.iso | en | |
dc.title | 茯苓免疫調節蛋白活化小鼠腹腔巨噬細胞之訊息路徑及促進 T 細胞活化與第一型 T 輔助細胞免疫反應 | zh_TW |
dc.title | A Novel Fungal Immunomodulatory Protein (PCP) Isolated from Poria cocos Induces Toll-like Receptor 4-dependent Activation in Mouse Peritoneal Macrophages and Promotes T Cell Activation and Th1 Immune Response | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 許先業,柯俊良,龔瑞林,繆希椿 | |
dc.subject.keyword | 免疫調節蛋白,茯苓,巨噬細胞活化,TLR4,T 細胞分化,醣基化, | zh_TW |
dc.subject.keyword | immunomodulatory protein,Poria cocos,macrophage activation,TLR4,T cell development,glycosylation, | en |
dc.relation.page | 131 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2009-08-18 | |
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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