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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張喜寧,許輔 | |
| dc.contributor.author | Hui-Hsin Chang | en |
| dc.contributor.author | 張慧欣 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:20:51Z | - |
| dc.date.copyright | 2005-08-01 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-27 | |
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Phytochemistry 48(8):1357-1360. Yu, S. J., Tseng, J. 1996. Fu-Ling, a Chinese herbal drug, modulates cytokine secretion by human peripheral blood monocytes. Int. J. Immunopharmacol. 18(1):37-44. Zakany, J., Chihara, G. and Fachet, J. 1980. Effect of lentinan on the production of migration inhibitory factor induced by syngeneic tumor in mice. Int. J. Cancer 26(6):783-788. Zhang, J., Wang, G., Li, H., Zhuang, C., Mizuno, T., Ito, H., Mayuzumi, H., Okamoto, H. and Li, J. 1994. Antitumor active protein-containing glycans from the Chinese mushroom songshan lingzhi, Ganoderma tsugae mycelium. Biosci. Biotechnol. Biochem. 58(7):1202-1205. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24288 | - |
| dc.description.abstract | 茯苓 (Poria cocos) 為我國重要藥用蕈菌之一。本研究以茯苓菌核為材料，經由茯苓原料破碎、硫酸銨蛋白沉澱及透析所得粗蛋白質萃取液後，再以DE-52陰離子交換及FPLC層析純化濃縮而得茯苓免疫調節蛋白PCP。膠體過濾管柱層析顯示，茯苓蛋白PCP分子量約35.6 kDa；經蛋白質膠體電泳分析測得茯苓蛋白PCP具兩個單元蛋白，其分子量約為14.3 kDa及20.3 kDa。於等電聚焦電泳發現其pI值約為5.2，並取得茯苓蛋白PCP之胺基酸組成及兩單元蛋白之N-端胺基酸序列。
另一方面，利用細胞融合技術製作出3株能辨識茯苓蛋白PCP之單株抗體，進一步確認其抗體之專一性、抗體亞型及抗原決定區，並以免疫組織染色法偵測得知，茯苓蛋白PCP主要表現於茯苓菌絲的細胞壁、細胞膜及細胞核等部位中。 體外免疫調節試驗發現，茯苓蛋白PCP可以直接活化RAW 264.7巨噬細胞分泌 nitric oxide (NO) 與 TNF-a，均有顯著的成效，亦提高巨噬細胞之iNOS、TNF-a、IL-1b、IL-6、IL-12以及IL-18 mRNA的表現，於PMB與LAL試驗顯示排除茯苓蛋白PCP受到LPS污染的可能性。 此外，茯苓蛋白PCP可刺激小鼠淋巴細胞之增生及細胞代謝活性，亦提高細胞激素IFN-g 分泌量；流式細胞分析結果顯示，茯苓蛋白PCP能藉由提高淋巴細胞S及G2/M時期細胞比例，以達到促進T細胞及B細胞之增生，並可調節IL-2、IL-4、IL-5、IFN-g 的mRNA表現量，然而茯苓蛋白PCP能促進IFN-g 的分泌，但不會產生細胞激素IL-4，由上述結果顯示，茯苓蛋白PCP能活化小鼠脾細胞，並初步推測能促使T細胞分化為Th1細胞。 綜合本研究試驗結果得知，茯苓蛋白為免疫刺激物，可以提昇宿主相關免疫反應，極具醫藥及臨床發展之潛力。 | zh_TW |
| dc.description.abstract | A new immunomodulatory protein (PCP) is purified from the dried sclerotium of an therapeutic fungi, Poria cocos (Schw.) Wolf, by extraction using 5 % cold acetic acid in the presence of 0.1 % 2-mercaptoethanol, followed by ammonium sulfate fractionation, DE-52 anion-exchange and FPLC chromatography. Gel filtration chromatography shows that PCP has a molecular mass of 35.6 kDa. SDS-PAGE electrophoresis reveals that PCP contains two subunits with molecular mass of 14.3 and 21.3 kDa, respectively. Based on these results, we suggest that PCP is a heterodimer protein. Additionally, isoelectric focusing electrophoresis shows that the pI value of PCP is around 5.2. The amino acid composition and N-terminal amino acid sequences of these two subunits of PCP are also obtained.
Three hybridoma clones, which secrete monoclonal antibodies recognizing PCP, are obtained. The specificity and isotype of these monoclonal antibodies are also confirmed. Immunocytochemical analysis suggests that PCP mainly expressed on cell wall, cell membrane, and nucleus of Poria cocos sclerotium. PCP directly activates RAW 264.7 macrophages and enhances the production of both nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) by LPS-induced cells, and increases the mRNA expression of iNOS, TNF-a, IL-1b, IL-6, IL-12, and IL-18 of the cells. Polymyxin B (PMB) and LAL tests show that the capability of PCP activating macrophages is not due to LPS contamination. Furthermore, PCP activates murine splenocytes, markedly enhances cell proliferation and metabolization of the cells, and also increases their secretion of gamma-interferon (IFN-g) in vitro. Flow cytometry analysis reveals that PCP significantly promotes cell proliferation in both T cells and B cells by increasing S and G2/M phases of DNA content. Additionally, PCP up-regulates the mRNA expression of IL-2, IFN-g, IL-4, and IL-5. However, PCP stimulates IFN-g but not IL-4 secretion in murine splenocytes. Taken together, these findings suggest that PCP can activate murine splenocytes and drive their Th1 development. This study suggests that PCP is an immune stimulant and can strengthen the immune response of its host and having medicinal capability. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:20:51Z (GMT). No. of bitstreams: 1 ntu-94-R92628107-1.pdf: 1764097 bytes, checksum: fc2ee88e7587aef353179dac5260a621 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 中文摘要 1
英文摘要 3 第一章 研究背景 5 第一節 序言 5 第二節 第二節茯苓之生理活性 6 一、 茯苓之簡介 6 二、 茯苓之形態特徵 7 三、 茯苓之化學組成 8 四、 茯苓中已知機能性之成分 9 五、 茯苓之藥理作用 12 第三節 食藥用菇類之生理活性 15 一、菇類生理活性功能 15 二、菇類免疫調節活性蛋白 18 第四節 單株抗體 21 一、單株抗體的起源背景 24 二、單株抗體之原理 25 三、單株抗體之應用 26 第五節 研究動機與架構 28 第二章 材料與方法 31 第一節 茯苓蛋白之萃取 31 一、蛋白萃取 32 二、離子交換樹脂層析 33 三、快速蛋白質層析系統分析 33 四、透析 34 五、蛋白質濃度測定 34 第二節 茯苓蛋白之生化特性 35 一、SDS-膠體電泳法 37 二、Native-膠體電泳法 39 三、醣蛋白染色分析 40 四、等電點電泳分析 41 五、血液凝集活性試驗 42 六、胺基酸組成分析 43 七、蛋白質胺基酸序列分析 43 第三節 茯苓免疫調節蛋白之單株抗體分析 44 一、單株抗體之製備 46 二、單株抗體之生產 50 三、酵素連結免疫分析法 51 四、西方轉漬分析 53 五、各株單株抗體抗原決定區之分析 54 六、抗體亞型之測定 56 七、茯苓之免疫組織染色 57 第四節 免疫調節活性試驗 58 一、茯苓蛋白對小鼠巨噬細胞之影響 62 1、活化巨噬細胞產生一氧化氮試驗 62 2、細胞激素TNF-a 之測定 63 3、反轉錄聚合酶連鎖反應 64 4、Polymyxin B (PMB)試驗 67 5、Limulus amebocyte lysate (LAL)試驗 67 二、茯苓蛋白對小鼠淋巴細胞之影響 69 1、小鼠脾細胞之取得 69 2、淋巴細胞增生試驗 69 3、細胞代謝活性分析( MTT / XTT assay) 71 4、細胞激素IFN-γ之測定 73 5、細胞激素IL-4之測定 74 6、淋巴球增生及細胞流式儀之分析 75 7、反轉錄聚合酶連鎖反應 77 第五節 統計分析 79 第三章 結果 80 第一節 茯苓蛋白PCP之純化及生化特性 80 一、茯苓蛋白PCP之純化及離子交換樹脂層析 80 二、SDS-膠體電泳分析 80 三、醣蛋白染色分析 80 四、快速蛋白質層析系統與Native-膠體電泳分析 81 五、蛋白質濃度測定 83 六、等電點電泳分析 83 七、血球凝集活性分析 83 八、胺基酸組成分析 84 九、蛋白質N端胺基酸序列分析 84 第二節 茯苓免疫調節蛋白單株抗體之製作 85 一、單株抗體細胞株之製作 85 二、陽性融合細胞株之篩選 86 三、單株抗體之生產 87 四、西方轉漬分析 88 五、各種單株抗體抗原決定區之分析 89 六、抗體亞型之測定 90 七、茯苓之免疫組織染色 90 第三節 茯苓蛋白PCP的免疫調節活性 91 一、茯苓蛋白PCP對RAW264.7巨噬細胞之影響 92 1、一氧化氮 (nitric oxide) 產量之分析 92 2、細胞激素TNF-a 產量之分析 93 3、細胞激素mRNA表現量之影響 94 4、Polymyxin B (PMB)試驗 96 5、Limulus amebocyte lysate (LAL)試驗 98 二、茯苓蛋白PCP對小鼠脾細胞之影響 99 1、淋巴細胞增生試驗 99 2、細胞代謝活性分析( MTT / XTT assay) 100 3、細胞激素IFN-γ的測定 101 4、淋巴球增生之流式細胞儀分析 102 5、細胞激素mRNA表現量之影響 103 6、細胞激素IFN-γ及IL-4之測定 106 第四章 討論 108 參考文獻 115 圖與表 130 | |
| dc.language.iso | zh-TW | |
| dc.title | 茯苓免疫調節蛋白的純化與生理活性之探討 | zh_TW |
| dc.title | Studies on the Purification and Bioactivity of the Immunomodulatory Protein PCP from Poria cocos | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 柯俊良,沈立言,繆希椿 | |
| dc.subject.keyword | 茯苓,免疫調節蛋白,單株抗體,生理活性, | zh_TW |
| dc.subject.keyword | Poria cocos,immunomodulatory protein,monoclonal antibody,bioactivity, | en |
| dc.relation.page | 162 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2005-07-27 | |
| 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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