臺灣博碩士論文加值系統

分別從靈芝 (Ganoderma tsugae)、金針菇 (Flammulina velutipes) 純化出來的真菌類免疫調節蛋白 FIP-gts 和 FIP-fve，皆具有免疫調節的功能及60% 相同序列的胺基酸。在此研究中，利用FIP-gts 接上FITC 螢光物質來追蹤對HPBMCs (Human peripheral blood mononuclear cells)之影響，發現大部分的螢光位在淋巴球細胞細胞質內。在分析 FIP-gts 和 FIP-fve 誘導 HPBMCs 產生細胞激素時，預先處理鈣離子螯合劑 EDTA 會抑制 FIP-gts 和 FIP-fve 誘導之 IFN-g 的表現；預先處理 PKC (a, bI, bII and g) 的抑制劑 G06976，發現也可抑制FIP-gts 和 FIP-fve 誘導之 IFN-g 的表現及細胞增生的情形，由此顯示鈣離子在FIP-gts 和 FIP-fve 活化 HPBMCs 的過程中扮演一個相當重要的角色。在分析 GST-Der p 2 刺激肺泡巨噬細胞產生 NO 及 pro-inflammatory cytokines時，發現 FIP-gts 可以抑制 NO 的產生，而無法抑制 TNF-a 的表現；而FIP-fve則無抑制的效果。另一方面，評估 FIP-fve 對於塵蟎過敏原 GST-Der p 2或 OVA 致敏性氣喘 (Allergic asthma) 的減敏效果。我們發現經餵食 FIP-fve 的小鼠，似乎可以抑制 OVA 所誘發支氣管過度反應(AHR, Airway hyperresponsiveness)。遺憾的是我們無法測得血清中IL-4、TNF-a、IFN-g 的增加及肺部沖洗液中NO、IL-4、TNF-a 的增加。另外一方面，評估FIP-gts 和 FIP-fve 蛋白的穩定性，經100℃加熱1分鐘仍保有活性；利用人工模擬胃液和腸液測試蛋白分解的情形，都有可能通過胃壁到達小腸且發現 FIP-gts 比 FIP-fve 更容易被分解掉；在蛋白活性保存的評估，發現冷凍乾燥 (lyophilize)及冷凍乾燥(添加蛋白含量的70％ sucrose) 能夠有效保存蛋白活性。另外，探討FIP-gts及 FIP-fve 形成雙體的情形中，我們用 glutaraldehyde (蛋白連結劑) 發現FIP-gts 會形成dimer ，而FIP-fve 則以 homodimer 和polymer 的形式存在 。為了進一步探討FIP-gts及 FIP-fve在生物體內之影響，在我們實驗室已成功製備出FIP-gts 單株抗體，但 FIP-fve 由於製備出來的抗體效價不夠，故必須再進一步篩選細胞，進而純化抗體。

Two fungal immunomodulatory proteins (FIPs) have been isolated, purified from the edible mushroom Ganoderma tsugae and Flammulina velutipes and designated FIP-gts and FIP-fve, respectively. They have immunomodulatory activities and 60% identical amino acid sequence. Using FIP-gts conjugated with FITC to trace the influence of FIP-gts in human peripheral blood mononuclear cells (HPBMCs), we found that most fluorescence located on cytoplasm in lymphocytes. Analyzing the cytokines induced by FIP-gts or FIP-fve in HPBMCs, pre-treatment of EDTA was inhibited the secretion of IFN-g stimulated by FIP-gts or FIP-fve. Pre-treatment of G06976 (PKC (a, bI, bII and g) inhibitor) were also inhibited the secretion of IFN-g and cell proliferation stimulated by FIP-gts or FIP-fve. It is revealed that calcium plays a pivotal role on FIP-gts or FIP-fve-activated HPBMCs. Analyzing NO and inflammatory cytokines induced by GST-Der p 2 in alveolar macrophages, we found that FIP-gts could retard NO production but no effects on TNF-a production. FIP-fve did not have inhibitory effects. On the other part, developing allergy of house dust mite GST-Der p 2 or OVA-sensitized animal model, we evaluated whether FIP-fve could attenuate the symptoms of allergic asthma. Measuring AHR (airway hyperresponsiveness), we found that after sensitized with OVA induced AHR in Balb/c mice, after fed with FIP-fve AHR decreased to normal level. Unfortunately, we did not observe IL-4、TNF-a and IFN-g in serum were increased in GST-Der p 2 or OVA-sensitized Balb/c mice. We also did not observe NO、IL-4 and TNF-a in BAL (bronchoalveolar lavage) were increased. On the other part, we evaluated the stability and activity of FIP-gts and FIP-fve. They still maintain protein activity through 100℃ heat 1 min. Using SGF (simulated gastric fluid) and SIF (simulated intestinal fluid), we found that FIP-fve was more resistant to digestion than FIP-gts. We also found that lyophilized and lyophilized (contain sucrose, protein/sugar ratio 2：3) were effectively used for preservation and storage of protein activity. To study the dimer formation of FIP-gts or FIP-fve, we found that FIP-gts could become dimer and FIP-fve become dimer or polymer in the presence of glutaraldehyde. To further investigate the influence of FIP-gts or FIP-fve in organism, we successfully prepared monoclonal antibody to FIP-gts but we did not get FIP-fve monoclonal antibody.

頁次
謝誌 2
目錄 4
壹、 中文摘要 10
貳、 英文摘要 12
參、 縮寫表 14
肆、 緒論 16
一、具生理機能性的菇類蛋白 16
二、真菌類免疫調節蛋白 (FIPs) 17
三、體內與細胞激素相關之免疫反應 18
四、IFN-g 的相關調控 20
五、過敏性氣喘與衛生假說 22
六、肺泡巨噬細胞與氣喘的致病機轉 24
七、發炎中介物質與氣喘的關係 25
八、一氧化氮 25
伍、 研究動機 29
陸、 實驗材料與方法 31
一、儀器 31
二、藥品 32
三、實驗動物來源 33
柒、 實驗方法 34
一、細胞培養 34
二、SDS-聚丙烯醯胺板膠電泳法 36
三、流式細胞儀分析細胞週期 38
四、細胞激素之活性測定 39
五、FIP-gts 與 FIP-fve 在模擬消化液穩定性之測定 41
六、FIP-gts 與 FIP-fve 對熱穩定之活性評估 41
七、FIP-gts 與 FIP-fve 形成 monomer/dimer 之測定及活性分析 42
八、Ca2+ 對 FIP-gts 與 FIP-fve 刺激 HPBMCs 產生IFN-g 之影響 43
九、FIP-gts 之螢光測定 44
十、FIP-fve 保存方法及活性評估 44
十一、FIP-fve 之單株抗體製作 45
十二、塵蟎蛋白 Der p 的粗萃取 50
十三、重組 GST-Der pⅡ1-129 刺激NR8383 之影響 51
十四、塵蟎致敏動物模式的建立 56
捌、 結果 58
一、探討 FIP-gts 所引起免疫調節反應之作用位置 58
二、探討 FIPs 誘發 IFN-g 產生之訊號傳遞路徑 58
(1)、處理不同濃度 EDTA 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 產生IFN-g 的影響 58
(2)、處理不同濃度 G06976 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 產生IFN-g 的影響 59
(3)、處理不同濃度 G06976 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 增生的影響 59
(4)、預處理EDTA 和G06976 抑制 FIP-gts 或 FIP-fve 活化 PKCa 蛋白表現之影響 60
三、單株抗體的製作 60
(1)、免疫計畫 60
(2)、骨髓瘤細胞培養 60
(3)、細胞融合及陽性細胞株的篩選 60
(4)、抗體的大量生成 61
(5)、腹水的取得 61
(6)、酵素連結免疫分析法 61
(7)、西方點墨法 61
四、利用 native Der p 萃取物純化 Der p 粗蛋白液 61
五、探討 FIP-gts 抑制重組蛋白 GST-Der pⅡ1-129 刺激肺泡巨噬細胞誘發產生 nitric oxide 之影響 62
(1)、Der p extract 刺激肺泡巨噬細胞釋放nitric oxide 之關係 62
(2)、GST-Der pⅡ1-129 刺激肺泡巨噬細胞釋放nitric oxide 與pro-inflammatory cytokine 之劑量關係 63
(3)、GST-Der pⅡ1-129 刺激肺泡巨噬細胞釋放nitric oxide 與pro-inflammatory cytokine 之時間關係 63
(4)、FIP-gts 抑制 GST-Der pⅡ1-129 刺激肺泡巨噬細胞釋放nitric oxide 與pro-inflammatory cytokine 之關係 64
六、致敏小鼠之動物實驗 64
(1)、支氣管過度反應的測量 65
(2)、致敏小鼠 BAL 中 nitric oxide 和血清中細胞激素的變化 65
七、FIP-gts 和 FIP-fve 蛋白之高溫測試 65
八、模擬 FIP-gts 和 FIP-fve 在胃及腸道之蛋白穩定性 66
九、FIPs 保存方法的應用及評估 67
十、FIP-gts 和 FIP-fve 蛋白質 monomer/dimer 之測定及釋放細胞激素之能力 67
(1)、以 Glutaraldehyde 進行 cross-linking 實驗 67
(2)、刺激 HPBMCs 產生 IFN-g 之活性測試 68
玖、 討論 69
拾、 圖表說明 80
圖1 利用FIP-gts 接上 FITC 觀察對HPBMCs 之作用位置 80
圖2 處理不同濃度 EDTA 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 產生IFN-g 的影響 81
圖3 處理不同濃度 G06976 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 產生IFN-g 的影響 82
圖4 處理不同濃度 G06976 對於 FIP-gts 或 FIP-fve 誘導 HPBMCs 增生的影響 83
圖5 EDTA 和G06976 抑制 FIP-gts 或 FIP-fve 活化 PKCa 蛋白表現之影響 84
圖6 FIP-fve 單株抗體製作之免疫流程 85
圖7 測試 anti-FIP-fve Ab 對 FIP-fve 之 titer 86
圖8 anti-FIP-fve Ab 對 FIP-fve 辨識之影響 87
圖9 Der p 粗蛋白萃取液之製備流程說明圖 88
圖10 Der p 誘導 NR8383 中 NO 產生的情形 89
圖11 處理不同濃度 GST-Der pⅡ1-129 誘導 NR8383 中 NO 及 TNF-a 產生的情形 90
圖12 處理不同時間 GST-Der pⅡ1-129 誘導 NR8383 中 NO 及 TNF-a 產生的情形 91
圖13 處理不同濃度與不同時間 GST-Der pⅡ1-129 誘導 NR8383 中 iNOS 蛋白表現情形 92
圖14 處理不同濃度 GST-Der pⅡ1-129 誘導 NR8383 中 iNOS 及 IL-1b mRNA表現情形 93
圖15 處理不同濃度 FIP-gts 對於 GST-Der pⅡ1-129 誘導 NR8383 產生 NO 的影響 94
圖16 處理不同濃度 FIP-gts 對於 GST-Der pⅡ1-129 誘導 NR8383 產生 TNF-a 的影響 95
圖17 處理不同濃度 FIP-gts 對於 GST-Der pⅡ1-129 活化 NR8383 中 iNOS 蛋白表現情形 96
圖18 GST-Der pⅡ1-129 及 OVA 致敏小鼠之流程圖 97
圖19 偵測 Balb/c 小鼠肺功能之儀器 98
圖20 餵食 FIP-fve對於GST-Der pⅡ1-129 及 OVA 致敏Balb/c 小鼠引起支氣管過度反應之影響 99
圖21 餵食 FIP-fve對於GST-Der pⅡ1-129 及 OVA 致敏Balb/c 小鼠肺部沖洗液中誘發 NO 之影響 100
圖22 餵食 FIP-fve對於GST-Der pⅡ1-129 致敏Balb/c 小鼠血清中誘發 IL-4、TNF-a、IFN-g 之影響 101
圖23 餵食 FIP-fve對於OVA 致敏Balb/c 小鼠血清中誘發 IL-4、TNF-a、IFN-g 之影響 102
圖24 FIP-gts 或 FIP-fve 經由 100℃ 加熱對於誘導 HPBMCs 產生IFN-g 的影響 103
圖25 FIP-gts 和 FIP-fve 在模擬胃液中分解之情形 104
圖26 FIP-gts 和 FIP-fve 在模擬腸液中分解之情形 105
圖27 評估 FIP-fve 活性保存之方法 106
圖28 FIP-gts 及 FIP-fve monomer/dimer 之測定 107
拾壹、 參考文獻 108
拾貳、 附圖與附表 118

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