真菌類免疫調節功能蛋白抗發炎及穩定性之研究

Anti-inflammatory and Stability of Fungal Immunomodulatory Protein

王文賢(Wen-Hsien Wang)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2005年)

摘要

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。

關鍵字

真菌類免疫調節蛋白

並列摘要

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.

並列關鍵字

FIP

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