| Summary: | 碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 93 === 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.
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