| Summary: | 碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 94 === 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. Using six different carbohydrate to trace which is the FIP-gts and FIP-fve binding receptor on human peripheral blood mononuclear cells (HPBMCs) plasma membrane by competition assay for receptor strategy. We could not find that which receptor involved in endocytosis for FIP-gts and FIP-fve. Analyzing the IFN-gamma induced by FIP-gts or FIP-fve in HPBMCs, pre-treatment of Nifedipine (L-type Ca2+ channel blocker) could not inhibit the secretion of IFN-gamma stimulated by FIP-gts or FIP-fve. Pre-treatment of BAPTA-AM which is chelator of intracellular Ca2+ decrease the secretion of IFN-gamma 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 for activation and proliferation of HPBMCs. HPBMCs were treated with different components of Shuang Hor Supreme Lingzhi such as polysaccharide, ganoderan, triterpenoids and FIP-gts. We found FIP-gts has the best immunomodulatory activity for inducing IFN-gamma cytokines in HPBMCs. Moreover, our results demonstrated that T-box transcription factor (T-bet), which is essential for the induction of IFN-gamma expression during Th1 development, was activated and raised by FIP-gts and FIP-fve. A series of truncated mutants were construct such as reFIP-fve1-114, reFIP-fve 1-103, reFIP-fve 14-103, reFIP-fve 14-114, reFIP-fve Δ13-27-103, reFIP-fve Δ13-27-114, reFIP-fve 28-103 and reFIP-fve 28-114. The results indicated that reFIP-fve1-114 and reFIP-fve1-103 owned partial activity of native FIP-fve by analying expression of IFN-gamma in HPBMCs. Except for reFIP-fve1-114, other fusion protein completely loss activity at hemagglutinating and cell proliferation. To further investigate the influence of FIP-gts or FIP-fve in organism, we had raised successfully monoclonal antibody to FIP-gts. It was induced type I diabetes mellitus by Streptozotocin, FIP-fve could not reduce the blood glucose concentration in ICR mice. Mice were induced type II diabetes mellitus by Streptozotocin, FIP-gts and FIP-fve could reduce the blood glucose concentration and FIP-fve was useful for modify the blood glucose concentration. It was inhibited cell proliferation, migration and invasion by FIP-gts in lung carcinoma A549 cell. Promote A549 tumor development in nude mice. It was inhibited cell proliferation by FIP-gts in Lewis Lung carcinoma cell using cell count and soft agar experiment, but FIP-gts could not inhibit LLC tumor development in C57BL/6 mice.
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