研究真菌類免疫調節蛋白質之免疫調節活性

Immunomodulatory activity of Fungal Immunomodulatory Proteins

胡哲榮(Che-Jung Hu)

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(2006年)

https://doi.org/10.6834/CSMU.2006.00018

摘要

已知從松杉靈芝 (Ganoderma tsugae)、金針菇 (Flammulina velutipes)純化出來的真菌類免疫調節蛋白質命名為FIP-gts與FIP-fve，具有免疫調節的功能，在本研究中使用6種不同的醣類及其磷酸化醣類，利用競爭接受器的策略，追蹤FIP-gts與FIP-fve是透過哪種位在細胞膜上的醣類接受器進到HPBMCs (Human peripheral blood mononuclear cells) 內，但無法找到真正FIP-gts與FIP-fve的接受器。在分析FIP-gts與FIP-fve誘導HPBMCs產生IFN-gamma時，預先處理細胞膜上鈣離子通道的阻斷劑Nifedipine (L-type Ca2+ channel blocker)，無法抑制IFN-gamma的表現；不過在預先處理鈣離子細胞內的螯合劑BAPTA-AM，則是可以有效的降低IFN-gamma的產生；利用PI染色與流式細胞儀分析，發現BAPTA-AM會減少FIP-gts與FIP-fve刺激HPBMCs細胞週期當中的S 期比例，由此可知鈣離子在FIP-gts與FIP-fve活化HPBMCs與增生過程中扮演了一個很重要的角色。在靈芝的不同成分：多醣體 (ganoderan)、高多醣靈芝 (polysaccharide)、高三萜 (triterpenoids)與FIP-gts當中，FIP-gts造成HPBMCs產生IFN-gamma的活性最好。另一方面FIP-gts與FIP-fve皆能促進T-bet (T-box transcription factor) 的表現，其為Th1細胞產生IFN-gamma必備的轉錄因子，說明了FIP-gts與FIP-fve的確會刺激Th1細胞產生細胞激素。此外構築re-FIP-fve蛋白，包含reFIP-fve 1-114、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、reFIP-fve 28-114重組融合蛋白，發現reFIP-fve 1-114與reFIP-fve 1-103保有部分native FIP-fve活性，皆可刺激HPBMCs產生IFN-gamma，其他組別的蛋白則只刺激產生少許的IFN-gamma而已；在紅血球凝集方面，只有reFIP-fve 1-114有凝集活性；在細胞週期的變化中，也只有reFIP-fve 1-114有增加HPBMCs細胞週期由G0/G1期進入S期的比例，至於其他構築的reFIP-fve蛋白皆沒有對細胞週期產生明顯的影響。為了進一步探討FIP-gts與FIP-fve在生物體內的影響，利用Western blot證明出已經成功先製備出具有專一性的FIP-gts單株抗體。由Streptozotocin誘發第一型糖尿病的ICR小鼠中，給予FIP-fve無法降低血糖的濃度，不過在第二型糖尿病的ICR小鼠中，給予FIP-gts與FIP-fve都能降低血糖的濃度，尤其是FIP-fve改善的效果較明顯。本實驗室之前從細胞實驗得知FIP-gts能抑制A549細胞生長、移動(migration)與侵襲(invasion)，本研究從裸鼠實驗中卻發現FIP-gts與FIP-fve無法抑制A549腫瘤生長。另一方面發現FIP-gts從細胞計數與soft agar實驗可以抑制 Lewis Lung carcinoma cell (LLC)的生長，但動物實驗觀察到有無餵食FIP-gts的C57BL/6小鼠，腫瘤生長差異並不大，也說明FIP-gts 無法抑制LLC腫瘤生長。

關鍵字

真菌類免疫調節蛋白質

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. 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.

並列關鍵字

FIP-fve ； FIP-gts ； calcium ； diabetes mellitus

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