巴西洋菇發酵物對BALB/c鼠之非特異性免疫調節及其免疫調節蛋白純化與生理活性之探討

Abstract

本研究以黑豆為發酵基質之巴西洋菇發酵產物為樣品進行體外及 體內之免疫活性分析，實驗內容主要包含三部分，首先探討以黑豆為 發酵基質之巴西洋菇發酵樣品於體外實驗對人類白血病細胞株 (U937) 之生長抑制作用及刺激鼠巨噬細胞RAW 264.7，觀察其分泌TNF-α 及 Nitric oxide 之活性，藉以選出最具提升免疫活性之樣品濃度後再進行 動物模式評估，將樣品濃度區分為低、中及高劑量分別為 150、300 及600 mg/kg bw，依據健康食品之非特異性免疫調節功能評估法探討 巴西洋菇發酵產物對Balb/c 小鼠的非特異性免疫調節能力。最後，針 對巴西洋菇發酵產物生理活性成分利用Con A Sepharose 4B 親合性管 柱層析進行初步純化免疫調節蛋白。 非特異性免疫功能評估結果顯示，樣品並不會影響小鼠正常生長 情形及組織重量；於中、高劑量可增加血清中 IgG 抗體分泌量及增加 小鼠脾臟中毒殺型 T 細胞比例；三組劑量皆具有刺激脾臟細胞增生的 能力及顯著增加脾臟細胞之細胞激素IL-2、IFN-γ及TNF-α分泌量；各 劑量組血液中單核細胞球或嗜中性白血球之吞噬能力顯著提升；高劑 量組顯著增加小鼠脾臟中自然殺手細胞之毒殺能力。 體外實驗發現經親和性管柱純化所得之免疫調節蛋白與人類週邊 血液單核細胞培養一天後，以流式細胞儀分析細胞表面抗原結果顯示 2 CD3(+) T細胞及CD56(+)自然殺手細胞比例顯著增加。樣品蛋白質濃度 為7.4 μg/mL 及 11.1 μg/mL 與人類周邊血液單核細胞培養一天收集 條件培養液，與人類白血病細胞培養五天後之細胞生長抑制率分別達 87.7 %及91.3 %；樣品組之U937細胞CD11b及CD14陽性表現率顯著高 於控制組，顯示巴西洋菇發酵產物中免疫調節蛋白條件培養液具有誘 導U937細胞分化為成熟單核球及巨噬細胞的能力。此外免疫調節蛋白 進行 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) 分析後約估分子量為32 kDa，可被Con A 親和性管柱吸附，經 醣蛋白染色推測其含多醣及蛋白質成分，經由血球凝集活性試驗發現 對人類O型及B型紅血球不具有凝集活性。

In the present research, submerged fermentates of Agaricus blazei (AB), cultured under various conditions, were first screened based on the observation of the U937 cell growth inhibition and the activity of stimulating mouse macrophage cell line RAW 264.7 cells to secret TNF-α and Nitric oxide. The dosages which had the highest bioactivities in vitro were evaluated for the non-specific immunomodulatory effects of fermentates in balb/c mice by oral administration of low (150 mg/kg/ bw/day), medium (300 mg/kg/ bw/day), and high (600 mg/kg/ bw/day) dosages on the basis of the regulations of “Health-Promoting Food” of FDA, Taiwan. The purification and immunobioactivities of immunomodulatory proteins from submerged fermentates of Agaricus blazei were also investigated. Evaluation of the non-specific immunomodulatory effects showed that all the three dosages did not affect the growth and tissue weight of Balb/c mice.The medium and high dosages were significantly (P<0.05) enhancing the antibody (IgG) secretion and increasing CD8+ cytotoxic T cells of splenocytes. All the three dosages were effective in promoting splenocytes proliferation and cytokines (TNF-α, IL-2 and IFN-γ) secretion (P<0.05). Phagocytosis activities of blood granulocytes or monocytes were significantly improved at all the three dosages. The high dosage significantly (P<0.05) enhances the cytotoxic activity of natural killer cells of splenocytes. Incubating the immunomodulatory proteins with human peripheral blood mononuclear cells (MNCs) for one day revealed that the positive percentages of cell surface marker CD3 (T cell) and CD56 (NK cell) were 4 increased by flowcytometry analysis. The immunomodulatory proteins (7.4 and 11.1 μg/mL) were used to stimulate MNCs for one day to prepare conditioned medium, then incubated the U937 cells with the medium for five days to evaluate growth inhibition. The U937 cells growth inhibition reached 87.7 % and 91.3 % respectively. In addition, the expression of monocyte-associated cell surface marker CD11b and CD14 on U937 cells showed that the positive percentages of CD11b and CD14 were much increased after immunomodulatory proteins treatment, revealing that the immunomodulatory proteins have the effect of antiproliferation and differentiation of U937 cell line. SDS-PAGE electrophroresis found that the purified immunomodulatory proteins sized about 32 kDa. PAS staining revealed that the immunomodulatory proteins might be consist of polysaccharides and proteins. The hemagglutining activity assay showed that the immunomodulatory preteins were not lectin.