草菇免疫調節蛋白之生物可及性與其對小鼠免疫細胞的影響

Abstract

草菇 (Volvariella volvacea) 為光炳菇科 (Pluteaceae) 小包腳菇屬 (Volvariella) 真菌，文獻指出新鮮草菇子實體中分離出的草菇免疫調節蛋白 (Fungal immunomodulatory protein—Volvariella volvacea, FIP-vvo) ，其分子量為12.7 kDa，由112個胺基酸所組成，並對小鼠脾細胞具有免疫調節活性。本研究以新方法純化 FIP-vvo，並研究其生物可及性與對小鼠免疫細胞的調控活性。第一部分探討熱、酸鹼與胃腸環境對FIP-vvo的影響，結果發現FIP-vvo在 20-90°C與pH 2-11中依然具有凝血活性 (hemagglutination activity)，為熱與酸鹼穩定的蛋白質。在個別模擬胃及腸道消化模式中，FIP-vvo僅部分被胃蛋白酶消化，而不會被胰酶消化；於模擬連續式胃腸道模式中，經由胃及腸道各消化兩小時後，FIP-vvo並不會降解。第二部分探討FIP-vvo對小鼠免疫細胞的調節活性，結果顯示FIP-vvo與小鼠腹腔巨噬細胞及脾細胞中子細胞群 CD3+、CD4+、CD8+ 和CD45R/B220+ 的細胞表面有顯著結合能力。FIP-vvo與小鼠腹腔巨噬細胞共培養，並不會有促細胞增生作用，且對細胞表面CD80、CD86和MHC class II的表現沒有顯著影響，僅在高濃度FIP-vvo的刺激下，小鼠巨噬細胞會產生IL-10和TNF-α。然而，FIP-vvo與小鼠CD90.2+ T 細胞共培養，會促進T細胞增生比例高達50%，也會增強T細胞表面分子標記CD25、CD28和CD69的表現，分別上升95.9%、47.3% 與96%，且經由FIP-vvo刺激的小鼠CD90.2+ T細胞會產生細胞激素IFN-γ、IL-2、IL-4、IL-10、IL-13和TNF-α。FIP-vvo 也能直接促進T細胞子群中的輔助CD4+ T細胞與毒殺CD8+ T細胞的增生與IFN-γ 的產生，且會增強CD4+ T細胞中T-bet、GATA-3和Foxp3的mRNA的表現，分別增加18、5.5與2倍，但不會對RORγt的基因表現有影響，表示FIP-vvo對小鼠T細胞的活化作用包含Th1、Th2與Treg細胞。最後，測定FIP-vvo於小鼠CD19+ B細胞的影響，結果顯示FIP-vvo能夠促進小鼠CD19+ B細胞增生，並且增強細胞表面CD25、CD28、CD40、CD69、CD80、CD86和MHC class II的表現。而受FIP-vvo刺激的小鼠CD19+ B細胞會產生IL-2和TNF-α 這兩種細胞激素。以上結果顯示FIP-vvo 對T細胞與B細胞均具有活化作用，可作為未來研究FIP-vvo對此兩種細胞的活化機制的基礎。

Volvariella volvacea is an edible paddy straw mushroom, belonging to the family of Pluteaceae and the genus of Volvariella. FIP-vvo has been reported as an immunomodulatory protein isolated from V. volvacea and been proved that it could induce cytokine gene expression and proliferation of murine splenocytes. The aim of this study was to find a new way to purify FIP-vvo, and to investigate its bioaccessibility and immunomodulatory functions toward different murine immune cells, such as macrophages, T cells and B cells. First, we tested the bioaccessibility of FIP-vvo. The results indicated that FIP-vvo was not a glycoprotein and showed hemagglutination activity (HA) against red blood cells of BALB/c mice. The HA of FIP-vvo was stable between 20 and 90°C and also maintained between pH 2 to 11. Moreover, FIP-vvo was partially digested by pepsin in simulated gastric fluid (SGF), but not digested by pancreatin in simulated intestinal fluid (SIF). After two-step stimulated gastrointestinal digestion, FIP-vvo still remained un-digested within 120 minutes. Second, the immunomodulatory function on the immune cells was examined. FIP-vvo exhibited significant binding to peritoneal macrophages and also showed strong binding capability to CD3, CD4, CD8 and CD45R/B220 subpopulations of the splenocytes. These results demonstrated that FIP-vvo might have the ability to activate macrophages, T cells and B cells. Therefore, the mitogenic potential of FIP-vvo on these immune cells was examined, and the cytokines production and the expression of surface markers of these cells were further observed. It was found that FIP-vvo could not induce the proliferation of peritoneal macrophages and did not enhance the CD80, CD86 and MHC class II expression. FIP-vvo could not stimulate NO and IL-10 production by murine peritoneal macrophages, but showed significant TNF-α production when the concentration of FIP-vvo was higher than 2.5 μg/mL. Moreover, the results of influence on T cells indicated that 0.63 μg/mL FIP-vvo induced 50% CD90.2+ T cell proliferation and stimulated IFN-γ, IL-2, IL-4, IL-10, IL-13 and TNF-α production. FIP-vvo also up-regulated CD25, CD28 and CD69 expression, which increased by 95.9%, 47.3% and 96.0%, respectively. T cells were subdivided into CD4+ and CD8+ T cell subsets and FIP-vvo could induce both CD4+ and CD8+ T cells proliferation and IFN-γ production. CD4+ T cells were also subdivided to Th1, Th2, Th17 and Treg cells; FIP-vvo could enhance the mRNA level of T-bet by 18-fold, GATA-3 by 5.5-fold, Foxp3 by 2-fold but not influence on RORγt gene expression. Finally, the effect of FIP-vvo on B cells proliferation and activation was explored. FIP-vvo could directly induce approximately 80% of murine CD19+ B cells proliferation and CD19+ B cells stimulated by FIP-vvo produced IL-2 and TNF-α but could not produce IFN-γ, IL-4 and IL-10. FIP-vvo up-regulated the expressions of CD25 and CD69, increasing by 24.82% and 60.9%, respectively, and expression level of CD40, CD80, CD86 and MHC II also increased by 60.9%, 41.9%, 10.0% and 19.3%, respectively. These results built up a basis for further research regarding activation mechanism on macrophages, T cells and B cells by FIP-vvo.