探討舞菇免疫調節蛋白 GFP 之純化及其增強小鼠 Th1 免疫反應與抗腫瘤作用

Abstract

舞菇 (Grifola frondosa) 是日本傳統藥用食材，為中藥豬苓 (Grifola umbellate) 近親。本研究由舞菇中純化出免疫調節蛋白 (G. frondosa protein, GFP)。由分子篩濾層析得知在自然狀態下 GFP 分子量為 83 kDa，而在 SDS-PAGE 膠體電泳方面，還原狀態下 GFP 分子量為 41.1 kDa，由分子篩濾與 SDS-PAGE電泳結果推測 GFP 可能為雙元蛋白，且 GFP 非醣蛋白。以 N 端定序分析 GFP 胺基酸組成發現 GFP 內含兩種蛋白，因此推論 GFP 為分子量 83 kDa 的異質雙元體蛋白，且單元蛋白分子量約為 41.1 kDa。由體外試驗得知，GFP 可活化脾細胞分泌 IFN-γ 與提升細胞存活率，並促使脾細胞中 T 細胞與自然殺手細胞表面分子 CD25、CD69 與 NKG2D 表現量增加。在 T 細胞方面，GFP 無法直接活化 CD90.2+ T 細胞分泌 IFN-γ，需藉助樹突細胞分泌 IL-12，間接刺激 CD90.2+ T 細胞分泌IFN-γ。在自然殺手細胞方面，GFP 可直接活化 CD49b+ 自然殺手細胞分泌IFN-γ。在樹突細胞方面，GFP 藉由 TLR4 受器使之成熟並表現 MHC I、MHC II 和 CD86 與分泌 IL-12 和 IL-6。此外由動物試驗可知，經 GFP 活化的樹突細胞可抑制 LLC-1 小鼠肺腫瘤細胞生長率達 92.3%，且有較高效價的特異性抗體，表示後天免疫反應有被活化。由上述結果可知，GFP 為舞菇中新發現的免疫調節蛋白，且能夠趨化免疫反應導向 Th1 與抑制 LLC-1 小鼠肺腫瘤生長的效用，具有開發成商品或藥品的潛力。

Grifola frondosa is a Japanese traditional medicine and is phylogenetically close to a Chinese traditional medicine, Grifola umbellate. A novel immunomodulatory protein (G. frondosa protein, GFP) was purified form the fruiting bodies of G. frondosa. Size-exclusion chromatography and SDS-PAGE electrophoresis results indicated that the native GFP (83 kDa) was a dimer consisted of 41.1 kDa subunits and was not a glycoprotein. By the analysis of N-terminal amino acid sequence, we discovered that the subunits of GFP were different proteins. Therefore, we concluded that GFP was a non-glycosylated heterodimer protein with subunits having the same molecular weight. GFP enhanced cell viability and induced IFN-γ production by murine splenocytes in vitro. In murine splenocytes, we observed an increase in expression of CD25, CD69 on T cells and NKG2D on natural killer (NK) cells. GFP could directly stimulated IFN-γ production by CD49b+ NK cells. However, GFP could not directly stimulate T cells. The IL-12 produced by GFP-stimulated bone marrow derived dendritic cells (BMDCs) was required for GFP-induced T cells activation. GFP also increased the expression of major histocompatibility complex (MHC) class I, II, CD86 molecules and induced IL-12, IL-6 production by BMDCs in a TLR4-dependent manner. Finally, GFP-treated BMDCs vaccines inhibited tumor growth and increased tumor-specific antibody production in a LLC-1 murine tumor model. Taken together, these studies characterized a new immunomodulatory protein, GFP, which triggered a Th1-dominant immune response and suppressed tumor growth. These results provided new pharmaceutical and commercial potential of G. frondosa.