靈芝發酵液之免疫活性分析及以靈芝多醣處理人類單核細胞之蛋白質體學探討

Immunomodulatory Activities of Ganoderma lucidum Fermentation Products and Proteomic Analysis of Human Mononuclear Cells Following Treatment with Ganoderma lucidum Polysaccharide

楊舒秦(Shu-Chin Yang)

指導教授 ： 吳瑞碧

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00839

摘要

靈芝在中國傳統醫學中一直佔有極崇高的地位，被視為是滋補強壯、扶正固本的珍貴藥材。本實驗以豆科植物為發酵基質之靈芝發酵液為實驗材料，由細胞實驗篩選出具有免疫活性之靈芝發酵液樣品，進入動物實驗以評估體內非特異性免疫調節作用；另將酒精沈澱後之靈芝多醣刺激人類單核細胞觀察細胞增生能力，並以蛋白質體學探討受靈芝多醣刺激後之免疫細胞內蛋白質表現的變化。實驗結果顯示，靈芝發酵液GL-93-12-21具有促進人類單核細胞分泌細胞激素的能力，並對人類白血病細胞U937產生生長抑制之效果，且其酒精沈澱多醣能活化巨噬細胞RAW 264.7分泌NO及TNF-α，根據多醣分析結果此靈芝多醣特性與文獻指出之活性多醣特徵相符。經動物實驗觀察體內非特異性免疫調節反應，此批靈芝發酵液具有促進小鼠抗體IgG之生成、促進脾臟細胞中T及B淋巴細胞之增生、增加細胞激素TNF-α、IL-2及IFN-γ分泌量、活化單核細胞吞噬能力及促進自然殺手細胞活性之功效。另實驗將靈芝多醣刺激人類單核細胞後觀察到細胞增生現象，並以蛋白質體學研究細胞內蛋白質表現的變化，結果共有19個表現量差異性在50 % 以上之蛋白質點經由質譜儀（MALDI-Q-TOF）被鑑定出，除了一個為表現量下降外，其餘皆為表現量上升之蛋白質點。經資料庫比對及蛋白質功能鑑定分析結果顯示，細胞骨架及其結合蛋白與核糖核酸蛋白表現量的上升，推論與靈芝多醣刺激免疫細胞的活化及增生有相關。此外，annexin A6及伴護子（chaperone）表現量的增加，可能對於B淋巴細胞的活化與其膜上接受器之表現有所影響。

關鍵字

靈芝 ； 免疫調節 ； 人類單核細胞 ； 蛋白質體學

並列摘要

Ganoderma lucidum is one of the most well known Chinese medicine in ancient times and is considered as the most valuable plant for health and longevity promotion. In this study, we investigated the effects of Ganoderma lucidum liquid fermentation broth on immunomodulatory activities in vitro and in vivo. After an in vitro experiment, we selected the most active fermentation product for experiments on non-specific immune response in BALB/c mice. In addition, we observed the MNC proliferation effect and performed the proteomic analysis of the protein expression by human mononuclear cells (MNC) treated with ethanol precipitated Ganoderma lucidum polysaccharide. Results show that Ganoderma lucidum fermentation product GL-93-12-21 stimulates human mononuclear cells to secrete cytokines and inhibit the human myelocytic lymphoma U937 cell growth. Moreover, the ethanol precipitated polysaccharide stimulats NO and TNF-α production in RAW 264.7 macrophage, and the polysaccharide characteristics are similar with bioactive polysaccharides that have been reported. In vivo studies showed that oral administration of Ganoderma lucidum fermentation product GL-93-12-21 on BALB/c mice significantly (P＜0.05) increases the secretion of IgG, the proliferation of T- and B-lymphocyte, the level of TNF-α、IL-2 and IFN-γ production, the phagocytosis effect of monocytes and the cytotoxicity of nature killer cells. Results also show that Ganoderma lucidum polysaccharide stimulates the proliferation of human mononuclear cells and changes the protein expression. There are 19 protein spots (1 down-regulated and 18 up-regulated) differentially expressed more than 1.5-fold, which was identified by quantitative image analysis and matrix assisted laser desorption ionization-quadrupole-time of flight (MALDI-Q-TOF) mass spectrometry. The Swiss-Prot database search indicated that increased expression of cytoskeleton and ribonucleoprotein is associated with the immune cell activation and proliferation. Furthermore, the increase of annexin A6 and chaperon protein expression, may play a part in activating of B lymphocyte and assembling of B cell receptor.

並列關鍵字

Ganoderma lucidum ； Immunomodulatory ； Human mononuclear cells ； Proteomics

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