山藥塊莖主要儲藏性蛋白質免疫調節活性研究

Studies on the immunomodulatory activities of dioscorin, the major storage protein.

劉燕雯(Liu, Yen-Wenn)

指導教授 ： 徐鳳麟 ； 侯文琪

臺北醫學大學/藥學院/藥學系(博士班)/博士(2008年)

摘要

中文摘要
本研究探討山藥儲藏性蛋白質dioscorin的體內及體外免疫調節活性。以RAW 264.7細胞株評估dioscorin對於一氧化氮的產生、吞噬反應、氧化噴發以及分泌細胞激素 (IL-1β, IL-6, TNF-α) 的影響；並以BALB/c小鼠的脾細胞與腹腔吞噬細胞進行體外實驗，探討dioscorin對於腹腔吞噬細胞的吞噬活性與腹腔吞噬細胞以及BALB/c、C57BL/6兩種品系小鼠脾細胞在dioscorin刺激後產生細胞激素的影響。將dioscorin與人類單核球共同培養，分析單核球分泌細胞激素 (IL-1β, IL-6, TNF-α) 的濃度。體內實驗將dioscorin管灌餵食BALB/c小鼠，探討餵食期間小鼠周邊血中淋巴球分布及血中多形性顆粒球與單核球的吞噬能力，腸道分泌性抗體IgA的濃度於每週收集一次糞便以ELISA法分析濃度，最後犧牲小鼠分析脾臟中淋巴球分布、自然殺手細胞活性、與dioscorin餵食對Th-1相關細胞激素（IL-2、IFN-γ）與Th-2相關細胞激素（IL-4、IL-6、IL-10型）分泌的影響。實驗結果顯示，dioscorin能藉由促進RAW 264.7細胞株表現iNOS而產生一氧化氮 (2.5-3.5 μM)，促進RAW 264.7的吞噬能力達1.5-2.0倍。對於RAW 264.7細胞及人類單核球分泌細胞激素方面發現有顯著性促進IL-1β, IL-6, TNF-α分泌。Dioscorin促進小鼠腹腔吞噬細胞的吞噬能力達1.1倍，促進腹腔巨噬細胞IL-6分泌 (6.0-9.0 ng/mL)；在小鼠脾細胞的體外刺激，發現dioscorin能促進脾細胞增生，對於PHA所誘導的細胞增生具有協同促進，為PHA的1.4倍。Dioscorin培養BALB/c脾細胞48小時後，Th-2相關細胞激素(IL-6與IL-10)具有濃度依順性上升，Th-1相關細胞激素（IL-2）則為濃度依順性下降；而Dioscorin對於另一品系C57BL/6小鼠脾細胞培養48小時後，Th-1相關細胞激素（IL-2）反而具有濃度依順性上升，顯示dioscorin對於促進小鼠脾細胞分泌細胞激素具有雙向性。將dioscorin與人類單核球共同培養，發現dioscorin能促進IL-1β, IL-6, TNF-α的分泌。在體內實驗結果顯示，餵食dioscorin能使增加小鼠周邊血中B細胞與自然殺手細胞數目與脾臟淋巴細胞當中的自然殺手細胞數目；促進小鼠先天性免疫活性，包括吞噬及自然殺手細胞活性；餵食dioscorin小鼠脾細胞對於PHA誘導的細胞增生能力比控制組小鼠脾細胞高；對於Th-2型細胞激素，包括：IL-4與IL-10分泌促進較明顯，另外IFN-γ的濃度在餵食組小鼠明顯較高；小腸壁上的Peyer’s patces的數目在餵食dioscorin後明顯增加，腸道分泌性抗體IgA濃度隨著餵食的時間增加而上升。由上述實驗結果顯示，dioscorin具有體內、體外的免疫調節活性。

關鍵字

山藥 ； 主要儲藏性蛋白質 ； 免疫調節

並列摘要

Abstract
The major storage protein of yam (Dioscorea alata cv. Tainong No.1) tuber, dioscorin, was used to investigate the immunomodulatory activities by a series of in vitro and in vivo assay systems. Initially, the in vitro assays included nitric oxide production, stimulated phagocytosis, and cytokine production (TNF-α, IL-1β, and IL-6) of RAW 264.7 and peritoneal macrophages from BALB/c mice after being treated with different concentrations of dioscorin in the presence of polymyxin B. The spleen cells of BALB/c and C57BL/6 mice treated with dioscorin were used to perform the effects of dioscorin on cytokine production. The effect of dioscorin on cytokines production from human monocytes was also investigated. The in vivo immunomodulatory effects of dioscorin on BALB/c mice were studied by orally administration of dioscorin under the dose of 2.5 mg/kg/day and 20 mg/kg/day for 21days. The lymphocyte subpopulation in peripheral blood and stimulated phagocytosis of polymorphonuclear cells and monocytes were analyzed by flow cytometry at day0, 7, 14, and 21. The feces of mice were collected once a week for measuring the concentration of sIgA in gastrointestinal tract. After being sacrificed at day 21, spleens were removed and used to prepare splenic cells and to analyze the stimulated proliferation, cytotoxicity of natural killer cells, and cytokine production. The results of in vitro assays showed that dioscorin could increase the NO production through iNOS activation and cytokines (including IL-1β, IL-6, and TNF-α) production of RAW 264.7 cells and enhanced the phagocytosis and oxidative burst. Dioscorin also stimulated the proliferation of BALB/c mice spleen cells and synergistically enhanced the proliferative activity compared to the PHA alone. T helper type 2 cytokines, IL-6 and IL-10, were stimulated and the T helper type 1 cytokine, IL-2, was inhibited in dioscorin cultured with BALB/c mice splenic cells. However, IL-2 production was increased dose-dependently of dioscorin cultured with C57BL/6 mice splenic cells. Cytokines production on mice splenic cells showed that dioscorin might regulate the immune responses through the T helper type 2 responses and exhibit dual effects on T helper type 1 responses. Dioscorin increased the cytokines (including IL-1β, IL-6, and TNF-α) production of human monocytes. The results of in vivo experiments showed that dioscorin might enhance the innate immunity of BALB/c mice, including stimulated phagocytic activity and natural killer cell activity, and the profile of cytokine secretion toward T helper type 2 responses. The number of Peyer’s patches and secreted IgA concentration were increased in mice fed with dioscorin. From the results of above-mentioned, we concluded that dioscorin exhibited immunomodulaory activity both in vitro and in vivo.

並列關鍵字

dioscorin ； yam storage protein ； immunomodulatory

參考文獻

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