鴻喜菇免疫調節蛋白 HMP 於活化小鼠樹突細胞及呼吸道過敏模式之研究

Study on a Novel Immunomodulatory Protein (HMP) from Hypsizigus marmoreus which Induces Activation of Dendritic Cells and Alleviates Allergic Airway Inflammation in Murine Model

黃千育(Chien-Yu Huang)

指導教授 ： 許 輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10640

摘要

鴻喜菇 (Hypsizigus marmoreus) 又名 bunashimeji，為亞洲地區受歡迎的食用菇類之一，文獻指出鴻喜菇具有藥理療效如抗癌、抗氧化、抗病毒等，但其免疫調節之活性物質尚未釐清。本研究利用陰離子交換樹脂與快速蛋白液相層析系統，純化取得分子量約12 kDa純蛋白稱之鴻喜菇免疫調節蛋白 (H. marmoreus immunomodulatory protein, HMP)，經希氏高碘酸染色得知 HMP 非醣蛋白，並由胺基酸定序得知此蛋白 N 端胺基酸序列為 ATT-WKTSK。
第一部分活性試驗結果顯示，HMP可誘導小鼠脾細胞分裂及產生 IFN-γ，然而， HMP 無法直接刺激 CD90.2+ T 細胞增生和 IFN-γ 產生，此結果顯示 HMP 無法直接活化 T 細胞，而抗原呈獻細胞可能在T 細胞活化反應中扮演重要角色。
第二部分試驗探討 HMP 對小鼠骨髓衍生樹突細胞 (bone marrow derived dendritic cells, BMDCs) 之影響。我們發現 HMP 能誘導 BMDCs 表面分子 CD40, CD80, CD86 和 MHC class II 表現量增加，並促使 BMDCs 分泌促發炎細胞激素 IL-6 及 IL12-p70。再者，在混合淋巴細胞反應中，HMP 能增強 BMDCs 抗原呈獻之能力，並活化特異性 DO11.10 CD4+ T 細胞分泌 IFN-γ。因此，在體外試驗得知 HMP 能先活化 BMDCs，再進一步刺激 T 細胞走向 Th1 反應之能力。
第三部分以雞卵蛋白 (Ovalbumin, OVA) 來誘發小鼠產生強烈 Th2 反應呼吸道過敏之模式，發現 HMP 能降低小鼠血清中 OVA 特異性 IgE並提升 OVA 特異性 IgG2a。此外，小鼠肺灌洗液 (bronchoalveolar lavage fluid, BALF) 中所測得 IL-4, IL-5 及 IL-13 之含量降低，而 IFN-γ 含量增加。BALF 細胞中與發炎相關細胞之嗜酸性球數量降低，並透過小鼠肺切片染色分析得知 HMP 能減緩肺細胞浸潤。由此試驗可推斷，HMP 在體內可使 T 細胞走向 Th1 反應而扭轉 OVA 誘導 Th2 反應。
綜合以上結果，鴻喜菇免疫調節蛋白 HMP，於體外試驗中具有先刺激樹突細胞後再活化 Th1 反應，HMP 更可於體內試驗中增強 Th1 反應而減緩呼吸道過敏之現象，因此顯示其具有免疫調節功效之潛力。

關鍵字

鴻喜菇 ； 免疫調節蛋白 ； 骨髓衍生樹突細胞 ； T 細胞 ； 混合淋巴細胞反應 ； 呼吸道過敏

並列摘要

Bunashimeji mushroom (Hypsizigus marmoreus) is one of the most popular edible mushrooms in Asia. Recent studies showed that H. marmoreus had pharmacological functions such as antitumor, antioxidant and antivirus properties; however, the compound which possessed immunomodulatory abilities had not been clarified. In this study, we have purified the H. marmoreus protein via DE-52 anion-exchange gel firstly and further utilized fast protein liquid chromatography (FPLC) system with Hitrap-Q column. The single protein having a molecular mass of around 12 kDa was named H. marmoreus immunomodulatory protein (HMP). In addition, PAS staining showed HMP was not a glycoprotein. Moreover, N-terminal amino acid sequence of HMP, ATT-WKTSK, was determined by Edman degradation.
To evaluate the immunomodulatory capability of HMP, we observed that it stimulated splenocytes cell division and IFN-γ production. Nevertheless, HMP was failed to promote purified CD90.2+ T cell proliferation and IFN-γ secretion. This revealed that HMP was unable to elicit T cells activation directly and the antigen presenting cell (APC) might be a key factor in T cell activating reaction.
Next, we investigated the activation of HMP on murine bone marrow derived dendritic cells (BMDCs). HMP could up-regulate CD40, CD80, CD86 and MHC class II expression and induce the secretion of proinflammatory cytokines IL-6 and IL-12p70 by BMDCs. Furthermore, in a mixed lymphocyte reaction (MLR), HMP enhanced antigen presentation ability of BMDCs and activated IFN-γ production by purified DO11.10 CD4+ T cells. Therefore, HMP had the potential to induce BMDCs maturation and provoke Th1 response in vitro.
In murine model of OVA-induced Th2 response allergic airway inflammation, HMP was capable of restraining the level of OVA-specific IgE and increase OVA-specific IgG2a in mice sera. Additionally, in mice bronchoalveolar lavage fluid (BALF), IL-4, IL-5 and IL-13 were reduced and IFN-γ was up-regulated by peritoneal injection with HMP. In the presence of HMP, amount eosinophil also was decreased in mice BALF. HMP caused alleviation of inflammatory cell infiltration in mice lung tissues. These results suggested HMP could skew OVA-indued Th2 response to Th1 response in vivo.
In conclusion, a novel immunomodulatory protein purified from H. marmoreus could induce Th1 response development via BMDCs activation in vitro and ameliorate allergic airway inflammation in vivo. These findings supported the Th1 driving ability of HMP and indicated its immunoregulatory potential.

並列關鍵字

Hypsizigus marmoreus ； immunomodulatory protein ； bone marrow derived dendritic cell ； T cell ； mixed lymphocyte reaction ； allergic airway inflammation

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