Ganoderma microsporum 免疫調節蛋白GMI 在動物體之免疫佐劑與抗微生物感染活性之研究

Antimicrobial and adjuvant functions of the immunomodulatory protein GMI from Ganoderma microsporum in animals

游耀萱(Yao-Hsuan Yu)

指導教授 ： 陳俊任

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02767

摘要

由不同真菌純化出與 LZ-8序列相近的免疫調節蛋白質，通稱為 FIPs (fungal immunomodulatory proteins)，其中 GMI 是近年來成功選殖自小孢子靈芝 (Ganoderma microsporum) 的免疫調節蛋白質。本實驗室發現 GMI 刺激小鼠巨噬細胞株 RAW264.7 分泌 TNF-α，增加一氧化氮生成，並增進吞噬作用 (phagocytosis)，因此本篇研究以小鼠感染李斯特菌 (Listeria monocytogenes) 的模式，探討 GMI 對抗微生物感染之能力，結果顯示先給予小鼠 GMI 可以有效的降低脾臟和肝臟的菌數量，抑制細菌的感染。此外在動物實驗中，利用 GMI 免疫調節蛋白質作為免疫佐劑，以卵清蛋白 (OVA) 為抗原對小鼠進行免疫，發現 GMI 可增加抗原專一性的免疫球蛋白 IgG1 和 IgG2a及 Th 1 反應細胞激素之 IFN-γ 生成。以 OVA 刺激免疫過後之小鼠脾臟細胞，能刺激專一性的細胞增生，增加CD4和CD8 T細胞表面活化標誌CD44表現量，並引發專一性的CD8 T細胞毒殺作用：提升 granzyme B在CD8 T細胞中表現量及殺死表現有 OVA257-264 peptide (MHC class Ι epitops) 的目標細胞。進一步以 OVA 與GMI進行免疫，可使 MO5 腫瘤 (表現 OVA 之 B16 黑色素瘤) 之生長受到抑制。 雖然 GMI 在小鼠體內具有免疫佐劑活性，但以 GMI 刺激小鼠骨髓細胞衍生之樹突細胞 (BMDC) 之結果顯示， GMI 不能刺激增加樹突細胞的表面分子 CD40、CD80、CD86、MHC II 的表現量，也無法讓樹突細胞分泌 TNF-α 。由本研究結果顯示，選殖自小孢子靈芝之GMI蛋白，藉由巨噬細胞的活化，可增加小鼠抵抗微生物感染的能力。 此外 GMI 可作為免疫佐劑，促使免疫反應趨向 Th1反應，並活化CD4和CD8 T細胞，引發專一性的細胞毒殺作用。

關鍵字

免疫調節蛋白質 ； 佐劑

並列摘要

The immunomodulatory protein GMI was recently cloned from Ganoderma microsporum. We have previously found that GMI could stimulate murine monocyte macrophage RAW264.7 cells to produce TNF-α and NO and enhanced phagocytosis. In this study, we first evaluated the potential antimicrobial activity of GMI against Listeria monocytogenes infection in mice, and we found that GMI pretreatment significantly reduced the bacterial loads in liver and spleen after L. monocytogenes infection. We also investigated whether GMI could function as an adjuvant in vivo. Mice immunized with the OVA antigen and GMI generated OVA-specific IgG1 and IgG2a, and after OVA restimulation, the splenocytes underwent proliferation, produced IFN-γ, and had enhanced expression of CD44 on both CD4 and CD8 T lymphocytes. GMI and OVA immunization also induced an OVA-specific CTL response, as demonstrated by higher levels of granzyme B expression in CTLs and specific lysis of OVA257-264 peptide-pulsed target cells. Furthermore, mice immunized with OVA and GMI were also protected from MO5 (OVA-transfected B16 melanoma) tumor challenge. Although GMI stimulates the activation of macrophages, interestingly we could not detect a stimulatory effect of GMI on bone marrow-derived dendritic cells (BMDCs). Taken together, our results demonstrated that GMI could stimulate an innate immune response which protected mice from L. monocytogenes infection as well as function as an adjuvant that promoted the activation of Th1 and CTL responses.

並列關鍵字

fungal immunomodulatory protein ； Ganoderma microsporum ； antimicrobia l ； Listeria monocytogenes ； adjuvant

參考文獻

10.林采蔆: 靈芝屬免疫調節蛋白 GMI 與 GFO-1 基因之選殖與 Pichia pastoris 之異源表現. 台灣大學; 2005.

2.Sasaki T, Arai Y, Ikekawa T, Chihara G, Fukuoka F: Antitumor polysaccharides from some polyporaceae, Ganoderma applanatum (Pers.) Pat and Phellinus linteus (Berk. et Curt) Aoshima. Chem Pharm Bull (Tokyo) 1971, 19(4):821-826.

3.Moradali MF, Mostafavi H, Ghods S, Hedjaroude GA: Immunomodulating and anticancer agents in the realm of macromycetes fungi (macrofungi). Int Immunopharmacol 2007, 7(6):701-724.

4.Takashi Kubota YA: Structures of Ganoderic Acid A and B, Two New Lanostane Type Bitter Triterpenes from Ganoderma lucidum (FR.) KarstHelv Chim Acta 1982, 65:611-619.

5.Kim HWaBKK: Biomedicinal triterpenoids of Ganoderma lucidum (Curt.: Fr.) P Karst.(Aphyllophoromycetideae) . Intl J Med Mushrooms 1999, 1:121-138.