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  • 學位論文

靈芝免疫調節蛋白 Ling Zhi-8 活化小鼠骨髓衍生性樹突細胞及刺激免疫細胞產生介白素-17A 之探討

Immunomodulatory Protein Ling Zhi-8 of Ganoderma lucidum Activates Mouse Bone Marrow-derived Dendritic Cells and Stimulates IL-17A Production in Immune Cells

指導教授 : 許輔
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摘要


靈芝自古以來被認為是上藥之藥,性平無毒,具有多方面的效果,其中的活性成分及其功效已受到相當的關注。研究顯示純化自 Ganoderma lucidum 之靈芝免疫調節蛋白 Ling Zhi-8 (LZ-8) 能刺激樹突細胞、巨噬細胞及 T 淋巴細胞的活化或增生,促使細胞產生細胞素及提升細胞表面分子的表現量。在抗原呈現細胞的部份,前人研究發現 LZ-8 能透過細胞表面的 TLR4 受器活化人類樹突細胞,但對小鼠巨噬細胞的活化卻與 TLR4無關。因此本論文使用重組 LZ-8 蛋白 (rLZ-8 ),以 TLR4 缺陷型小鼠骨髓衍生性樹突細胞進行實驗,希望能釐清前人相矛盾的結果。本研究結果顯示雖然受 rLZ-8 刺激後,相較於原生型骨髓衍生性樹突細胞,TLR4 缺陷型樹突細胞之細胞素產生量會顯著降低,但是 rLZ-8 仍能促使 TLR4 缺陷型小鼠骨髓衍生性樹突細胞表面分子表現量提昇,且無論表面是否具 TLR4 受器,骨髓衍生性樹突細胞皆能與 FITC 螢光標定的 rLZ-8 蛋白結合。此外,TLR4 缺陷型樹突細胞受 rLZ-8 刺激後,也仍具有促使 T 細胞增生之活性。上述結果顯示 rLZ-8 於細胞表面的受器並非只侷限於單一的 TLR4 受器。 針對 T 細胞反應的部分,除了已知的第一型輔助性 T 細胞 (type 1 helper T cell, TH1) 反應以及調節性 T 細胞 (regulatory T cell, Treg) 反應,在本研究中亦發現 rLZ-8 能刺激免疫細胞產生介白素-17A (interleukin-17A, IL-17A)。進一步探討顯示,於小鼠脾臟細胞中,rLZ-8 所誘發的 IL-17A,主要可能來自表型特徵為 CD3e- CD4- B220- CD11c- NK1.1- 的先天性 IL-17 產生細胞,且這群細胞亦無法特異性辨識 α-GalCer: CD1d 複合物,因此目前結果仍無法判定其為何種細胞。不過直接以 rLZ-8 刺激分離出的 CD4+ T 細胞,亦可以促使 IL-17A 的產生,雖然胞內染色結果並不明顯,但由於 rLZ-8 刺激 IL-17 產生之能力會受維他命 A 酸所抑制,顯示 rLZ-8 的刺激除了能促使先天性 IL-17A 產生細胞活化,可能也與 TH17 的反應相關,後續仍需以 real-time PCR 確認受 rLZ-8 所活化之 IL-17A 產生細胞所具有的特性。此外,未來的研究可繼續深入探討 rLZ-8 的刺激,與 TH1、Treg 相關反應以及 IL-17A 產生細胞之間的關係,進一步釐清 LZ-8 蛋白於免疫細胞上的受器。

並列摘要


Reishi, also known as Ling-Zhi, are biosidiomycetous fungi. Because of it poceesed various biological activeties in different aspects and with little or no side effects, this fungi were used as traditional herb for over two millennia in Asia. Ling Zhi-8 (LZ-8), an immunomodulatory protein purified from Ganoderma lucidum has been revealed to induce cell proliferation, cytokine production and surface marker expression in various immune cells including dendritic cells, macrophages and T lymphocytes. Previous report showed that LZ-8 induced cytokines production in human monocyte-derived dendritic cells through Toll-like receptor 4 (TLR4). However, the others researches claimed that LZ-8 could activate TLR4-/- mouse peritoneal macrophages. Hence, we used recombinant LZ-8 (rLZ-8) and mice bone marrow-derive dendritic cells (BM-DCs) to elucidate the conflicting results. Although cytokine produtions induced by rLZ-8 were reduced in TLR4-/- BM-DCs in our study, surface markers expression still enhanced on TLR4-/- BM-DCs in response to rLZ-8 stimulation. In addition, FITC-labeld rLZ-8 could bind to BM-DCs regardless of wether the cells equipped TLR4 or not. Moreover, rLZ-8 activated TLR4-/- BM-DCs induced T cell proliferation as WT BM-DCs did. Thus we suggested that TLR4 was not the only receptor for rLZ-8 on the surface of APCs. On T lymphocytes, stimulation of LZ-8 has been reported to induce TH1 cells or Treg cells response. In the present study, we proposed that mice splenocytes or isolated CD4+ T cells stimulated with rLZ-8 also resulted in IL-17A production. In mice splenocytes, rLZ-8 induced IL-17A producing cells principally belonged to non T cell population. These cells might be a kind of innate IL-17A producing cell which was characterized as CD3e-, CD4-, B220-, CD11c-, NK1.1- and could not react with α-GalCer: CD1d complex. Although IL-17A intracellular staining of splenocytes or isolated CD4+ T cells stimulated with rLZ-8 were not remarkable, the production of IL-17A did detect by ELISA in the culture supernatant; Besides, IL-17A production in CD4+ T cells directily stimulated with rLZ-8 was inhibited in the presence of retinoic acid. These resulte indicated that rLZ-8 might induce TH17 response, in addition to activating innate IL-17A producing cells. Further studuies by real-time PCR should be carried out to confirm the properties of rLZ-8 inducde IL-17A producing cells. The relationship between TH1, Treg and TH17 response induced by rLZ-8, and the unknown receptor other than TLR4 can still be topics of our future studies.

參考文獻


葉鎮豪。2010。靈芝蛋白 Ling Zhi-8 鍵結於奈米金粒子促進巨噬細胞及脾細胞免疫調節能力與其粒徑依賴性之探討。國立台灣大學生物資源暨農學院園藝學研究所碩士論文。
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高雅玲(2015)。市售靈芝產品品管指標之研究〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2015.02058

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