金針菇免疫調節蛋白FIP-fve活化小鼠樹突細胞調節T細胞免疫反應及作為腫瘤疫苗佐劑之活性

Immunomodulatory Protein FIP-fve of Flammulina velutipes Activates Murine Dendritic Cells to Regulate T Cell Immune Responses and Acts as a Cancer Vaccine Adjuvant

巫沛樺(Pei-Hua Wu)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/碩士(2016年)

https://doi.org/10.6342/NTU201602333

摘要

金針菇 (Flammulina velutipes) 為常見的食用菇菌之一，也被證明具有多種保健功效。FIP-fve為從金針菇分離出的免疫調節蛋白。文獻指出FIP-fve具有活化小鼠脾細胞、巨噬細胞及外周血單核球細胞等多種免疫細胞之能力，但目前尚無針對樹突細胞之相關研究。本研究之目的為探討FIP-fve是否能夠活化小鼠樹突細胞並調節T細胞免疫反應，以及探討FIP-fve作為樹突細胞疫苗佐劑之潛力。
首先，體外試驗中發現 FIP-fve 可以刺激小鼠骨髓衍生性樹突細胞 (bone marrow-derived dendritic cells, BMDCs) 表現 CD40、CD80、CD86 及 MHC (major histocompatibility complex) class II，且能誘導樹突細胞產生 TNF-α, IL-1β, IL-6 , IL-12及IL-10 等細胞激素，在 TLR4 缺陷小鼠中則發現 FIP-fve 刺激樹突細胞之效果明顯受到抑制，證明其活化路徑很可能是透過細胞表面之 TLR4 受體。另一方面，研究發現 FIP-fve 需要透過抗原呈獻細胞來活化 T 細胞，在 T 細胞與樹突細胞同時存在下，FIP-fve 可使T細胞產生大量 IFN-γ，且能使T細胞之活化分子標誌 CD25 及 CD69 表現量上升。利用對 OVA 具特異性之 DO11.10 小鼠的 CD4+ T 細胞與經過FIP-fve活化之樹突細胞共同培養，發現經FIP-fve活化之樹突細胞可以增強 OVA 特異性 CD4+ T 細胞增生，且能使 T 細胞產生 TH1 免疫反應。在體內試驗的部分發現以FIP-fve 與 OVA 刺激之樹突細胞作為疫苗免疫小鼠，可以誘發小鼠體內產生 OVA特異性免疫反應，另外在 MO5 黑色素瘤小鼠模式中，以 FIP-fve 作為佐劑可以增強罹癌小鼠產生抗腫瘤免疫反應以抑制腫瘤生長。藉由上述結果證明FIP-fve 活化之樹突細胞能夠調節 TH1 細胞分化，增強抗原特異性 T 細胞免疫反應，且作為樹突細胞疫苗佐劑可以增強疫苗的抗腫瘤效果。

關鍵字

金針菇 ； 免疫調節蛋白 ； 骨髓衍生性樹突細胞 ； 樹突細胞疫苗 ； 疫苗佐劑

並列摘要

Golden needle mushroom (Flammulina velutipes) is one of the most common edible mushrooms and has been demonstrated to have many health benefits. FIP-fve is an immunomodulatory protein isolated from F. velutipes and was proved to have an ability to activate many immunocytes such as murine splenocytes, macrophages and hPBMCs. The aim of this study was to demonstrate whether FIP-fve could activate murine dendritic cells and further regulate T cell immune responses, and to investigate its activity as a vaccine adjuvant.
The results indicated that FIP-fve could up-regulate surface marker expression of CD40, CD80, CD86, and MHC calss II, and induce cytokines production of TNF-α, IL-1β, IL-6, IL-12, and IL-10 by BMDCs in vitro. We then found that the ability of FIP-fve to activate DCs was remarkably limited in TLR4 knock out mice. These results demonstrated that FIP-fve activated murine DCs mainly through the TLR4 pathway. Furthermore, FIP-fve needed the presence of antigen-presenting cells (APCs) to activate T cell. In the presence of DCs, FIP-fve could induce great amount IFN-γ production and up-regulate the expression of CD25 and CD69 by T cells. Moreover, we isolated CD4+ T cell from OVA-specific DO11.10 mice and cultured with OVA pulsed FVE-treated DCs and OVA antigen. Our data showed that FVE-treated DCs enhanced OVA-specific CD4+ T cell proliferation and IFN-γ production, and demonstrated that FIP-fve could induce TH1 response. On the other hand, we used FIP-fve as a vaccine adjuvant, and found that FIP-fve could induce OVA-specific immune response in vivo. Immunization of mice with OVA pulsed FVE-treated DCs induced antitumor response in a murine MO5 melanoma model and suppressed the tumor growth. According to these findings, we demonstrated that FIP-fve could regulate TH1 cell differentiation through DC maturation and boost antigen-specific immune responses, and enhanced the effect of antitumor response by acting as a dendritic cell vaccine adjuvant.

並列關鍵字

Flammulina velutipes ； immunomodulatory protein ； bone marrow-derived dendritic cells ； dendritic cell vaccine ； vaccine adjuvant

參考文獻

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