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質之探討

Multivalency of Ling Zhi-8 coated Gold Nanoparticles Enhances the Immunomodulatory Response on Macrophages and Splenocytes As a Function of Size

葉鎮豪(Chen-Hao Yeh)

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(2010年)

https://doi.org/10.6342/NTU.2010.10777

摘要

園產品功能性成分進行奈米化處理，根據其粒徑、形狀以及材料性質而在調控功能性成分的消化吸收與生物可利用性上具有不同的利用價值。然而，就分子層次而言，粒徑不同之奈米化園產品與細胞間之交互作用仍屬未知。靈芝菌絲體中萃取出之免疫調節蛋白LZ-8 (Ling Zhi-8)，經酵母菌重組後以單體之形式存在，其分子量約為15 kDa。本研究中，利用靜電吸附原理 (electrostatic adsorption) 將LZ-8鍵結構築於奈米金粒子 (gold nanoparticles) 上，獲得大小分布於10奈米至100奈米的穩定LZ-8奈米金粒子，其LZ-8蛋白的鍵結量與奈米金粒子之大小成正比。
LZ-8奈米金粒子處理小鼠腹腔巨噬細胞可以提升LZ-8刺激巨噬細胞產生IL-1β與IL-10之能力，而流式細胞儀分析經GNP-LZ-8刺激之小鼠巨噬細胞，則顯示多價構築LZ-8於奈米金粒子上，可以提升LZ-8刺激細胞表面分子MHC class II, CD80, CD86的表現量增加，其中以30nm以上GNP-LZ-8的效果較為顯著。巨噬細胞的顆粒性增加則與奈米金粒子之大小成正相關。與同濃度的LZ-8測試組比較，LZ-8奈米金粒子處理小鼠脾細胞可以明顯提升脾細胞增生與刺激IL-2、INF-γ細胞激素分泌之能力，其中以50nm與30nm大小的GNP-LZ-8效果最為顯著；流式細胞儀分析經GNP-LZ-8刺激之小鼠脾細胞，顯示構築LZ-8於奈米金粒子上，可以提升LZ-8刺激CD3+與CD3-細胞表面CD25分子表現量增加與顆粒性上升，其中同樣以50與30nm的LZ-8奈米金粒子效果較佳。上述結果顯示，LZ-8奈米金粒子所引發的免疫調節活性在吞噬與非吞噬細胞中具有不同的規模依賴性。而奈米化處理除考量其生物傳遞特性外，其粒徑之大小，亦會影響LZ-8之生物活性，此一發現也許對奈米技術在園產品上的應用與園產品功能性之探討有所助益。

關鍵字

奈米金粒子 ； 靈芝 ； LZ-8 ； 免疫調節蛋白 ； 巨噬細胞活化 ； 脾細胞活化

並列摘要

Nano-scaled functional ingredients with different sizes, shapes and material properties have many applications in biological transport and hence the bioavailability. In spite of what has been achieved so far, cellular response between nano-scaled functional ingredient and target cells remains mostly unknown. Immunomodulatory protein named Ling Zhi-8 (LZ-8) extracted from the mycelia of Ganoderma lucidum has been cloned and expressed at Saccharomyces cerevisiae expression system as a recombinant protein rLZ-8. In the present study, suspensions of gold nanoparticles (GNPs) with stable surface rLZ-8 coating were prepared using electrostatic adsorption strategy. As size distribution from 10 to 100 nm, the quantity of rLZ-8 binding on GNPs was obviously correlated with particle size, which significantly enhanced the multivalency of rLZ-8 being conjugated on GNP surface.

Treatment of mouse peritoneal macrophages with GNP-LZ-8 significantly increased IL-1β cytokine production and surface CD80/86, MHC class II molecule expression as compare with the univalent free form LZ-8. Although the induction of cytokine and surface molecule activities was enhanced for all GNP-LZ-8, particle with larger size exhibited greater difference on both cytokine production and surface costimulatroy molecule expression on macrophages. Furthermore, side scatter (SSC) variation of macrophages after GNP-LZ-8 uptake revealing a granularity enhancement as well as an increase of GNP-LZ-8 size. GNP-LZ-8 treatment further indicated the enhancement of cell proliferation, IL-2 and INF-γ cytokine production as compare to univalent free form rLZ-8 on splenoctes. Moreover, free form rLZ-8 could activate the surface expression of CD25 on CD3+ splenocytes, while GNP-LZ-8 treatment elicited more significant upregulation on both CD3+ and CD3- cells. Among splenocyte treated with various sizes GNP-LZ-8, the greatest difference was observed in 30 and 50 nm GNP-LZ-8-treated cells. Taken together, these data implied the induction of diverse size-dependent immunomodulatory responses between phagocyte and non-phagocyte.

Base on these results, nano-size modification should no longer be consider only as a passive rule of delivery or bio-transportation, but could also play an active role in mediating biological effects. The findings presented here may assist in the design and development of nano-scaled functional ingredient.

並列關鍵字

gold nanoparticles ； Immunomodulatory protein ； Ganoderma lucidum ； Ling Zhi-8 ； macrophage activation ； splenocyte activation

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