許多研究指出機能性脂質(functional hydrocarbons)之攝取與人類健康的相關性，而一部分的機能性脂質也對人體有調節免疫系統之功效。其中，二十碳五烯酸(eicosapentaenoic acid, EPA)屬於ω-3脂肪酸，能從魚油當中攝取；花生四烯酸(arachidonic acid, AA)屬於ω-6脂肪酸，富含於肉類與植物油中。角鯊烯(squalene)、角鯊烷(squalane)與姥鮫烷(pristane)為不溶於水的脂質，能夠從魚肝油取得。研究指出，從飲食中獲取適量的ω-3和ω-6脂肪酸，能夠幫助免疫系統的調節，其攝取比例對於免疫反應具有平衡作用；此外，攝取過多短鏈姥鮫烷脂質將導致體內嚴重的自體免疫反應。有必要進一步釐清機能性脂質當中，ω-3和ω-6脂肪酸與脂質碳鏈長度對於免疫反應所造成的影響。
樹突狀細胞(dendritic cells, DCs)於身體的先天性與後天性免疫中扮演重要的角色，我們規劃利用C57BL/6小鼠的骨髓樹突狀細胞(bone marrow-derived dendritic cells, BMDCs)模型來評估機能性脂質對於免疫調節的影響。然而，因為上述機能性脂質幾乎不溶於水的緣故，我們透過乳化技術將其製備成奈米乳液，藉此能讓不溶於水的物質均勻分散於水中、增加與細胞的接觸機會。粒徑分析顯示，以音波振動處理搭配擠壓程序，能夠將乳液粒徑調整至奈米等級粒子(100 nm)。我們將這些含有機能性脂質之奈米乳液刺激樹突狀細胞，於結果中並未造成顯著的影響。接著我們以脂多醣內毒素(lipopolysaccharide, LPS)來活化樹突狀細胞，藉此評估這些機能性脂質對樹突狀細胞活化的影響。結果中發現，二十碳五烯酸降低了功能性分子標誌(functional marker)的表現與發炎細胞激素(proinflammatory cytokine)的生產。同時也注意到花生四烯酸在樹突狀細胞活化方面卻有相反的影響，例如花生四烯酸提升了其CD86的表現。角鯊烯與角鯊烷雖然擁有相同的碳鏈長度但是因雙鍵的飽和度不同，透過兩者比較得知其飽和度差異並不會改變對樹突狀細胞的影響。角鯊烷與姥鮫烷為飽和脂肪，但姥鮫烷的碳鏈較短，結果也指出碳鏈長短差異對樹突狀細胞沒有明顯的改變。接下來，我們透過卵清蛋白(ovalbumin, OVA)作為抗原，以探討樹突狀細胞同時攝取抗原與機能性脂質的影響。結果顯示，二十碳五烯酸不僅降低了功能性分子標誌的表現，也減少了樹突狀細胞的數量。相反地，花生四烯酸提升了其CD86與MHCII的表現並顯著的增加樹突狀細胞的數量。除此之外，於角鯊烯、角鯊烷與姥鮫烷的結果中顯示，飽和度差異與碳鏈長短對同時攝取抗原的樹突狀細胞沒有明顯的影響。
我們進一步驗證二十碳五烯酸與花生四烯酸對於體內樹突狀細胞的影響。我們將卵清蛋白搭配二十碳五烯酸和花生四烯酸以肌肉注射方式接種於C57BL/6小鼠體內。結果顯示樹突狀細胞會被誘發並聚集於引流淋巴結(draining lymph node, dLN)當中。值得注意的是，二十碳五烯酸會抑制樹突狀細胞的匯聚，而花生四烯酸卻能促進其聚集到引流淋巴結。此體內實驗分析可呼應了前面骨髓樹突狀細胞實驗中兩者對樹突狀細胞相反的影響，也能更了解二十碳五烯酸與花生四烯酸的在體內的作用。
總歸而言，我們的研究著重探討含有機能性脂質之奈米乳液的免疫調節功效。透過對樹突狀細胞的評估，能更加瞭解當中對免疫功能造成的影響，也能夠幫助探討這些機能性脂質對健康的關聯性。

Many studies described the correlation between functional hydrocarbon and human health. Some of the functional hydrocarbons exert biological effect that is beneficial to the immune system. Eicosapentaenoic acid (EPA) belongs to ω-3 fatty acids and is the component of fish oil; arachidonic acid (AA) belongs to ω-6 fatty acids and is abundant in vegetable oils and meat. Squalene (C30H50), squalane (C30H62) and pristane (C19H40) can be found in the liver oil of shark. Squalene and squalane have the same carbon chain length. Squalane and pristane are completely saturated while squalene is unsaturated. Studies demonstrated the dietary intake of ω-3 and ω-6 fatty acids improves the regulation of immune system. Besides, an overdose of pristane could lead to severe autoimmune response. To clarify the biological effects from the functional hydrocarbons, we investigate the effects of ω-3 and ω-6 fatty acids to immune responses. We also need to understand the influence of carbon chain length and saturation level on the immune responses.
Dendritic cells (DCs) play critical roles in both innate and adaptive immune responses. For this reason, we employed C57BL/6 murine bone marrow-derived dendritic cells (BMDCs) as the in vitro DC model. However, the hydrocarbons mentioned above are poorly soluble in water. We introduced an emulsifying system to formulate these hydrocarbons into emulsions. Thereby, the hydrocarbons could disperse in water so as to facilitate the interactions with DCs. Notably, by sonication and extrusion, we can control the size of the emulsion at about 100 nm, which is consistent with the definition of nanoemulsion. We first treated DCs with these hydrocarbon-based nanoemulsions. However, the DC activation was not significantly altered. We then stimulated DCs with these nanoemulsions plus lipopolysaccharide (LPS), a molecule on the cell membrane of bacteria, to study whether these hydrocarbons inhibit LPS-induced DC activation. The results show EPA inhibited the expression of functional marker and the production of proinflammatory cytokine. However, we found AA exert opposite effects on CD86 expression compared with EPA. The results of squalene and squalane show the saturation level barely alter the influence on DCs. In addition, the results of squalane and pristane also prove the carbon chain length did not alter the influence to DCs. After that, we stimulated DCs with ovalbumin (OVA) to mimic the interaction between DCs and antigen. EPA inhibited the DC number and the expression of functional markers. In contrast, AA increased the number of DCs and the expression of functional markers such as CD86 and MHCII. The results from squalene, squalane and pristane show the saturation level and carbon chain length did not alter the influence to OVA-treated DCs, which are consistent with the LPS-treated DCs.
We further evaluate their effects on DCs in vivo. After intramuscular injection of OVA in C57BL/6 mice, the result shows the DCs recruited to draining lymph nodes (dLNs) within 24 hr postinjection. Remarkably, EPA attenuated the recruitment of DCs to dLNs while AA enhanced the recruitment. This opposite effect of EPA and AA are correlate with the BMDC experiments.
Overall, our study focuses on the immunomodulatory activity of the nanoemulsions based on the functional hydrocarbons. Through the evaluation of DC function, the influence of these functional hydrocarbons to the immune responses could be deeply understood and help us to explore their health benefits.

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