- 學位論文
薑黃與柳橙胞外囊泡特性與其細胞生物活性之研究
Characterization of turmeric and citrus-derived extracellular vesicles and investigation of biological activities in cell models
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摘要
植物來源性胞外囊泡 (Plant-derived extracellular vesicles，簡稱PDEVs) 如其名為從植物分離出來的胞外囊泡，雖然PDEVs相關的研究剛起步沒有多少年，但已有研究指出其具有多種生物活性。近年來，從食用蔬果到植物根莖皆有發表成功分離出胞外囊泡的成果。薑黃是物種Curcuma longa的根莖，常用於烹飪和草藥中，為咖哩橘黃色的色素來源之一，薑黃的研究在學界已持續多年，已知這類橘黃色植物根莖具有多種生物活性，包括抗發炎、抗癌等，然而關於薑黃來源性胞外囊泡 (TEVs) 的研究相當少。此外，柑橘類水果常見於日常生活飲食中，以富含維生素 C 並具抗氧化及抗發炎功效聞名，已有研究成功從中分離出柑橘類胞外囊泡 (CEVs)，但從不同品種柑橘類分離出的胞外囊泡之間是否有差異則不明確。因此在本次研究中，我們目標從薑黃與不同品種柳橙中分離出胞外囊泡，並分析其性質和生物活性。藉由切向過濾法成功分離出 TEVs，CEVs 則是利用超高速離心與蔗糖梯度離心法純化得到，後續我們測定粒徑分布與界達電位來了解不同胞外囊泡間的奈米性質差異。分析不同成熟度來源的 TEVs 中發現了小片段RNA，且未成熟 TEVs 薑黃素(Curcumin, CU)含量較高。在細胞實驗中，TEVs 可以抑制大腸癌細胞株的增殖，然而CEVs 則否，同時從成熟薑黃中分離出的 TEVs 在抑制癌細胞生長的效果比未成熟薑黃的效果好。除此之外，成熟來源的 TEVs 在不殺死巨噬細胞的情況下，能夠抑制經 LPS 誘導後的 NO 產生量，但再製成空載體後這些現象就消失。這些結果得出 TEVs 具有抗癌與抗發炎應用的潛力，我們也發現薑黃的成熟差異及內容物在本研究中影響 TEVs 的生物活性。在未來，影響 TEVs 功能的關鍵因素及 CEVs 其他的生物活性需要更深入的研究探討。
並列摘要
Plant-derived extracellular vesicles (PDEVs) are kind of extracellular vesicles isolated from plants, which present the various bioactivities. Turmeric is the rhizomes of Curcuma longa which is commonly used in cuisine and herbs with various bioactivities, including anti-inflammatory, anticancer, etc. However, there is little research on the turmeric derived extracellular vehicles (TEVs). Citrus fruits are commonly found in the daily diet, and are known to be rich in vitamin C and have antioxidant and anti-inflammatory properties. Citrus derived extracellular vesicles (CEVs) have been successfully isolated, but it is not clear whether there are differences between the extracellular vesicles isolated from different citrus species. Therefore, in this study, we aimed to isolate EVs from turmeric and different varieties of citrus, and analyzed their characteristics and biological functions. The TEVs were successfully isolated by tangential flow filtration (TFF), and the CEVs were purified by ultra-centrifugation and sucrose gradient centrifugation. Particle size distribution and zeta potential were subsequently measured to understand the difference between different extracellular vesicles. Moreover, small-sized RNAs were found in TEVs, and TEVs from different maturity have differences in curcumin content. In cell experiments, TEVs could inhibit proliferation of colorectal cancer cells, but CEVs don’t have same effects. Interestingly, we noted that TEVs isolated from mature turmeric couldn’t suppress the growth of cancer cells. In addition, mature TEVs were able to suppress NO production after LPS induction without killing macrophages, but these phenomena disappeared when TEVs were remanufacture as vectors. These results concluded that the TEVs have the potential ability to apply in anticancer and anti-inflammatory, and we also found that different maturity and contents of TEVs affected the biological activity in this study. In the future, we would elucidate the key factors that contribute to the functions of TEVs, and the other biological functions of CEVs need more investigation.
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