臺灣博碩士論文加值系統

嗜中性白血球(neutrophil)對於宿主對抗外來微生物並且產生免疫反應的過程中，扮演了重要的角色。嗜中性白血球可藉由產生大量活性氧化物(reactive oxygen species, ROS)以及釋放顆粒球(granule)內酵素，產生殺菌的活性。先前研究發現受檳榔萃取液(areca nut extract, ANE)或檳榔嚼塊(areca quid, AQ)內所含成分黃樟素(safrole)處理的嗜中性白血球，其吞噬能力(phagocytosis)、釋放超氧化物(superoxide)以及殺菌等能力都會下降。顯示檳榔嚼塊成分會降低嗜中性白血球殺菌的功能，但對於檳榔嚼塊成分是否會影響嗜中性白血球細胞內活性氧化物的產生以及釋放顆粒球的能力還沒有深入的研究。本論文乃探討檳榔嚼塊成分對於嗜中性白血球細胞內活性氧化物產生和釋放顆粒球能力之影響。將自健康人周邊血液中所純化的嗜中性白血球，分別以不同濃度的ANE以及檳榔嚼塊內所含成分，包括檳榔鹼(arecoline)、黃樟素(safrole)和丁香酚(eugenol)處理後，以propidium iodide (PI)染色及流式細胞儀(flow cytometer)測定對細胞存活度的影響。此外，經檳榔成分處理後之嗜中性白血球，再以含或不含細胞鬆弛素B (cytochalasin B) 和N-formyl-methionyl-leucyl-phenylalanine (fMLP)之液體(CB/fMLP)活化刺激後，利用2'',7''-dichlorofluorescin diacetate來偵測細胞內活性氧化物的產生狀態，並進一步偵測骨髓過氧化酵素(myeloperoxidase, MPO)的釋放，做為釋放顆粒球之標記，來探討檳榔嚼塊成分對釋放顆粒球的影響。結果顯示以25 microgram/ml的ANE或2.5 mM的eugenol處理下會造成約50%的嗜中性白血球死亡。而arecoline和safrole在所有測試濃度之下，對於細胞存活度並無顯著的影響。此外，以檳榔嚼塊內所含成分分別處理嗜中性白血球30分鐘，並不影響細胞內活性氧化物產生，然而約在ANE (1.56 microgram/ml)、arecoline (0.016 mg/ml)、safrole (1.25 mM)或eugenol (1 mM)濃度下，嗜中性白血球被CB/fMLP活化而細胞內產生活性氧化物的能力則下降50 %。隨著ANE、arecoline、safrole和eugenol濃度增加，嗜中性白血球被CB/fMLP活化而釋放MPO酵素情形也有下降的趨勢。未被CB/fMLP活化的嗜中性白血球，在10 mg/ml的arecoline濃度下嗜中性白血球釋放MPO能力上升，而ANE、arecoline、safrole和eugenol在其他測試濃度下則無明顯的影響。本論文研究結果顯示檳榔嚼塊內的成分會影響嗜中性白血球正常活化功能，抑制嗜中性白血球被CB/fMLP活化產生細胞內活性氧化物量和釋放顆粒球的能力。由此可知，檳榔嚼塊內成分能藉由抑制嗜中性白血球的細胞內產生活性氧化物和釋放顆粒球的能力，進而影響到其殺菌的功能。

Neutrophils play important roles in host defense against external microorganisms and the process of immune response. Neutrophils carry out antibacterial activity by production of large amount of reactive oxygen species (ROS) and release of enzymes of granule. In previous studies, exposure of neutrophils to areca nut extract (ANE) or safrole which is one of the components of areca quid (AQ), reduces the phagocytosis, the production of superoxide anion and the bactericidal activity of neutrophils. The results suggest that the components of AQ reduced the antibacterial activity of neutrophils. Whether components of AQ can affect production of intracellular ROS and degranulation in neutrophils has not yet been studied. The goal of this study was to investigate the effects of components of AQ on intracellular ROS production and degranulation in neutrophils. Neutrophils that were isolation from peripheral blood of healthy volunteers were treated with different concentrations of the components of AQ, including ANE, arecoline, safrole and eugenol. The effects of these components on viability of neutrophils were determined by propidium iodide (PI) staining followed detected by the flow cytometer. Neutrophils were also treated with or without cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (CB/fMLP) after incubation with the components of AQ. Intracellular ROS production of neutrophils was determined using 2'',7''-dichlorofluorescin diacetate. The effects of components of AQ on granule release from neutrophils were determined using myeloperoxidase (MPO) activity assay. The results showed that the viability of neutrophils was approximately 50 % when 25 microgram/ml of ANE or 2.5 mM of eugenol was used. However, concentrations of arecoline or safrole need in this study did not affect the viability of neutrophils. Incubation of neutrophils with these components for 30 minutes did not affect the production of intracellular ROS. ANE (1.56 microgram/ml), arecoline (0.016 mg/ml), safrole (1.25 mM) or eugenol (1 mM) decreased approximately 50% of the intracellular ROS production of neutrophils that were treated with CB/fMLP. The ability of MPO release from CB/fMLP activated-neutrophils was inhibited by ANE, arecoline, safrole or eugenol in a dose-dependent manner. Arecoline (10 mg/ml) enhanced the release of MPO from neutrophils that were not treated with CB/fMLP, whereas other concentrations of arecoline, ANE, safrole or eugenol examined did not affect MPO release from neutrophils. The results in this thesis showed that various components of AQ affected normal activity function of neutrophils and inhibited intracellular ROS production and degranulation by neutrophils that were treated with CB/fMLP. In conclusion, the components of AQ inhibited intracellular ROS production and degranulation by neutrophils, that may lead to reduction of bactericidal activity of neutrophils.

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