利用原子力顯微鏡探討大蒜精油對類嗜中性球細胞機械特性的影響

Abstract

我們利用原子力顯微鏡(Atomic Force Microscopy)，量測類嗜中性球細胞經大蒜精油等不同藥物處理之後其彈性模數的變化，藉以比較具抗發炎效果的大蒜精油在發炎反應中對免疫主力嗜中性球的形變與遷移的影響，以探討細胞彈性模數的大小和細胞骨架的聚合與解離的難易程度的關聯性。 本實驗選用由人類骨髓癌細胞（HL-60）分化七天活化而成的類嗜中性球，因其具有嗜中性球的形變與遷移能力，且其存活力比嗜中性球強，因此常被用來研究嗜中性球的特性。嗜中性球是免疫系統中白血球的主力，其數量占各種白血球細胞總數的40~75%。當組織中的細胞受損或遭受病菌或病毒感染時，會擴散釋放出趨化分子，活化在血管中的嗜中性球，藉由形變從血管壁細胞間的縫隙間鑽出浸潤組織，然後循趨化分子的濃度梯度方向遷移到發炎之處，一路消滅病菌、病毒與受損的細胞。因此，嗜中性球的形變與遷移能力影響上述發炎反應的程度。 傳統上大蒜被認為是增進免疫能力、具有抗發炎反應的健康食品，許多文獻已報導其組成成分中的大蒜精油有抑制類嗜中性球遷移的效果。為驗證大蒜精油經由改變細胞骨架而抑制類嗜中性球的形變與遷移，並反映在個別細胞的彈性模數上，我們準備貼加了不同藥物的五組類嗜中性球： 10 μg/ml大蒜精油、5 μg/ml大蒜精油、對照組(不添加任何藥物)、細胞鬆弛素B（Cytochalasin B）10 μM與諾考達唑（Nocodazole）0.5 μM。因為細胞鬆弛素B已知能抑制細胞骨架聚合，而諾考達唑可以讓細胞骨架解離與聚合的作用減緩，僵化細胞骨架的現狀，所以可作為對照組檢驗細胞骨架與彈性模數的關係。 實驗時，我們利用原子力顯微鏡的探針逐一按壓上述五組類嗜中性球細胞，一共量測105個單一類嗜中性球，共738組表面的凹陷量與按壓力的關係。最後，我們根據赫茲模型（Hertz Model）擬合出各個細胞的彈性模數，亦即楊氏係數。結果，上述五組類嗜中性球的平均彈性模數分別為208.5 KPa、189.8 KPa、86.7 KPa、31.2 KPa和39.0 KPa，其中1 KPa = 103 N/m2。我們發現和未添加任何藥物的類嗜中性球的彈性模數相比，細胞鬆弛素B會減小類嗜中性球的彈性模數，而大蒜精油大幅增加類嗜中性球的彈性模數，且濃度愈濃、彈性模數愈大。此外，我們也發現類嗜中性球的彈性模數，在按壓之後有減小的趨勢，但加了諾考達唑的樣品其彈性模數的改變趨勢較小。 據此，我們推測大蒜精油有強化類嗜中性球細胞骨架強度的效果，不但增強類嗜中性球的彈性模數，而且也因而降低類嗜中性球變形從血管內鑽出浸潤到組織遷移的能力與數量。這個機制或許透露出大蒜能抗發炎反應的原因：阻止大量嗜中性球撲殺發炎區域內的正常細胞，避免引發更大發炎反應造成敗血症。此外，我們也看出類嗜中性球受外力刺激時，其彈性模數降低，變得柔軟而有助於形變與遷移的活化反應。雖然目前我們尚不明白大蒜精油增強嗜中性球細胞骨架強度的機制為何，不過本論文有趣的發現讓我們得以一窺發炎反應的堂奧，可供未來進一步研究嗜中性球細胞的彈性模數、細胞骨架、與不同藥物作用之間的關係。

While garlic is famous for its anti-inflammation effect, neutrophils are the most abundant type of white blood cells in mammals and form an essential part of the innate immune system. Both play two important roles in inflammation. Neutrophils are normally found in the blood stream. However, during the beginning phase of inflammation, neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation within minutes. They firstly deform their shapes and infiltrate through the blood vessels, then migrate through interstitial tissue, following chemical gradient signals (such as Interleukin-8 (IL-8) and C5a) in a process called chemotaxis. Since the deformation and migration of neutrophils highly involves the polymerization and depolymerization of their cytoskeleton, we assume the elastic modulus, Young’s modulus of neutrophil an effective index to quantify the influences of garlic oil on the mechanical properties of neutrophils. Using atomic force microscopy (AFM) in this research, we measured the elastic modulus, Young’s modulus, of a few groups of neutrophil-like cells which had been treated with garlic oil (GO), Cytochalasin B (CB) and Nocodazole (Noco), separately. By comparing the variation of the elastic moduli with different treatments, we study the influences of GO on the deformation and migration of neutrophils via cytoskeleton in the process of chemotaxis, based on the well known effects of CB and Noco on cytoskeleton. In the experiment, we pressed 105 neutrophil-like cells with the tip of an AFM and obtained 738 data of force-indent curves. Then, we analyzed the data with the Hertz model, As a result, we found that the statistically averaged elastic modulus of the neutrophil-like cells without any treatment, the control group, is 86.7 KPa (1 KPa = 103 N/m2). For comparison, the elastic moduli of the rest four groups of neutrophil-like cells under the treatment of 10 μg/ml GO, 5 μg/ml GO, CB and Noco are 208.5 KPa, 189.8 KPa, 31.2 KPa, and 39.0 KPa, respectively. Note that both CB and noco soften the cells while GO stiffens the cells; the higher the GO concentration, the stiffer the cells. In addition, the elastic modulus of neutrophil-like cells is found increasing with time. According to the results, we suggest that garlic oil would retard the deformation and migration of neutrophils in chemotaxis by stiffening the cytoskeleton of the cells, which reduces the response of an inflammation.