利用奈米技術從細胞模數及遷移行為探討大蒜精油及其含硫成分對類嗜中性球細胞遷移活性的影響

Abstract

發炎細胞「嗜中性球細胞」在發炎反應中的遷移行為一直是個被廣泛研究的基本生物問題，因 此，探討大蒜的抗發炎反應機制也成為重要的研究課題。然而，由於單一細胞難於操控，過去很難 從物理角度觀察嗜中性球在遷移行為中的物理變化並量化大蒜濃度對其影響。近幾年，我們和合作 的生物學家好奇大蒜精油的抗發炎反應機制可能和發炎細胞內細胞骨架的組裝與拆解有關。為此， 我們已先自製一台簡易型原子力顯微鏡，可以初步量測單一細胞的彈性模數。此外，我們也建立微 流道技術，已可初步追蹤單一細胞的遷移軌跡。因此，我們希望在這些技術之上，加入大蒜精油這 個新元素，以其不同濃度為實驗的控制變因，繼續研究此一課題。 在本計劃中，我們擬結合操控單一細胞的最新奈米技術：雷射鑷夾、原子力顯微鏡和微流道， 藉由觀察發炎細胞的兩個相關物理指標：彈性和速度，量測大蒜精油中的三種主要硫化合物成分分 別對於發炎細胞的彈性模數和遷移速度的影響，以量化這些變化和硫化合物濃度的關係。然後，我 們將比對細胞骨架與彈性模數和遷移行為的關連性，探討大蒜精油在發炎細胞抗發炎反應機制中的 角色與影響。 在本三年計畫中，我們擬採用癌化後比較強韌的「類嗜中性球細胞」作為實驗對象，並利用已 知具有拆解細胞骨架功能的細胞鬆弛素、nocodazole，和大蒜精油同步進行對照實驗。第一年，我們 將改善自製雷射鑷夾與原子力顯微鏡的功能，量測類嗜中性球的彈性模數，以量化不同濃度大蒜精 油對彈性模數之影響。第二年，我們將利用微流道技術量測類嗜中性球的遷移速度，以量化不同濃 度大蒜精油對遷移速度之影響。第三年，我們將利用此一平台，進一步分析大蒜精油中的三種主要 硫化合物成分：二烯丙基硫化物(dially sulfide, DAS)、二烯丙基二硫化物(diallyl disulfide,DADS)與二 烯丙基三硫化物(diallyl trisulfide, DATS)分別在不同濃度下對細胞彈性模數與遷移速度的影響。最 終，我們期望找出大蒜精油中這些硫化合物成分的最佳比例組合，以有助於未來新型抗發炎反應藥 物的研發。

The migration of neutrophiles in an inflammatory response is a fundamental biology problem which has been wildly studied. The anti-inflammatory mechanism of garlic oil is thus an important topic for investigation. However, it is difficult to observe the physical behavior of a neutrophile in migration and its influence caused by garlic oil because of the lack of the technique to manipulate a single cell. Recently, we are interested in the effect of garlic oil on cytoskeleton. In order to sudy this topic, we have built a simplfied atomic force microscope capable of measuring the biological Young’s modulus of a single cell. Additionally, we have developed a microfluidic system for single cells tracking. With these techniques, we propose to add garlic oil as a new parameter to study its influence at various concentrations on single neutrophiles in this project. In this proposal, we plan to integrate our latest nanotechniques, including optical tweezers, atomic force microscope, and microfluidics, to explore the relationship between the elasticity and the moving velocity of a single neutrophile as well as its influence caused by garlic oil treatment. Specifically, we will measure the biological Young’s modulus and the migrating velocity of a single neutrophile as a function of garlic oil with various concentrations. Further, we will analyze such effects caused by each of the three major sulfide components of garlic oil. In this three-year project, we will adopt neutrophil-like cell as our biological target for its toughness to experiment. We plan to use cytochalasin as a negative control and nocodazole as a positive one. In the first year, we will conduct precise measurement of biological Young’s modulus by strengthening our atomic force microscope. In the second year, we are to determine the migrating velocity of single neutrophil-like cells in a microchannel by utilizing the pattern recognition technique in image processing. Note that the two physical behaviors are mainly affected by the following three major sulfide components in garlic oil: dially sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In the third year, we will analyze the effects due to each of the three components by using the platform techniques established in the first two years. We expect to obtain an optimal combination of the three sulfide components for anti-inflammatory drug development in the future.