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標題: 奈米級黏土片及其衍生物在粒線體分離方法上之應用與探討
Isolation of Mitochondria Using Membranous Nanoscale Clay Platelets and Their Derivatives
作者: 蔡詠任
Tsai, Yung-Jen
關鍵字: 粒線體;mitochondria;奈米矽片;NSP
出版社: 生命科學系所
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粒線體是調控細胞功能與存活的核心胞器,生物學家早在1946年便發展出從細胞分離細胞粒線體的方法,利用低張(hypotonic)蔗糖水溶液使細胞吸水膨脹後,接著進行均質作用(homogenization)打破細胞膜,最後離心分離粒線體。然而低張溶液的環境與均質作用也會如同破壞細胞膜一樣地對粒線體造成傷害,如果可以增加均質細胞的效率,或許能提高分離粒線體的效率與粒線體的活性。運用奈米科技提供了可行的方向,材料學家發展出奈米黏土片(nanoscale clay platelets,NSP),又稱為奈米矽片(nanosilicate platelets)的相關技術。NSP是一種極薄的片狀材料,表面帶負電荷,具有貼附細胞膜或細菌細胞壁的特性;而將NSP表面離子以界面活性劑進行取代,即衍生出NSQ。NSQ若能貼附細胞膜並且以界面活性劑影響細胞膜的結構,如此一來就能增加細胞均質的效率。首先,trypan blue dye確認細胞經過NSP與NSQa的處理確會造成細胞膜破損;而利用場發式掃描電子顯微鏡(field-emission scanning electron microscope,FE-SEM)也可觀察到細胞因為NSP or NSQa貼附而造細胞外觀的改變。利用NSP與NSQa處理細胞後,分別於低張與微低張溶液以細胞均質器(dounce)均質細胞並分離粒線體,發現經過材料處理的組別都能比控制組獲得更多粒線體,但是NSQa處理的組別卻造成粒線體活性下降;考慮到不穩定的界面活性劑是否會從NSQ解離出來,後續實驗使用透析純化後的NSQ。比較NSP、數種經透析純化的NSQ以及未脫層的蒙脫土(montmorillonite,MMT)後,我們發現細胞經過NSP及NSQc處理後粒線體的分離效率及粒線體活性都有顯著的上升,而造成粒線體分離效率及活性降低的NSQa則提供了進一步對界面活性劑進行改質的方向。

Mitochondria are central organelles regulating biological functions and living of cell. Biologists developed a method to isolate mitochondria from cells since 1946. This process uses hypotonic sucrose buffer to make cells turgid, then homogenized cells to break the plasma membrane, finally the mitochondria are isolated by centrifuge. However, in this process the hypotonic environment and the homogenization damage the mitochondria when breaking the plasma membrane. We believe if we can improve the efficiency of homogenization, it is possible to increase the efficiency of mitochondrial isolation and the activity of isolated mitochondria. Nanotechnology is a workable solution because material scientists have developed nanoscale clay platelets (NSP, also called nanosilicate platelets). NSP are ultrathin and membranous materials with negative ionic charge on the surface and they are able to adhere to plasma membrane or bacterial cell wall. By replacing the surface ions of NSP with a surfactant, a hybrid derivatives called NSQ are created. It is assumed that NSQ can adhere to plasma membrane and effect membrane structure, thereby improving the efficiency of homogenization. It was confirmed by trypan blue staining that the plasma membrane broken after NSP and NSQ treatment. It was also observed under field-emission scanning electron microscope (FE-SEM) that the cell morphology changed because of NSP and NSQ adhering to the cell membrane. After NSP and NSQ treatment, the cells were homogenized using a dounce homogenizer and a hypotonic or minor hypotonic buffer to isolate mitochondria. It was found that the groups under NSP and NSQ treatment yielded more mitochondria, but the groups under NSQ treatment showed diminished mitochondrial activities. Concerning of deionization of unstable surfactants from NSQ, we purified NSQ by dialysis. Comparing PBS control with NSP, four kinds of NSQ and natural montmorillonite (MMT), it was found that efficiency of isolation and activities of mitochondria were both improved after both NSP and NSQc treatment. NSQa which diminished efficiency of isolation and activities of mitochondria also provided us with a direction for further research.
其他識別: U0005-1508201215533600
Appears in Collections:生命科學系所

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