Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98468
標題: 高純度粒線體大量萃取之離心式裝置
Development of a Novel Centrifugal Device for Massive Extraction of High-quality Mitochondria
作者: 陳頌慈
Sung-Tzu Chen
關鍵字: 粒線體治療
健康粒線體萃取
離心式微流道裝置
黃光微影製程
Mitochondrial therapy
healthy mitochondria extraction
centrifugal microfluidic device
photolithography
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摘要: 在大多數的真核細胞中,都擁有粒線體這一個具有獨特功能的胞器。除了做為「細胞的能量工廠」通過呼吸作用產生三磷酸腺苷(adenosine triphosphate, ATP)之外,粒線體還參與其他生理功能,諸如細胞分化、鈣離子的儲存、細胞代謝與細胞凋亡等過程。而當粒線體無法釋出足夠的能量或細胞有氧代謝過程中產生的活性氧化物質(Reactive oxygen species, ROS),皆會讓細胞產生病變。研究證實糖尿病、心血管疾病、帕金森氏症、老化導致的神經病變及多種癌症,皆與粒線體突變或老化降低ATP合成效能有關。目前有多種治療粒線體缺陷的方法被提出,如刺激粒線體新生、置換治療、基因治療等,但受損的粒線體是不可修復的,因此,使用從人體細胞提取健康之粒線體來恢復或取代病變之粒線體被提出,這類粒線體療法高度依賴大量且功能性良好的粒線體供應,故從細胞中有效提取健康之粒線體是非常需要的。 本文提出一可大量萃取高純度粒線體之離心式微流道裝置,以利粒線體療法發展之應用。本裝置主要分為兩部分,一為簡易的黃光微影製程及PDMS澆鑄來大量製造破壞細胞膜之微流道裝置,再搭配便於實驗室使用之離心式裝置,組裝後藉由離心力即可將大量懸浮細胞推過微流道裝置,破壞細胞膜,再經由差速離心法(differential centrifugation)萃取高純度無汙染之粒線體。 實驗結果顯示,「直線輻射狀之微流道結構」具有較佳之破壞細胞膜效率。並進一步以西方點墨法、流式細胞儀與TEM來分析所萃取之粒線體之蛋白質結構、純度、活性以及型態完整度,並與控制組之市售粒線體萃取套組以及全細胞裂解法進行比較。實驗結果發現利用離心式微流道裝置可在30分鐘內大量萃取高純度粒線體,並保持其之活性與功能。
In most eukaryotic cells, the mitochondria are unique organelles that act as the cell's 'power plant', producing adenosine triphosphate (ATP) through respiration. In addition, mitochondria also participate in other physiological functions such as cell differentiation, calcium storage, cell metabolism, and apoptosis, amongst others. When mitochondria are unable to release sufficient energy or when reactive oxygen species (ROS) are produced during cellular aerobic metabolism, the cells undergo pathological changes. Studies have confirmed that diabetes, cardiovascular disease, Parkinson's disease, aging-induced neuropathy, and multiple cancers are all associated with mitochondrial mutations or aging that reduce the efficiency of ATP synthesis. There are currently many methods for treating mitochondrial deficiencies, such as mitochondrial renewal, replacement therapy, gene therapy, etc.. Since damaged mitochondria are not repairable, using healthy mitochondria extracted from healthy human cells to replace damaged ones and restore mitochondrial functions is the trend of mitochondrial therapy. This type of mitochondrial therapy is highly dependent on the supply of a large number of well-functioning mitochondria; hence efficient extraction of healthy mitochondria from cells is highly sought after. This study proposes a centrifugal microfluidic device that can extract large quantities of high-purity mitochondria to facilitate the development and implementation of mitochondrial therapy. The fabrication of the device is divided into two major parts: one is a photolithography process, and the other is PDMS casting; which enables the production of a large number of microfluidic devices designed to disrupt and rupture the cell membrane. The device is then used in conjunction with a laboratory centrifuge to lyse the cells by forcing them through the microfluidic channels. The high-purity, contaminant-free mitochondria suspension is then obtained via differential centrifugation of the cell lysis. The experimental results show that the straight radial microchannel structure has the greatest cell lysis efficiency. Moreover, the structure, purity, activity, and integrity of the extracted mitochondria by the proposed device were examined by Western blotting, flow cytometry, and TEM. The results demonstrated that high-purity mitochondria could be extracted in large amounts using the centrifugal microfluidic device within 30 minutes, while also maintaining mitochondrial activity and function.
URI: http://hdl.handle.net/11455/98468
文章公開時間: 2021-08-16
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