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研究生:蕭語涵
研究生(外文):Yu-Han Hsiao
論文名稱:高品質高純度之功能性粒線體萃取之微流道裝置最佳化設計
論文名稱(外文):Design of an Effective Micro-channel Device for High-quality and High-purity Functional Mitochondria Extraction
指導教授:王國禎
指導教授(外文):Gou-Jen Wang
口試委員:廖國智張瑞芝
口試委員(外文):Kuo-Chih LiaoJui-Chih Chang
口試日期:2016-06-24
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生醫工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:74
中文關鍵詞:粒線體治療健康功能性粒線體萃取微流道裝置
外文關鍵詞:Healthy mitochondria extractionMicrofluidic deviceMitochondrial therapy
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近年研究證實帕金森氏症、中風、第二型糖尿病、心血管疾病、小腦萎縮症等疾病,皆與粒線體突變或老化降低ATP合成效能有關。故有多種治療粒線體缺陷方法被提出,臨床用常用之方法乃是給予粒線體受損細胞抗氧化藥劑,以抑制過多氧化壓力之產生。然而,此治療方式對於已受損之粒線體無法恢復其功能,故利用細胞可藉內噬作用攝取功能完整之粒線體之特性,用以恢復或取代受損粒線體功能之粒線體移植術被提出。粒線體移植術之成功關鍵在於如何有效取得大量健康且功能性完整之粒線體,提供有效之移植。故本研究提出一可有效大量萃取功能性完整粒線體之微流道裝置,以利粒線體移植術發展之應用。
本研究首先利用SolidWorks軟體設計7種具不同工作區之微流道裝置,並利用COMSOL軟體進行流場模擬,接著以黃光微影製程、澆鑄成型、氧電漿表面處理及封裝,製作不同架構之微流道裝置;再以注射幫浦控制工作流體速度,推動細胞液通過微流道裝置,破壞細胞膜,並經由差動離心萃取健康無汙染之功能性粒線體。
實驗結果顯示,「單一間距,菱角端長破壞距之微流道裝置」可透過一系列長度為290 um與流道間距為5 um之平行微流道架構,使細胞產生擠壓進而破裂,具有較佳之破壞細胞膜效率與較長使用壽命。接著以西方點墨法及流式細胞儀分析所萃取之粒線體之蛋白質結構、純度與膜電位,並與控制組之市售粒線體萃取套組以及IP Lysis Buffer法進行比較。實驗結果驗證本研究提出之微流道裝置所萃取之粒線體蛋白具相當完整結構,且各蛋白質複合體含量皆高於市售萃取套組所萃取之粒線體,粒線體之純度可高達86.2 %以上,且其電子傳遞鏈膜電位功能正常。

Recent studies have revealed that cells can ingest mitochondria via endocytosis in order to restore mitochondrial function. This new mitochondria therapy approach can be used to cure particular neuropathy-related diseases. Hence, obtaining high quality and healthy mitochondria is vital in mitochondria-based disease therapy. In this study, we propose the use of a microfluidic device to extract healthy and chemical-free mitochondria. The device was designed using commercially available COMSOL software and fabricated using soft lithography to produce the polydimethylsiloxane (PDMS) device, while C2-GFP cells were used to verify the efficiency of the proposed microfluidic device. The membrane protein structure (Complex I-V and Tom 20) of the extracted mitochondria were examined by Western blotting, while the purity of the extracted mitochondria and the mitochondrial membrane potential were measured by 10-N- Alkyl acridine orange (NAO) or tetramethylrhodamine ethyl ester (TMRE) staining in flow cytometry. Experimental results reveal that the complex I–V expression of the extracted mitochondria with the proposed device was close to the expression of the conventional isolation kit. Furthermore, the results of the Tom 20 expression demonstrate that our device was able to extract similar amounts of mitochondria as the conventional kit. The purity of the extracted mitochondria was found to be above 86%, and it was evidenced from the mitochondrial membrane potential results that the mitochondria retained normal mitochondrial activity after extraction by the microfluidic device. Through transmission microscopy (TEM) it was observed that the mitochondrial membrane was intact, which confirms that isolation of the mitochondria via the proposed microfluidic device does not damage or harm the membrane. These results confirm that it is possible to obtain healthy mitochondria without structural damage or loss of activity with the proposed microfluidic device.

誌謝 i
摘要 iii
Abstract v
目錄 vii
圖目錄 xi
表目錄 xv
第一章 緒論 1
1.1前言與研究動機 1
1.2研究目的 2
第二章 文獻回顧 4
2.1粒線體概述 4
2.1.1 粒線體結構與功能 4
2.1.2粒線體功能損壞與相關疾病治療 7
2.1.3粒線體移植治療 10
2.2粒線體萃取 12
2.2.1 機械法 13
2.2.2 非機械法 15
第三章 研究材料與方法 19
3.1材料、藥品與儀器 19
3.1.1材料 19
3.1.2溶液配方 21
3.1.3 耗材與儀器 21
3.2.實驗方法 22
3.2.1物理性破壞細胞膜之微流道裝置製作流程 22
3.2.2物理性破壞細胞膜微流道裝置設計 23
3.2.3流場變化模擬 26
3.2.4黃光微影製程 26
3.2.5 PDMS微流道製程 28
3.2.6氧電漿表面改質處理 30
3.3細胞培養 31
3.4萃取粒線體 32
3.4.1以微流道裝置萃取粒線體 32
3.4.2以市售粒線體萃取套組萃取粒線體 34
3.4.3以裂解液萃取粒線體 35
3.5粒線體之蛋白結構測試 36
3.5.1 粒線體蛋白濃度定量 36
3.5.2以西方點墨法分析粒線體膜蛋白結構 38
3.5.3電泳條帶定量分析 42
3.6微流道萃取粒線體之功能性分析 43
3.6.1粒線體之純度分析 43
3.6.2粒線體膜電位分析 44
3.6.3 粒線體形貌分析 46
第四章 實驗結果與討論 47
4.1. 微流道裝置 47
4.1.1. COMSOL流場模擬分析 47
4.1.2. 微流道裝置製備 51
4.2 微流道裝置破壞細胞膜效能評估 55
4.2.1具破壞細胞膜最佳效率之微流道裝置 56
4.2.2 微流道裝置平均使用時間評估 59
4.2.3最佳化微流道裝置選擇 60
4.3 微流道裝置萃取之粒線體結構、純度與功能性評估 61
4.3.1粒線體之複合蛋白結構完整性與組成含量評估 61
4.3.2微流道裝置萃取之粒線體功能性評估 64
第五章 結論與未來展望 68
文獻回顧 71


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