立體紙基外泌體核酸萃取裝置應用於檢測不同微環境下癌細胞所釋放之外泌體與外泌體微小核醣核酸之表現量

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

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姓名

賴祈宏(Chi-Hung Lai) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

立體紙基外泌體核酸萃取裝置應用於檢測不同微環境下癌細胞所釋放之外泌體與外泌體微小核醣核酸之表現量
(Three-dimensional paper-based exosomal nucleic acid extraction device for detection of exosomes and exosomal miRNAs released by cancer cell cultured in different microenvironment)

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摘要(中)

由細胞分泌的外泌體(exosome)含有細胞特異的核酸及蛋白質，已被證實參與腫瘤的發展、擴散及轉移的發生過程。其中微核醣核酸(microRNA) 在許多疾病的診斷以及治療上為一個非常具有潛力的生物標靶分子，藉由和其目標信使核糖核酸(messanger RNA)的互補序列結合誘發mRNA的降解以調節基因的表現。因為這些特性與功能，外泌體非常有潛力成為疾病檢測用的工具之一，尤其是腫瘤外泌體可能成為癌症早期診斷或術後檢測的判斷依據。由於外泌體能夠穩定存在於人體體液之中且帶有來自母細胞的生物分子，相較於傳統的組織切片(traditional biopsy)，從體液分析外泌體這類的液態切片(liquid biopsy)不僅降低了侵入風險，也能夠更即時的反應出腫瘤當下的情況，雖然目前外泌體的應用還停留在科學研究，尚未普遍於疾病的常規檢測上，但由於外泌體於臨床應用的潛力，有發展出相當多的外泌體的應用技術。但現今對於外泌體的分離純化或是生物分子的分析皆需要專業的操作人員、特殊的藥品、費時及昂貴的儀器，鑒於以上原因，使得外泌體及核酸檢測比較無法於一般檢驗室操作，更無法落實於Point-of-care testing。
在此研究中，本實驗室欲發展一套外泌體核酸萃取裝置，結合本實驗室對於核酸萃取的研究，以取代過往對於外泌體純化及核酸萃取繁瑣的操作流程。我們在裝置表面改植上特殊蛋白質，作為分離外泌體的機制；接著在裝置表面塗布上核酸吸附顆粒，藉此分離外泌體中的核酸。我們利用此外泌體核酸萃取裝置於檢測HCT116人類結腸癌細胞株於不同微環境(microenvironment)下外泌體釋放量及核酸表現量。實驗結果顯示在相同的實驗條件下，酸性環境下具有較多外泌體釋放量，也能成功地辨識不同樣品之間核酸表現量。
最後也將裝置應用於慢性傷口組織液的臨床檢測上並得到初步有意義的結果。期望藉由此外泌體核酸萃取裝置能夠使外泌體及核酸檢測，能夠更貼近POCT的應用。

摘要(英)

A rapid, microfluidic- extraction device was developed for the extraction of the exosomal nucleic acids of HCT116 colon cancer cell cultured in different microenvironment. Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. And currently require laborious off-chip exosome separation, nucleic acid extraction prior to detection. With advances in point-of-care testing (POCT) has been explored for nucleic acid detection. We definitely need to develop an inexpensive, robust, easy-to-use and compatible with downstream nucleic acid detection to replace the traditional method that need the expensive infrastructure and time-consuming process. Here, we have extracted nucleic acid from cell culture medium and clinical chronic wound samples using exosomal nucleic acid extraction devices. Captured exosomes were analyzed by SEM and qNano to check the morphology and size, respectively. Assessing the amount of exosomes captured using P-ELISA using antibodies conjugated to horseradish peroxidase to produce a colorimetric readout was accomplished within 30 min. Nucleic acid can be extracted by exosomal nucleic acid extraction devices and analyzed by real-time polymerase chain reaction (qPCR) to provide information for disease management. As a whole, we have already developed a exosomal nucleic acid extraction device to extract nucleic acid from HCT116 cultured in different microenvironment and chronic wound samples from the different patients in the different stages that employ an user-friendly procedure and encourage a quicker adoption of this technology in POCT.

關鍵字(中)

★ 外泌體
★ 微小核糖核酸
★ 反轉錄及時聚合酶鍊式反應
★ HCT116癌細胞
★ 紙基微流道
★ 慢性傷口

關鍵字(英)

★ exosome
★ microRNA
★ qPCR
★ HCT116 cancer cell line
★ Paper microfluidic
★ Chronic wound

論文目次

摘要 i
ABSTRACT iii
誌謝 iv
目錄 v
圖目錄 ix
表目錄 xiii
一、緒論 1
1.1 研究背景 1
1.2 論文架構 3
二、文獻回顧 4
2.1 核酸介紹 4
2.1.1 核酸分子 4
2.1.2 去氧核醣核酸 5
2.1.3 核醣核酸 6
2.1.4 微小核醣核酸 8
2.2 分子生物檢測平台 9
2.2.1 聚合酶鏈鎖反應(Polymerase chain reaction, PCR) 9
2.2.2 即時定量聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction, qPCR)檢測miRNA原理 11
2.3 酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA) 15
2.4 細胞外囊泡(Extracellular vesicles) 介紹 16
2.4.1 外泌體(Exosome) 17
2.5 Exosome分離方式 20
2.5.1 超速離心法(Ultracentrifugation-based isolation techniques) 20
2.5.2 沉澱法(Precipitation) 21
2.5.3 免疫法(Immunoaffinity capture-based techniques) 22
2.5.4 粒徑篩析法(Size-based isolation techniques) 22
2.6 Paper-based device 24
2.6.1 Paper-based immunoaffinity device 25
2.6.2 Paper-based ELISA 26
2.6.3 Paper-based nucleic acid extraction device 27
三、實驗藥品、儀器及方法 28
3.1 實驗藥品 28
3.1.1 細胞培養 28
3.1.2 外泌體純化濃縮 28
3.1.3 Paper-based immunoaffinity device 28
3.1.4 Paper-based ELISA 29
3.1.5 Paper-based nucleic acid extraction device 29
3.1.6 核酸萃取 29
3.1.7 即時聚合酶鏈式反應 30
3.2 儀器設備 31
3.3 實驗方法 32
3.3.1 細胞解凍 32
3.3.2 細胞培養 33
3.3.3 細胞培養於不同pH細胞培養液 34
3.3.4 細胞冷凍保存 36
3.3.5 微小核糖核酸萃取 37
3.3.6 外泌體純化濃縮 39
3.3.7 qNano 40
3.3.8 Paper-based immunoaffinity device 42
3.3.9 Paper-based ELISA 44
3.3.10 Paper-based nucleic acid extraction device 46
3.3.11 反轉錄及時聚合酶鍊式反應(qRT-PCR) 48
四、結果與討論 52
4.1 建立立體紙基外泌體核酸萃取裝置 52
4.1.1 Paper-based immunoaffinity device 應用於捕獲HCT116癌細胞之exosome 54
4.1.2 Paper-based nucleic acid extraction device應用於萃取HCT116 exosome之核酸 60
4.2 HCT116癌細胞培養於不同微環境 64
4.2.1 利用P-ELISA檢測HCT116癌細胞於不同微環境中exosome釋放量 65
4.2.2 HCT116癌細胞培養於不同微環境中外泌體微小核醣核酸表現量69
4.3 立體紙基外泌體核酸萃取裝置應用於慢性傷口(chronic wound)檢體 76
五、結論 83
六、未來展望 85
七、參考文獻 86
八、附錄 92
8.1 Paper-based nucleic acid extraction device 實驗參數 92
8.2 利用P-ELISA檢測慢性傷口組織液 95

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2020-7-21

推文