臺灣博碩士論文加值系統

　　目前，大部份的體外細胞研究採用二維培養，即讓細胞貼於培養皿底部平面，進行細胞培養與實驗研究，例如，在細胞遷移研究中，傷口癒合分析(Wound healing assay)是一個標準的分析方法，此分析是利用二維培養方式作為基礎，將細胞培養於培養皿中，當細胞生長至佈滿此細胞培養皿狀態之下，利用微量吸取管(tip)將細胞培養皿中間處刮出一條線，此線會將細胞群與細胞群做分隔，並觀察細胞群與細胞群之間往彼此方向做爬行的情形。此外眾多研究發現，細胞在人體中的影響因子是來至四面八方，並不是單純受到單一平面的方向影響，由此假設人體環境屬於立體空間(三維環境)，並非是屬於單純平面環境中(二維環境)，另外也多數研究指出細胞在二維環境培養與三維空間培養做比較，發現細胞在三維體外環境下培養，不論是細胞型態、抗藥性與受影響能力、侵襲與遷移能力等都接近細胞於體內微環境的表現，因此後期的許多研究開始朝向三維環境培養著手，將細胞培養於膠體中，為了讓細胞生存環境更接近於體內微環境，其中有學者提到三維微流道設計與應用可以保留三維環境培養的優點，另外更能模擬較精細的體內微環境，並且提高更多元的分析與應用，因此本研究會朝著三維微流道概念去做研究。
　　本研究將利用基質膠(Matrigel)作為細胞支架於微流道生醫晶片中，讓細胞處於模仿人體三維環境中進行傷口癒合分析之研究，本研究分為四大部分，第一部分為生晶片開發，利用軟硬體去設計與製作微流道生醫晶片的模具，接著將聚二甲基矽氧烷(PDMS)倒入於製作完成的模具中進行翻模，最後將翻好的成品與150 µm厚度的方形蓋玻片，放入於氧電漿機(O2 plasma)進行表面改質，才能讓彼此作黏合。第二部分，利用微流道生醫晶片預先設計好的兩端培養槽，添加不同或相同養分的培養基（DMEM），產生養分濃度梯度，去模擬當癌細胞於體內缺乏養分時，是否會往較營養區域作遷移，並將結果與相同養分環境下，癌細胞遷移能力做比較。第三部分，利用剛剛所提到的養分濃度梯度為基礎，再添加四種不同濃度的介白素Interleukin-6 (IL-6)與癌細胞於培養槽中，添加介白素(IL-6)用意是要去刺激癌細胞，並利用螢光顯微鏡觀測於不同介白素(IL-6)濃度去影響癌細胞之遷移速度能力。第四部分，最後將三種不同數量的纖維母細胞(HEL299-RFP)於培養於其中一側的培養槽中，癌細胞則是放於另一側，並利用螢光顯微鏡下觀測在不同數量的纖維母細胞影響癌細胞之下，癌細胞遷移能力的變化。
　　本研究開發創新之「在三維環境下之細胞傷口癒合分析」技術，利用此裝置能有效的幫助研究人員，並提供有效的三維環境下之癌細胞環境測試，所開發的三維微流道生醫晶片能用於生物醫學實驗室研究的實際應用。

　　Currently, most of the in vitro cell researches are based on two-dimensional (2D) culture. That is, biological cells are attached on the bottom surface of culture dish for culturing cells. For example, in the research of cell migration, wound healing assay is a standard ana-lytical method based on 2D culture. Cells are cultured in a culture dish and proliferate to confluence. A wound is then scraped by a microtip and separates the cell population. Cells move toward each other and cell migration is then observed. In addition, many studies have found that the influence factors of cells in human body are not only affected by one single dimension; but come from all directions. Therefore, the cells inhibit in a three-dimensional (3D) environment in native environment. On the other hand, cells express different properties, e.g., cell mor-phology, drug resistance, invasion, and migration, when they are cul-tured in 3D and 2D environment. Recently, biologists suggest that in vitro 3D culture can mimic the in vivo microenvironment.

　　In this study, a microfluidic chip was developed and Matrigel was used as cell scaffold. Cells cultured in 3D environment and invaded through a Matrigel filled microchannel in order to perform 3D wound healing assay. The research is divided into four parts. The first part is to develop a microfluidic biochip. By using a mold of microfluidic bi-ochip, polydimethylsiloxane (PDMS) chip could be replicated and bonded to a glass substrate by oxygen plasma treatment. In the second part, a nutrient gradient was generated in the microchannel of the bi-ochip. Cancer cells were guided and invaded to the direction with high concentration nutrient. The ability of cell invasion was studied. In the third part, cancer cells were stimulated by different concentrations of interleukin-6 (IL-6). The invasion ability was investigated and corre-lated to the IL-6 concentration. In the fourth part, due to fibroblasts can stimulate the invasion ability of cancer cells, cancer cells were co-cultured with fibroblasts and the invasion ability was studied.
 
　　In this study, "Three-dimensional wound healing assay" was de-veloped. This microfluidic biochip provides an effective approach for cancer cell invasion.

Keywords: Three-Dimensional cell culture; Microchannel biochip; In vitro cell culture; Cancer cell migration; Influence of Fibroblasts

指導教授推薦書
論文口試委員審定書
致謝 iii
中文摘要 iv
英文摘要 vi
目錄 viii
圖目錄 x
第一章 研究背景、動機及目的 1
1-1研究背景與動機 1
1-2研究目的 2
2-1細胞轉移 4
第二章 文獻回顧 4
2-2 IL-6刺激細胞，觀測於二維進行細胞Wound healing之變化 6
2-3正常細胞與癌細胞 7
2-4二維與三維環境比較 9
2-5文獻總結 11
第三章研究方法及實驗步驟 12
3-1-1生醫晶片設計 14
3-1-2生醫晶片製作 14
3-1-3生醫晶片應用說明 16
3-2子宮頸癌細胞於相同養分環境下之遷移能力 19
第四章 研究結果 24
4-1子宮頸癌細胞於相同養分環境下之遷移能力 24
4-2子宮頸癌細胞於不同養分梯度環境下之遷移能力 25
4-3利用不同濃度IL-6刺激子宮頸癌細胞之遷移能力比較 28
4-4不同數量的纖維母細胞誘導子宮頸癌細胞之遷移能力比較 33
第五章 結論 41
參考文獻 43
附錄 47


圖目錄
圖 1-1 Wound healing 前置作業[1]......................................................................... 2
圖 1-2 Wound healing 示意圖[2] ............................................................................... 2
圖 2-1 癌症腫瘤轉移過程[5]..................................................................................... 5
圖 2-2 二維環境觀測有無添加 IL-6 於細胞做 Wound healing [8].......... 6
圖 2-3 二維環境添加不同濃度 IL-6 於細胞做 Wound healing [9].......... 7
圖 2-4 癌細胞與纖維母細胞研究[15].................................................................... 8
圖 2-5 纖維母細胞與癌細胞及其他細胞關係[6]............................................. 8
圖 2-6 二維與三維細胞培養比較 [17]................................................................. 9
圖 2-7 二維與三維與三微微流道系統優缺點 [23]...................................... 10
圖 3-1 研究架構圖........................................................................................................ 13
圖 3-2 三維生醫晶片之設計..................................................................................... 14
圖 3-3 微流道生醫晶片製作過程........................................................................... 15
圖 3-4 微流道生醫晶片成品圖................................................................................ 16
圖 3-5 細胞培養箱.......................................................................................................... 17
圖 3-6 實驗流程圖.......................................................................................................... 18
圖 3-7 HeLa-GFP 於相同養分環境下之流程模擬圖..................................... 19
圖 3-8 HeLa-GFP 於不同養分環境下之流程圖............................................... 20
圖 3-9 IL-6 刺激子宮頸癌細胞於不同養分環境下之流程圖................... 22
圖 3-10 不同數量的 HeLa-GFP 誘導子宮頸癌細胞之流程圖 ................. 23
圖 4-1 HeLa-GFP 於相同養分環境結果螢光圖............................................... 25
圖 4-2 HeLa-GFP 於相同養分環境結果簡化圖與多組統計曲線圖...... 25
圖 4-3 HeLa-GFP 於不同養分環境結果螢光圖............................................... 26
圖 4-4 HeLa-GFP 於不同養分環境結果簡化圖與多組統計折線圖...... 26
圖 4-5 將 HeLa-GFP 於相同與不同養分環境之遷移能力結果比較..... 27
圖 4-6 添加 IL-6(12.5ng/ml)刺激 HeLa-GFP 結果螢光圖.......................... 28
圖 4-7 IL-6(12.5 ng/ml)刺激 HeLa-GFP 結果簡化與多組統計折線圖. 29
圖 4-8 添加 IL-6(25ng/ml)刺激 HeLa-GFP 結果螢光圖............................. 29
圖 4-9 IL-6(25 ng/ml)刺激 HeLa_GFP 結果簡化圖與多組統計折線圖30
圖 4-10 添加 IL-6(50 ng/ml)刺激 HeLa-GFP 結果螢光圖......................... 31
圖 4-11 IL-6(50 ng/ml)刺激 HeLa-GFP 結果簡化圖與多組統計折線圖
................................................................................................................................................. 31
圖 4-12 IL-6(0、12.5、25、50 ng/ml)刺激 HeLa-GFP 結果比較........... 32
圖 4-13 HEL299-RFP(2,500)誘導 HeLa-GFP 遷移能力結果螢光圖..... 34
圖 4-14 HEL299-RFP(2,500)誘導 HeLa-GFP 結果簡化圖......................... 34
與多組統計折線圖......................................................................................................... 34
圖 4-15 HEL299-RFP(5,000)誘導 HeLa-GFP 遷移能力結果螢光圖..... 35
圖 4-16 HEL299-RFP(5,000)誘導 HeLa-GFP 結果 ........................................ 35
簡化圖與多組統計折線圖.......................................................................................... 35
圖 4-17 HEL299-RFP(0、2,500、5,000)誘導 HeLa-GFP 結果比較...... 37
圖 4-18 將 HeLa-GFP 與 HEL299-RFP 進行不同條件 TGF-β1 分析.. 38
圖 4-19 添加不同濃度 IL-6 刺激 HeLa-GFP 進行 Transwell 實驗....... 38
圖 4-20 添加不同濃度 IL-6 刺激 HeLa-GFP 進行 Transwell 實驗統計
................................................................................................................................................. 39
圖 4-21 HeLa-GFP 進行與不同數量 HEL299-RFP 進行 Transwell 實驗
................................................................................................................................................. 39
圖 4-22 HeLa-GFP 進行與不同數量 HEL299-RFP 進行 Transwell 實驗
統計....................................................................................................................................... 40

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