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系統識別號 U0002-2807201114332100
中文論文名稱 防止HepG2肝癌細胞貼附之力學探討
英文論文名稱 A Mechanical Study on the anti-attachment of HepG2 Tumor Cells
校院名稱 淡江大學
系所名稱(中) 機械與機電工程學系碩士班
系所名稱(英) Department of Mechanical and Electro-Mechanical Engineering
學年度 99
學期 2
出版年 100
研究生中文姓名 徐智文
研究生英文姓名 Chih-Wen Hsu
學號 698370136
學位類別 碩士
語文別 中文
口試日期 2011-06-30
論文頁數 83頁
口試委員 指導教授-楊龍杰
委員-楊台鴻
委員-李慧芳
委員-吳文中
委員-康尚文
中文關鍵字 轉移  ECIS  細胞貼附  肝門靜脈 
英文關鍵字 metastasis  ECIS  cell adhesion  portal vein 
學科別分類 學科別應用科學機械工程
中文摘要 癌細胞透過入侵循環系統而達到轉移之過程,與細胞的脫離以及貼附機制有關,為了探討此一現象,本研究分為靜態實驗與動態實驗兩部分。靜態實驗利用電子細胞基質阻抗判斷技術(Electric Cell-substrate Impedance Sensing, ECIS)製作阻抗感測晶片,使用戊二醛交聯之明膠微圖案,吸引細胞貼附於電極上方,並監測HepG2細胞之形態與貼附變化。而藉由每隔1小時所得到的細胞貼附面積與阻抗變化之關係,可成為動態實驗參考之依據。
動態實驗藉由PDMS微流道內壁之設計,改變細胞的貼附條件,觀察癌細胞欲入侵之前的貼附行為以及阻塞之可能,並將HepG2肝癌細胞注入微流道內,模擬癌細胞經過微血管之情形。利用RIE以電漿轟擊PDMS,改變微流道內壁之粗糙度,進而改變細胞貼附之能力。癌細胞進入微流道時所表現之不同狀況,可反應該環境對癌細胞透過循環系統達到轉移的成功與否,也表示微血管於某種狀況下,阻塞不通的情形,並探討癌細胞透過肝門靜脈進入肝臟時,癌細胞貼附以及阻塞之情形,進而協助癌症之預防及治療,如飲食、藥物治療、基因治療等研究。
英文摘要 For metastasis of tumor cell invade into the circulatory system, it have to do with tumor-cell of detachment and attachment. This work presents the first part of a new framework for preventing the tumor-cell of carcinoma in situ transition from one organ to others. Using an ECIS (electric cell-substrate impedance sensing) chip coated with glutaraldehyde (GA)-crosslinked gelatin patterns suitable for cell attachment, the author monitor the cell adhesion situation not only by optical microscope but also by electrical means. This cell-culture experiment with 1-hour time resolution so far demonstrates that the attachment moment for HepG2 on gelatin surface is no longer than 4 hours after the cell dosing and these tumor cells cannot stay on GA-crosslinked gelatin surface for more than 7 hours.
The second part of experiment is dynamic. The author design an experiment and a microfluidic chip for investigating the relationship between the metastasis and the surface morphology of blood vessels. Using RIE (reactive ion etch) to change the inner wall morphology of PDMS. To different inner wall of PDMS roughness, the ability of cell adhesion is different. When tumor-cell through the microchannel, it may pass or get stuck. It’s mean that capillary may plugged by tumor-cell, or still smooth. Finally, to explore the adhesion of tumor-cell and blocking when tumor-cell into the liver through the hepatic portal vein. It can assist in the prevention and treatment of cancer, such as diet, drug, gene therapy research.
論文目次 目錄
中文摘要..................................................................................................... I
英文摘要....................................................................................................II
目錄...........................................................................................................III
圖目錄........................................................................................................V
表目錄...................................................................................................... IX
第一章 緒論...............................................................................................1
1-1 微機電系統...........................................................................1
1-2 研究動機...............................................................................2
1-3 文獻回顧...............................................................................6
1-3-1 癌細胞於循環系統之轉移.........................................6
1-3-2 肝硬化與癌細胞及循環系統的關係.........................8
1-3 研究目的.............................................................................13
1-4 研究架構.............................................................................15
第二章 肝癌細胞貼附之靜態實驗........................................................16
2-1 實驗設計.............................................................................16
2-2 電阻抗式感測晶片製作與改良.........................................18
2-3 實驗量測與結果.................................................................23
第三章 肝癌細胞貼附之動態實驗........................................................29
3-1 量測實驗設備架設說明.....................................................29
3-2 仿微血管之微流道設計.....................................................30
3-3 流動實驗設計.....................................................................32
3-4 微流道製作.........................................................................35
3-5 PDMS 微流道之表面粗糙化..............................................42
3-6 實驗量測結果.....................................................................63
第四章 結論與未來建議........................................................................67
4-1 本文貢獻之彙整..................................................................67
4-2 未來工作與建議..................................................................69
參考文獻...................................................................................................70
附錄A RIE 以氧氣電漿蝕刻Parylene N 之蝕刻率..............................76
附錄 B RIE 以氧氣電漿蝕刻Parylene C 之蝕刻率.............................77
附錄C 個人著作 “THE MINIUMUM TIME ESTIMATION FOR
INITIATING TUMOR-CELL ATTACHMENT”發表於國際傳感器會議
之全文。...................................................................................................79

圖目錄
圖1-1 癌細胞的轉移過程。....................................................................5
圖1-2 循環性癌細胞的生產過程與血管的SEM 圖。..........................7
圖1-3 共軛焦顯微鏡拍攝貼附於肺臟微血管之腫瘤細胞。................7
圖1-4 腫瘤細胞貼附於肝血竇(左圖)以及入侵至組織(右圖)。..........8
圖1-5 肝臟示意圖。................................................................................9
圖1-6 肝小葉示意圖。..........................................................................10
圖1-7 肝纖維化示意。(A)正常的肝血竇,藍色為星狀細胞,紫色為
Kupffer 細胞;(B)肝臟損傷後,星狀細胞增生以及週遭產生膠原組織
(黃色區域)。............................................................................................12
圖1-8 不同表面粗糙度對於親疏水性及細胞貼附的影響。..............14
圖1-9 細胞隨時間增加而增加貼附區域示意。..................................14
圖1-10 論文架構。................................................................................15
圖2-1 單一HeLa cell 細胞在不同接種時間貼附情形之量測結果。17
圖2-2 本實驗之感測電極與實體。......................................................18
圖2-3 感測晶片製作流程。..................................................................19
圖2-4 感測晶片製作流程(續)。...........................................................20
圖2-5 parylene C 包覆之電路板與晶片。.............................................22
圖2-6 細胞量測後電路板袘k之比較。..............................................22
圖2-7 感測電極編號。..........................................................................24
圖2-8 CH3 電極上方HepG2 細胞貼附之情形。.................................24
圖2-9 細胞貼附於CH3 電極上的電訊號。........................................25
圖2-10 CH3 電極上方HepG2 細胞貼附之情形。...............................25
圖2-11 細胞貼附於CH3 電極上的電訊號。.......................................26
圖2-12 CH3 電極上細胞貼附面積與阻抗變化量之關係。................27
圖2-13 CH5 電極上細胞貼附面積與阻抗變化量之關係。................28
圖3-1 實驗架設示意。..........................................................................29
圖3-2 實驗架設。..................................................................................30
圖3-3 微流道3D 模型。.......................................................................31
圖3-4 流道中間的觀察區 (單位:μm)。............................................32
圖3-5 SU-8 光阻厚度相對曝光劑量之對照表。.................................36
圖3-6 本實驗室真空烘箱(型號:DOV-30)。........................................37
圖3-7 PDMS 氧氣電漿表面處理化學狀態表示圖。...........................37
圖3-8 微流道製作流程圖。..................................................................39
圖3-9 完成顯影的SU-8 母模。............................................................40
圖3-10 OM 拍攝之SU-8 微流道。.......................................................40
圖3-11 SU-8 微流道觀察區之膜厚(量測區域為圖3-8 的A-A 線段)。
...................................................................................................................41
圖3-12 SEM 拍攝之PDMS 微流道。...................................................41
圖3-13 PDMS 微流道與Glass 接合。..................................................41
圖3-14 倒立式顯微鏡拍攝之微流道觀察區。....................................42
圖3-15 電漿蝕刻PDMS 之SEM 圖。.................................................43
圖3-16 本實驗室之反應離子蝕刻機(型號:SAMCO RIE1-C)。.....44
圖3-17 氟氧混合電漿蝕刻PDMS 13 min 之結果(參數為O2: 13
sccm;CF4: 37 sccm;80W)。...............................................................45
圖3-18 氧氣電漿蝕刻PDMS 14 min 之結果(參數為O2: 50 sccm;
80W)。.....................................................................................................46
圖3-19 Ra 與蝕刻時間的關係(左圖為CF4 O2 之混合氣體,右圖為O2
氣體)。.....................................................................................................47
圖3-20 表面接觸角量測儀。................................................................58
圖3-21 混合氣體電漿蝕刻PDMS 之時間與表面接觸角的關係。...59
圖3-22 CF4 與O2 混合氣體電漿蝕刻PDMS 之接觸角量測。...........60
圖3-23 O2 電漿蝕刻PDMS 之時間與表面接觸角的關係。...............61
圖3-24 O2 電漿蝕刻PDMS 之接觸角量測。.......................................62
圖3-25 微流道實驗影像擷取位置。....................................................63
圖3-26 微流道實驗影像擷取。............................................................64
圖A-1 載玻片上Parylene N 之量測點。.............................................76
圖A-2 氧氣電漿蝕刻Parylene N 之關係圖。.....................................76
圖B-1 載玻片上Parylene C 之量測點。..............................................77
圖B-2 氧氣電漿蝕刻Parylene C 之關係圖。......................................77
圖B-3 氧氣電漿蝕刻Parylene C 之關係圖。......................................78

表目錄
表1-1 癌細胞轉移前與轉移後之五年相對生存率。............................4
表2-1 CH3 電極之細胞貼附面積估算與阻抗變化量。......................27
表2-2 CH5 電極之細胞貼附面積估算與阻抗變化量。......................27
表3-1 電漿蝕刻PDMS 之參數。.........................................................43
表3-2 PDMS 表面粗糙度之參數。.......................................................47
表3-3 PDMS 以混合氣體電漿蝕刻後之表面形貌。...........................48
表3-4 PDMS 氧氣電漿蝕刻後之表面形貌。.......................................53
表3-5 CF4 與O2 混合氣體電漿蝕刻PDMS 之表面接觸角。.............59
表3-6 O2 電漿蝕刻PDMS 之表面接觸角。.........................................61
表A-1 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率。.........................76
表B-1 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率(100W,50sccm)。
...................................................................................................................77
表B-2 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率(80W,30sccm)。
...................................................................................................................78


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