組織工程用支架表面之細胞遷移行為研究

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姓名

張恆鐘(Heng-Chung Chang) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

組織工程用支架表面之細胞遷移行為研究
(Cell migration research in the surface of scaffold for tissue engineering)

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摘要(中)

組織工程用支架提供細胞貼附生長的環境，支架之材料選擇、外型結構與孔洞大小等優劣評估方式，是以最終細胞組織之生化檢測值為依據，然而細胞在支架表面生長與遷移行為也可視為一種評估方式，因此本研究以細胞生長與遷移過程之影像資訊，使用影像處理方式分析擷取出細胞在支架表面上的位移方向、移動速度等細胞生長訊息，以評估支架材料選擇、結構設計之好壞，也可進ㄧ步地探討生物反應器注流之剪應力對細胞生長的影響。
實驗中支架材料使用組織工程中常用的生醫材料:聚乳酸-甘醇酸(Poly-Lactic-Co-Glycolic Acid, PLGA)；細胞之選擇為老鼠軟骨細胞株(Immortalized Rat Chondrocyte, IRC)。為觀察活細胞在支架上分佈影像，採用基因改質之腺病毒(Adenovirus)感染細胞，使細胞帶有綠螢光蛋白(Green-Fluorometer-Protein, GFP)，進而能在倒立式顯微鏡下觀察。在本研究可分為三部份：1. 靜態培養研究：觀察細胞在支架上生長情況，得到細胞在支架上佈滿的過程。2. 細胞遷移研究：採用靜態培養支架上細胞、動態培養液注流支架表面細胞，以觀察細胞遷移情況，得到細胞在支架上遷移訊息。3. 生物反應器之注流研究：觀察不同流速對細胞生長的影響，進而了解細胞與剪應力間的關係，並找出細胞所能承受最大剪應力為何。結果發現細胞在78 μm/sec流速下會受剪應力影響而改變貼附型態，而56.75 μm/sec流速為細胞改變型態之臨界，其剪應力值為0.89 mPa，在28 μm/sec流速時細胞受流場影響平行排列。

摘要(英)

The tissue engineering of scaffold provided cell attachment conditions. Present to decide the scaffold materials, structure, and pore size methods by final biochemical examined values. Nevertheless, cell migration and proliferation process on scaffold surface is another one of method to decide scaffold design. We researching in cell migration process of information, using image processing method to find cell migration direction and move speed. This information also estimated that the bioreactor’s perfusion shear stress effect cell proliferation.
The experiment of scaffold use poly-lactic-glycolic acid (PLGA). It is common biomedical material using on tissue engineering. The immortalized rat chondrocyte (IRC) chooses as experiment cells. To observed the cells distribution on scaffold surface. Using transgenic of adenovirus infect cells that had Green-Fluorometer-Protein. The attachment of cells on scaffold surface would observe at Inversion Fluorescence Microscope. This research can be divided into three parts: 1. Static cell culture methods: Observe cell grow situation at support, it get cell’’s course of covering with the scaffold. 2. Cell migration of research: Using static cell culture of scaffold’’s surface cells or the perfusion of medium on scaffold plane, observed the cell migration situation. It will get cell migration direction and move speed. 3. The bioreactor’s perfusion research: Observed the changes of flow speed effect cell proliferation, and then understands the relation among cells and shearing stress, in addition finding out cells can bear the greatest shearing stress. Our findings showed that the cell had change morphology and attachment at a fluid velocity of 78 μm/sec. The cell had original morphology critical limit fluid velocity is 56.75 μm/sec. The shear stress value in the situation is 0.89 mPa. At a fluid velocity of 28 μm/sec, the cell arranged parallel with the flow direction.

關鍵字(中)

★ 剪應力
★ 注流式培養
★ 組織工程支架
★ 綠螢光蛋白
★ 細胞遷移

關鍵字(英)

★ Cell migration
★ Tissue engineering scaffold
★ Green-Fluorometer-Protein
★ Shear stress
★ Perfusion culture

論文目次

摘要　 I
Abstract　 II
誌謝　 III
目錄　 IV
表目錄　 VIII
圖目錄　 IX
符號說明　 XII
第1章　緒論 1
1-1　前言 1
1-2　研究動機與目的 1
1-3　文獻回顧 2
1-4　研究方法簡介 4
1-5　章節概敘 5
第2章　研究理論 6
2-1　活細胞具綠螢光原理 6
2-1-1　腺病毒之感染循環 6
2-1-2　基因改殖的腺病毒 6
2-2　計算流體力學分析支架表面剪應力 7
2-2-1　流體力學推導流程 7
2-2-2　軟體分析溝渠結構之流體力學 10
2-3　影像處理 12
2-3-1　細胞邊緣偵測 (Sobel Edge Detection) 12
2-3-2　形態學運算 13
2-3-3　元件連通標籤化 (Connected-Components Labeling)15
2-3-4　細胞遷移軌跡偵測 17
第3章　研究方法與設備架構 20
3-1　實驗設備說明 20
3-1-1　顯微鏡系統 20
3-1-2　影像擷取系統 21
3-1-3　活細胞連續培養系統 22
3-1-4　注流培養系統 24
3-2　支架材料與製作 26
3-2-1　支架材料 26
3-2-2　支架製作 26
3-3　細胞培養方法 28
3-3-1　細胞培養 28
3-3-2　活細胞具綠螢光蛋白處理 30
3-3-3　腺病毒感染劑量值測試 30
3-3-4　支架表面細胞固定與掃描式電子顯微鏡拍攝 31
3-4　細胞影像處理 32
3-4-1　軟體架構 32
3-4-2　綠螢光細胞影像輪廓處理流程 33
3-4-3　細胞遷移軌跡處理流程 33
3-5　實驗流程步驟 35
3-5-1　腺病毒劑量測試 35
3-5-2　觀察3D-PLGA支架細胞佈滿生長趨勢 35
3-5-3　細胞在不同表面曲率之支架下其生長情況 36
3-5-4　探討不同流體剪應力對細胞生長的影響 37
3-5-5　 IRC細胞在PLGA支架表面所能承受之最大剪應力 39
第4章　實驗結果與討論 41
4-1　腺病毒劑量測試實驗結果 41
4-2　 3D-PLGA支架上細胞佈滿生長趨勢實驗結果 42
4-3　細胞在不同表面曲率之支架下其生長情況實驗結果 46
4-3-1　 3D-PLGA支架 46
4-3-2　 2D-PLGA支架 49
4-3-3　 3D-PLGA與2D-PLGA實驗結果討論 51
4-4　探討不同流體剪應力對細胞生長的影響實驗結果 51
4-4-1　流速78μm/sec注流實驗 51
4-4-2　流速44μm/sec注流實驗 54
4-4-3　流速28μm/sec注流實驗 58
4-4-4　三種流速實驗結果討論 62
4-5　 IRC細胞在所能承受最大剪應力實驗結果 63
4-5-1　以52.5 μm/sec流速觀察細胞 63
4-5-2　以56.75 μm/sec流速觀察細胞 64
4-5-3　以78.0 μm/sec流速觀察細胞 65
4-5-4　實驗結果討論 66
第5章　結論與未來展望 67
5-1　結論 67
5-2　未來展望 67
參考文獻　69
附錄一藥品配製方法 72

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指導教授

曾清秀(Ching-Shiow Teseng)

審核日期

2007-10-11

推文