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論文中文名稱:利用立體繪圖方式(3D Plotting)製作水凝膠纖維中包覆細胞之立體孔洞狀支架以供皮膚修復 [以論文名稱查詢館藏系統]
論文英文名稱:Cell Embedded in Fibers of Porous Scaffold through 3D Plotting Fabrication to Repair Skin [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:100
出版年度:101
中文姓名:吳瑋文
英文姓名:Wei-Wen Wu
研究生學號:99688011
學位類別:碩士
語文別:中文
口試日期:2012-07-13
論文頁數:75
指導教授中文名:林忻怡
口試委員中文名:蔡偉博;王孟菊;鍾仁傑
中文關鍵詞:立體繪圖褐藻酸鈉皮膚修復
英文關鍵詞:3D plottingalginateskin repairL-929
論文中文摘要:組織工程中,細胞均生長於支架表面,與其在生理環境中四周均被細胞間質包覆的情況有很大差異。且在種植細胞的過程中易產生細胞分布不均的問題,造成只有局部性的細胞表現(如分泌胞外基質)。本實驗在支架成型的過程中,將材料和細胞in situ混勻,使材料和細胞共同成型,可使細胞四周均勻被材料包覆(更相似於天然組織),並解決細胞在支架中分布不均的問題。進一步亦探討均勻生長於支架纖維內之細胞和生長在支架纖維表面上之細胞的細胞生長與表現上的差異。
因支架成型的過程不能對其中的細胞產生傷害,故以褐藻酸鈉作為支架材料,以溫和之氯化鈣進行交聯,透過立體繪圖方式製作支架。實驗組為將細胞與3%褐藻酸鈉混合後共同成型。對照組則為將細胞種植在已製作好3%褐藻酸支架上。由螢光染色觀察細胞存活率,發現兩組細胞在隔天大部分皆可存活下來;光學顯微鏡下可看到實驗組的細胞呈現小球狀分布於支架纖維之中;共軛焦系統所模擬的立體模型顯示出實驗組的細胞較對照組更加均勻。透過細胞定量與細胞活性測

試中,發現兩組的細胞皆隨時間穩定上升,且實驗組在初始期間內生長較對照組快速,可能是細胞均勻分佈而有更大的空間增長。在基因表現上,實驗組的生長因子(fibroblast growth factor)和纖維結合素(fibronectin)的表現皆明顯高於對照組。兩組的一型膠原蛋白(type I collagen)表現均不高,但實驗組單位DNA產生的膠原蛋白量較多。
實驗證明均勻生長於支架纖維中的纖維母細胞,由於更均勻分佈於支架中,可更有效地利用空間及取得養分,且生長環境更接近正常的生理環境,能夠有較快的細胞生長和較佳基因表現。
論文英文摘要:The situation is different between cell growing on scaffold surface in tissue engineering and cell growing in intercellular substance in physiological environment. Moreover, the unequal cell distribution on the scaffold results local cell expression (ex. secrete extracellular matrix). In order to improve cell distribution, we fabricate a cell-embedded (L-929) scaffold which mimics natural skin by well-mixing materials and cells in situ and co-dispensing through 3D plotting. We further investigate the difference of cell growth and performance between cells uniformly growing in the fibers of scaffold and cells growing on the fibers of scaffold.
For avoid hunting cells on the process of 3D plotting, a dispensed scaffold of cell-containing alginate was gently crosslinked with calcium chloride. The experimental group (Co) was co-dispensed a scaffold with well-mixed cells and 3% alginate; the control group (S) was seeding cells on the 3% alginate scaffold. Live/dead staining appeared the cells were mostly survival in the next day; the cells of Co were observed as small spheres in the fibers by microscope; Confocal system showed that cell distribution of the Co scaffold was obvious more uniform than S. DNA quantitation and MTT assay. It appeared the cells of both groups were increased steadily over time, but the cell growth rate of Co was faster than S before Day 7. Because the cells of Co were full over the scaffold and grew in more available space. Quantitative RT-PCR revealed higher mRNA expression of fibroblast growth factor (Fgf11) and fibronectin (Fn1) on Co. Although the expression of type I collagen (Col1a1) was unapparent on both groups, but the product of collagen per 1 ng DNA on Co was higher than S.
Our results suggest that L-929 uniformly growing in the fibers of scaffold is more similar to normal physiological environment. Effectively utilizing growth space and obtaining nutrients results higher cell growth and gene expression. Hence, it may be useful for future potential application in skin repair.
論文目次:中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 IX
表目錄 XII
第一章 緒論 1
第二章 文獻回顧 3
2-1 傷口愈合過程 3
一、纖維蛋白凝塊形成(fibrin colt formation): 4
二、發炎反應(inflammation): 4
三、肉芽組織(granulation tissue)、傷口縮合(wound contraction)和表皮細胞移行再生(reepithelialization): 4
四、胞外基質再構(extracellular matrix reorganization): 6
2-2 水膠 7
2-3 褐藻酸鈉 9
2-4 立體繪圖方式(3D Plotting) 11
2-5 水膠在傷口敷材上的應用 13
第三章 實驗材料與方法 15
3-1-1 細胞來源 15
3-1-2 細胞培養用藥品 15
3-1-3 實驗藥品 16
3-1-4 儀器設備 20
3-1-5 藥品及溶液製備 21
3-2 實驗方法 23
3-2-1 實驗設計 23
3-2-2 實驗流程 23
3-2-3 立體繪圖系統架設 24
3-2-4 製作褐藻酸鈉-細胞支架 25
3-2-5 細胞型態與分布 26
一、光學顯微鏡 26
二、細胞存活螢光染色 26
三、掃描式電子顯微鏡 27
四、共軛焦螢光顯微鏡 27
五、Haematoxylin & Eosin染色(H&E) 27
3-2-6 細胞增生(DNA定量) 28
3-2-7 細胞代謝活性(MTT assay)測定 29
3-2-8 細胞基因表現(Real-Time quantitative PCR) 29
一、Total RNA萃取 29
二、RNA反轉錄為cDNA 30
三、Real-Time quantitative PCR 31
3-2-9 胞外基質表現(膠原蛋白定量) 33
3-2-10 統計分析 34
第四章 實驗結果與討論 35
4-1 細胞型態與分布 35
一、光學顯微鏡 35
二、細胞存活螢光染色 36
三、掃描式電子顯微鏡 39
四、共軛焦螢光顯微鏡 49
五、H&E染色 51
4-2 細胞增生(DNA定量) 56
4-3 細胞代謝活性(MTT assay)測定 58
4-4 細胞基因表現(Real-Time quantitative PCR) 60
4-5 胞外基質表現(膠原蛋白定量) 63
第五章 結論 65
附錄一 DNA定量標準曲線 66
附錄二 Primer相關測試 67
附錄三 Collagen定量標準曲線 69
參考文獻 70
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