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論文中文名稱:RGD改質後之海藻酸鈉水凝膠對包埋在內的纖維母細胞生長與分化之影響 [以論文名稱查詢館藏系統]
論文英文名稱:In Vitro Studies of Fibroblasts Embedded in Hydrogelic Fibers Made of RGD-modified Alginate [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:102
出版年度:103
中文姓名:李彥瑩
英文姓名:Yen-Ying Li
研究生學號:101688012
學位類別:碩士
語文別:中文
口試日期:2014-07-01
論文頁數:86
指導教授中文名:林忻怡
口試委員中文名:黃聲東;王大銘
中文關鍵詞:海藻酸鈉RGD胜肽纖維母細胞
英文關鍵詞:alginateRGD peptidefibroblastrapid protoyping
論文中文摘要:在組織工程中,支架通常是水膠做成的是具有多孔性、可解性,海藻酸鈉是無毒、溶於水的天然高分子材料,所以可以與細胞混合,讓細胞較均勻的分布,RGD胜肽會與某些細胞表面受體結合促進細胞貼附、生長,為了讓細胞在水凝膠纖維中生長能夠生長得更好,所以使用RGD胜肽改質海藻酸鈉,再用快速原型機製作立體結構的水凝膠纖維,比較在改質前後海藻酸鈉水凝膠內細胞生長的差異性。
研究發現,在DNA定量中,可以看從第四天到第十四天有改質過的海藻酸鈉中DNA含量比沒改質的高;由細胞存活螢光染色觀察細胞存活率,可以知道從第一天到第十四天兩組支架內的大多數的細胞接存活下來,在第十四天RGD改質過的海藻酸鈉有看到比較細長的形狀,在細胞貼附實驗中,發現細胞在第二天開始呈現梭狀,而且RGD-alginate海藻酸鈉薄膜上的呈現梭狀的細胞明顯比較多。
結果顯示,我們有成功接枝RGD胜肽到海藻酸鈉上,提供更適當的條件讓細胞生長。在未來,希望RGD海藻酸鈉可以運用在傷口敷料上,讓細胞可以再更適合的環境與條件,幫助傷口癒合。
論文英文摘要:In tissue engineering, it is important that scaffolds require porosity, biocompatibility and degradability. Alginate is a natural material to fabricate scaffold. RGD peptide that can promote cell adhesion, cell growth and help wound healing is combined with some surface receptors of cell.
For improve cell distribution, we mix the alginate and cells together. We modified alginate with RGD and embedded cells in hydrogel fibers made of RGD-alginate scaffold using rapid prototyping. Cell morphology and proliferation inside the RGD-alginate hydrogel were examined and compared to cells embedded in alginate.
In this study, we knew the DNA quantification of the RGD-alginate is higher than alginate . Live/dead stain showed all cells are alive in two scaffolds. HE stain appeared the cell’s morphology no significant difference in two kinds of scaffolds. Results show that RGD-alginate scaffold is more stable for cell to growth.
論文目次:目錄
摘要 I
ABSTRACT II
誌謝 III
目錄 IV
表目錄 VII
圖目錄 IX
第一章 緒論 1
1.1 前言 1
1.2研究目的 3
第二章 文獻回顧 4
2.1組織工程 4
2.2 傷口癒合 5
2.3傷口敷料演變 7
2.4快速原型系統 8
2.5 海藻酸鈉 9
2.6 RGD胜肽 11
2.6.1 RGD胜肽介紹 11
2.6.2 RGD改質方法 13
第三章 實驗材料與方法 15
3.1實驗材料 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 21
3.1.5藥品及溶液配製 23
3.2實驗方法 27
3.2.1實驗設計 27
3.2.2實驗流程 27
3.2.3快速原型機架設 28
3.2.4 RGD海藻酸鈉改質 29
3.2.5製作褐藻酸鈉支架 30
3.2.5.1溶液配製 30
3.2.5.2支架製作 31
3.3物理性質測試 33
3.3.1螢光胜肽接枝海藻酸鈉 33
3.3.1.1螢光纖維 33
3.3.1.2螢光接枝效率 34
3.3.2 支架大小與纖維直徑 35
3.3.2.1支架大小 35
3.3.2.2纖維直徑 35
3.4培養液降解 36
3.5細胞貼附 37
3.6細胞在支架上分布 38
3.6.1細胞存活螢光染色 38
3.6.2H&E染色 39
3.6.3掃描式電子顯微鏡 39
3.7細胞增生 40
3.8細胞基因表現 41
3.9統計分析 45
第四章 實驗結果與討論 46
4.1物理性質測試 46
4.1.1螢光胜肽接枝海藻酸鈉 46
4.1.1.1螢光纖維 46
4.1.1.2螢光接枝效率 48
4.1.2支架大小與纖維直徑 49
4.1.3 培養液降解 51
4.2細胞貼附 53
4.3細胞在支架上分布 55
4.3.1細胞存活螢光染色 55
4.3.2 H&E染色 58
4.3.3掃描式電子顯微鏡 63
4.4細胞增生 70
4.5細胞基因表現 72
第五章 結論 75
參考文獻 76
附錄一 DNA定量標準曲線 81
附錄二 GRGDYK- FITC螢光標準曲線 82
附錄三 改變清洗海藻酸鈉步驟 83
表目錄
表3.1 細胞培養用所需藥品 15
表3.2 配製食鹽緩衝溶液(PBS)所需藥品 16
表3.3 製備樣品所需藥品 16
表3.4細胞存活染色之kit 17
表3.5細胞增生之kit 17
表3.6 H&E染色所需藥品 18
表3.7 抽RNA所需藥品 18
表3.8 RNA反轉錄成cDNA kit 19
表3.9 Real-Time quantitative PCR kit 19
表3.10實驗所需其他藥品 20
表3.11儀器設備 21
表3.12配製DMEM培養液(1L) 23
表3.13 配製10×食鹽緩衝溶液(10×PBS) 24
表3.14配製0.1M MES buffer 24
表3.15配製0.25% papain buffer(500ml) 25
表3.16配製Hematoxylin染色用試劑 25
表3.17配製Acid-Ethanol退染用溶液 26
表3.18配製Eosin染色用試劑 26
表3.19樣品製作參數 32
表3.20實驗設計組別 32
表3.21 GRGDYK- FITC螢光標準曲線 34
表3.22 DNA標準曲線濃度 40
表3.23 RNA反轉錄成cDNA反應小管中成分含量 42
表3.24 RNA反轉錄成cDNA之反應溫度與時間 42
表3.25各基因primer資訊 43
表3.26 RT-PCR反應時反應管中各成分含量 43
表3.27各基因的PCR反應步驟與溫度 44
圖目錄
圖2.1發炎期 6
圖2.2增生期 6
圖2.3成熟期 6
圖2.4海藻酸鈉結構 9
圖2.5 海藻酸與鈣離子形成Egg-box結構 10
圖2.6 RGD胜肽結構式 12
圖2.7 EDC結構式 13
圖2.8海藻酸鈉與GRGDY胜肽反應式 13
圖3.1 實驗流程圖 27
圖3.2快速原型機架設圖 28
圖3.3水凝膠樣品製作示意圖 29
圖4.1用螢光顯微鏡觀察,有GRGDYK-FITC接枝海藻酸鈉 47
圖4.2 RGD-alginate與Alginate水凝膠外觀 49
圖4.3 RGD-alginate與Alginate水凝膠的平均纖維直徑 50
圖4.4 RGD-alginate與Alginate的水凝膠進行培養液降解 52
圖4.5 用光學顯微鏡觀察纖維母細胞在RGD-alginate與Alginate薄膜上的生長情形 54
圖4.6 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行Live/Dead螢光染色拍照,放大倍率為40X 56
圖4.7 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行Live/Dead螢光染色拍照,放大倍率為100X 57
圖4.8 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行H&E染色拍照,放大倍率為100X 59
圖4.9 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行H&E染色拍照,放大倍率為200X, 60
圖4.10 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行H&E染色拍照,放大倍率為400X, 61
圖4.11 纖維母細胞生長在RGD-alginate水凝膠的第一層表面,進行H&E染色拍照,放大倍率為400X 62
圖4.12纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行正面掃描式電子顯微鏡拍照,放大倍率為100X; 64
圖4.13纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行正面掃描式電子顯微鏡拍照,放大倍率為200X 65
圖4.14 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行正面掃描式電子顯微鏡拍照,放大倍率為400X, 66
圖4.15 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行反面掃描式電子顯微鏡拍照,放大倍率為100 67
圖4.16 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行反面掃描式電子顯微鏡拍照,放大倍率為200X 68
圖4.17 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行反面掃描式電子顯微鏡拍照,放大倍率為400X 69
圖4.18 將纖維母細胞埋在RGD-alginate與Alginate水凝膠中,進行DNA定量 71
圖4.19 將纖維母細胞埋在RGD-alginate與Alginate水凝膠之Fn-1基因之相對基因表現量 73
圖4.20 將纖維母細胞埋在RGD-alginate與Alginate水凝膠Fgf-11基因之相對基因表現量 74
附錄1 DNA定量標準曲線 81
附錄2 GRGDYK- FITC螢光標準曲線 82
附錄3-1 將纖維母細胞埋在RGD-alginate與Alginate水凝膠中,進行DNA定量 84
附錄3-2 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行正面掃描式電子顯微鏡拍照,放大倍率為100X 85
附錄3-3 纖維母細胞生長在RGD-alginate與Alginate水凝膠中,進行正面掃描式電子顯微鏡拍照,放大倍率為400X 86
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