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論文中文名稱:幾丁聚醣纖維中均勻包覆褐藻酸纖維改善幾丁聚醣支架之細胞與藥物傳送效率 [以論文名稱查詢館藏系統]
論文英文名稱:Chitosan homogenous coating alginate fiber for improving the cell in fiber and promoting the efficiency for drug release [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:化學工程研究所
畢業學年度:103
畢業學期:第二學期
中文姓名:張藏文
英文姓名:Tsang-Wen Chang
研究生學號:102738018
學位類別:碩士
語文別:中文
口試日期:2015/07/23
指導教授中文名:林忻怡
口試委員中文名:蔡偉博;王孟菊
中文關鍵詞:海藻酸鈉、幾丁聚醣、骨母細胞、藥物釋放、抗發炎、共培養
英文關鍵詞:alginate, chitosan, osteooblast, rapid protoyping, anti-flammation, coculture
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立體多孔性,海藻酸鈉是無毒、溶於水的天然高分子材料,所以可以與細胞均勻混合,提供細胞生長環境,其缺點是機械性質較差而且與細胞互動性較弱。而幾丁聚醣也常做成多孔性支架以作為細胞培養的基材,雖然有較好的機械性質以及較好的細胞互動性但在配製溶液時需要在酸性環境下才可將幾丁聚醣溶解,因此無法將細胞直接包覆在殼聚醣溶液中。為了改善褐藻膠的缺點,利用離子交聯的方式在褐藻酸纖維外包覆一層幾丁聚醣以提升機械強度與細胞生長,也以通樣的方式來提升藥物釋放的效率。
本研究將褐藻酸分別與氯化鈣以及含幾丁聚醣的氯化鈣進行離子交聯,並以快速成形系統做成多孔性支架;實驗以抗發炎藥雙氯芬酸鈉(diclofenac)作為藥物傳輸系統中的承載藥物,研究其釋放性質(藥物載量、藥物釋放率、抗發炎測試、qPCR),並對包覆幾丁聚醣含量、細胞活性測試進行探討。
論文英文摘要:In tissue engineering, it is important that scaffolds require porosity, biocompatibility and degradability. Alginate is a not only non-toxic but water-solublenatural material to fabricate scaffold. It can uniform mix with cell and provide growth environment, but it is poor for mechanical properties and weakly interact with cell.Chitosan is also good biomaterial. It is made porous 3D-scaffold. Although it have good mechanical properties and better interact with cell, preparations need acid environment. The cell is harmful at acid environment. In order to make better alginate scaffold, we use advantage both biomaterials to make the coating scaffold and we can use it to improve drug release efficiency.
In this study, alginate could do ion-crosslinkedwith calcium chloride or chitosan in calcium chloride, and using rapid prototyping systems made of porous scaffold; experimental anti-inflammatory drug diclofenac as the carrying drugs for drug delivery system.Containing drugs and osteoblast scaffold co-cultured with macrophage to study the release properties (drug loading, drug release rate, anti-inflammatory tests, qPCR), and coated with chitosan content , cell viability and toxicity testing are discussed
論文目次:摘要 I
ABSTRACT II
誌謝 IV
目錄 V
表目錄 XI
圖目錄 XIV
第一章緒論 1
1.1 前言 1
1.2研究目的 2
1.3研究架構 3
第二章文獻回顧 4
2.1組織工程 4
2.2 免疫系統與發炎 5
2.3骨母細胞 6
2.3巨噬細胞(MACROPHAGE) 7
2.4 酯多醣體(LPS) 8
2.5 抗發炎藥-雙氯芬酸鈉(DICLOFENAC)簡介 9
2.6快速原型系統 10
2.5海藻酸鈉 11
2.5.1材料介紹 11
2.5.2褐藻酸鈉纖維支架 13
2.6幾丁聚醣 14
2.6.1 幾丁聚醣介紹 14
2.6.2 幾丁聚醣性質 16
2.7幾丁聚醣與褐藻酸鈉交聯材料 17
第三章 實驗材料與方法 19
3.1實驗材料 19
3.1.1細胞來源 19
3.1.2細胞培養用藥品 19
3.1.3實驗藥品 21
3.1.4儀器設備 27
3.1.5藥品及溶液配製 29
3.2實驗方法 41
3.2.1實驗設計 41
3.2.1.1製作褐藻膠纖維支架 42
3.2.1.2製作幾丁聚醣包覆褐藻膠纖維支架 42
3.2.1.3幾丁聚醣包覆褐藻膠纖維支架細胞增生 42
3.2.1.4含藥支架藥物釋放 42
3.2.1.5決定雙氯芬酸鈉藥物濃度 42
3.2.1.6尋找配製藥物的初始濃度 42
3.2.1.7巨噬細胞發炎培養 43
3.2.1.7抗發炎測試 43
3.2.2實驗流程 44
3.2.3快速原型機架設 44
3.2.4巨噬細胞繼代 46
3.2.5製作褐藻酸鈉支架 47
3.2.5.1溶液配製 47
3.2.5.2支架製作 48
3.2.6巨噬細胞發炎培養 50
3.2.7共培養-抗發炎 50
3.3 幾丁聚醣包覆褐藻膠支架 51
3.3.1螢光拍攝 51
3.3.2 幾丁聚醣定量 51
3.3.3有幾丁聚醣包覆- SEM拍攝褐藻膠支架截面 51
3.3.4適合細胞培養中和鹼濃度-細胞增生 52
3.3.4 細胞存活螢光染色(Live & dead) 52
3.4支架藥物釋放 53
3.4.1決定氯化鈣(w/v)%濃度參數 53
3.4.2含雙氯芬酸鈉(diclofenac)支架的藥物釋放 53
3.4.3含雙氯芬酸鈉(diclofenac)支架的藥物載量 53
3.4.4決定細胞培養用的雙氯芬酸鈉(diclofenac)藥物濃度 54
3.4.4.1巨噬細胞含抗發炎藥培養 54
3.4.4.2骨母細胞含抗發炎藥培養 54
3.4.5尋找合適的初始配製雙氯芬酸鈉(diclofenac)藥物濃度 54
3.5鹼性磷酸酶測定與DNA測定 55
3.5.1測定前處理 55
3.5.2鹼性磷酸酶測定 55
3.5.3 細胞增生測定 55
3.6抗發炎因子測定 56
3.6.1樣品存放 56
3.6.2一氧化氮測定(Griess assay) 56
3.6.3發炎因子腫瘤壞死因子(TNF-α)測定 56
3.6.4發炎因子介白素第六型(IL-6)測定 56
3.7鈣定量 57
3.7.1前處理 57
3.7.2測定 57
3.8細胞基因表現 58
3.9統計分析 62
第四章 實驗結果與討論 63
4.1 選擇製作褐藻膠支架的氯化鈣(W/V)% 63
4.2 幾丁聚醣包覆3% 褐藻酸鈉支架 64
4.2.1 螢光拍攝 64
4.2.2 幾丁聚醣定量(Ninhydrin assay) 65
4.2.3 有幾丁聚醣包覆褐藻膠支架之截面 65
4.2.4 適合細胞培養中和鹼濃度-骨母細胞增生 66
4.2.3 適合細胞培養中和鹼濃度-細胞增生-續 67
4.2.3 適合細胞培養中和鹼濃度-細胞增生-續 68
4.3 細胞在支架上分布 69
4.3.1 細胞存活螢光染色 69
4.4 含不同濃度雙氯芬酸鈉(DICLOFENAC)細胞培養 73
4.4.1 巨噬細胞-含LPS 1μg/ml刺激培養 73
4.4.2 巨噬細胞-一氧化氮測定 74
4.4.3 巨噬細胞-腫瘤壞死因子(TNF-alpha)測定 75
4.4.4 骨母細胞 76
4.5 含雙氯芬酸鈉褐藻膠纖維支架藥物釋放 77
4.6含雙氯芬酸鈉(DICLOFENAC)褐藻膠纖維支架藥物載量 78
4.7 尋找初始藥物配製濃度 79
4.7.1 支架雙氯芬酸鈉(diclofenac)藥物含量測定 79
4.7.2 支架藥物釋放總含量 80
4.8 共培養-支架抗發炎 81
4.8.1 細胞增生 81
4.8.2 鹼性磷酸酶(ALP)活性 84
4.8.3 鈣定量 86
4.8.4 發炎因子-一氧化氮(NO)測定 87
4.8.5 發炎因子- 第六型介白素(IL-6)測定 88
4.8.7 支架附著上巨噬細胞-細胞增生 91
4.9 基因表現 92
4.9.1 基因相對量計算方式 92
4.9.2 基因表達-TIMP2 92
4.9.3 基因表達-MMP13 93
4.9.3 基因表達-OPN 94
第五章 結論 95
附錄一 DNA定量標準曲線 96
附錄二 一氧化氮標準曲線 97
附錄三 P-NITROPHENOL標準曲線 98
附錄四鈣定量標準曲線 99
附錄五 TNF-Α ELISA KIT實驗步驟(含標準曲線) 100
附錄六 IL-6 ELISA KIT 實驗步驟(含標準曲線) 102
附錄七 幾丁聚醣標準曲線 104
附錄八 PRIMER測試 105
參考文獻 108
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