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研究生:古皓夫
研究生(外文):Ku, Haofu
論文名稱:白蛋白、彈性蛋白、聚賴氨酸修飾的幾丁聚醣-聚麩胺酸支架對培養牛膝軟骨細胞生長與凋亡的效應
論文名稱(外文):Effect Of Chitosan/gamma-PGA Scaffold Surface-modified By Albumin, Elastin, Poly-L-lysine On The Cultivation And Apoptosis Of Bovine Knee Chondrocytes
指導教授:郭勇志郭勇志引用關係
指導教授(外文):Kuo, Yungchih
口試委員:朱一民徐善慧蔡瑞瑩
口試委員(外文):Chu, ImingHsu, ShanhuiTsai, Juiying
口試日期:2012-07-25
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:134
中文關鍵詞:幾丁聚醣聚麩胺酸彈性蛋白人類血清白蛋白聚賴氨酸牛膝軟骨細胞細胞凋亡
外文關鍵詞:chitosangamma-PGAelastinhuman serum albuminpoly-L-lysinebovine knee chondrocytesapoptosis
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本研究以幾丁聚醣(chitosan)、聚麩胺酸(gamma-PGA)兩種生醫材料以不同的質量比例混合,結合山梔子(genipin)交聯劑,經由冷凍乾燥法(freeze-drying)製備可生物利用性的三維立體孔洞支架(scaffold),並對支架孔隙度(porosity)、膨潤度(swelling ratio)、機械強度性質(modulus)、細胞存活率(viability)做材料特性分析。支架表面再以三種不同材料白蛋白(albumin)、彈性蛋白(elastin)、聚賴氨酸(poly-L-lysine)進行雙成份混合修飾,提供體外(in vitro)良好培養環境,提升牛膝軟骨細胞(chondrocyte)增生以及胞外基質(extracellular matrix, ECM)分泌的效益。實驗結果顯示,支架中幾丁聚醣:聚麩胺酸組成比例為1:3時為佳的孔隙度、抗壓強度、延展性質,進行牛膝軟骨細胞體外培養以此比例為最佳效果。此外,發現聚麩胺酸的比例增加時,膨潤性質上升表示支架表面抓住水分子的能力增加,但是孔隙度以及孔洞大小的影響在體外培養後細胞數(DNA)的增生以及聚葡萄醣胺(glycosaminoglycans, GAGs)與第二型膠原蛋白(type II collagen)的分泌開始產生下降趨勢。研究中以白蛋白、彈性蛋白、聚賴氨酸對支架進行表面修飾,三成份中各取雙成份依體積比例混合,結果顯示表面改質以白蛋白:彈性蛋白比例1:3經體外4週培養,細胞數量的增生及聚葡萄醣胺的分泌、產生第二型膠原蛋白皆有最佳的結果。細胞凋亡(apoptosis)的檢測當中,發現以彈性蛋白最不易產生細胞凋亡的情況,以粒線體(mitochondria)損傷的染色結果顯示,軟骨細胞體外培養在彈性蛋白的環境中,受損的情形較低。實驗結果證明,本研究製備的幾丁聚醣-聚麩胺酸支架進行表面修飾能夠有效的提升牛膝軟骨細胞生長與分泌胞外基質,改善軟骨組織的再生能力。
The purpose of this study is to blend chitosan with gamma-PGA in different quantity proportion and combine with genipin (crosslinker) ,then producing bioavailable scaffold through freeze-drying and analyzing material properties of porosity, swelling raito, modulus and viability. The surface of scaffold will be modified with albumin, elastin and poly-L-lysine to provide good environment for in vitro cultivation, promote chondrocyte growth and benefit for extracellular matrix (ECM) secretion. The result of experiment reveals that when chitosan and gamma-PGA form with the ratio of 1:3 in the scaffold has the optimal porosity, pressure resistance and extensibility. It’s the most appropriate condition for cultivating bovine knee chondrocytes. Besides, when the ratio of gamma-PGA raises, the increase of swelling ratio equivalent to the enhancement of hydrophilic ability on scaffold surface. However, due to the effect of porosity and pore size, chondrocytes cell numbers, secreted glycosaminoglycans and produced type II collagen shown a descendant tendency. The experiments utilize surface-modified scaffold with a pair of components of albumin, elastin and poly-L-lysine then mixed in volume ratio. The result indicated that when the albumin and elastin mixed with the ratio of 1:3 for four weeks, the increase chondrocytes cell numbers, secretion glycosaminoglycans and production type II collagen has the optimal condition. In the apoptosis detection, elastin is the most long-lived one to appear the phenomenon of cell death. As the result of mitochondria damage revealed, chondrocytes damaged slightly when in vitro cultivation with elastin surrounded. It is concluded that utilizing chitosan/gamma-PGA scaffold can be effectively promoting the growth of bovine knee chondrocytes, secreting extracellular matrix and improving the regeneration ability of cartilage tissues.
中文摘要 I
英文摘要 III
目錄 V
圖目錄 XII
表目錄 XIV
符號說明 XV
研究動機與目的 XVII
第一章文獻回顧 1
1.1 軟骨組織 1
1.2 組織工程 2
1.3 生醫材料的應用 3
1.4 生物反應器 4
1.5 幾丁聚醣與聚麩胺酸 5
1.6 交聯劑山梔子(Genipin) 8
1.7 表面修飾材料 9
1.7.1 彈性蛋白 9
1.7.2 人類血清白蛋白 10
1.7.3 聚賴氨酸 11
1.8 細胞凋亡 12
第二章 實驗儀器、材料、方法及步驟 15
2.1 實驗儀器(依筆劃排列) 15
2.2 實驗器材(依筆劃排列) 19
2.3 實驗材料 22
2.3.1 支架的製備 22
2.3.1.1 聚麩胺酸膠體的製備 22
2.3.1.2 幾丁聚醣膠體的製備 24
2.3.1.3 交聯劑 24
2.3.2 支架表面改質 24
2.3.2.1 彈性蛋白 24
2.3.2.2 人類血清白蛋白 25
2.3.2.3 聚賴氨酸 25
2.3.3 支架特性分析 26
2.3.3.1 孔隙度測試 26
2.3.3.2 支架對細胞活性測試 26
2.3.3.3 貼附率測試 26
2.3.3.4 支架結構與孔洞分析 27
2.3.4 軟骨細胞取得以及細胞與支架共培養 28
2.3.4.1 培養基的配製 28
2.3.3.2 軟骨細胞的分離 29
2.3.4.3 軟骨細胞冷凍保存 29
2.3.4.4 軟骨細胞的植入 30
2.3.5 軟骨細胞培養後生化分析 30
2.3.5.1 分解液 30
2.3.5.2 細胞數的分析(DNA定量分析) 31
2.3.5.3 聚葡萄糖胺定量分析(GAG定量分析) 31
2.3.5.4 膠原蛋白定量分析(Collagen 定量分析) 32
2.3.6 組織切片 33
2.3.7 組織染色 34
2.3.7.1 蘇木紫-伊紅染色(Hematoxylin-eosin, H&E) 34
2.3.7.2 藏紅染色(Safranin-O) 34
2.3.7.2 膠原蛋白第二型免疫染色及螢光定量 35

2.3.8 細胞凋亡 36
2.3.8.1 Caspase-3檢測 36
2.3.8.2蛋白質檢測 36
2.3.8.3 Mitochondria染色 36
2.4 實驗方法與步驟 38
2.4.1 支架的製備 38
2.4.1.1幾丁聚醣-聚麩胺酸膠體製備 38
2.4.1.2支架成型 38
2.4.1.3支架表面改質製備 39
2.4.1.3.1 彈性蛋白 39
2.4.1.3.2 白蛋白 39
2.4.1.3.3聚賴氨酸 40
2.4.2 支架特性分析 41
2.4.2.1 孔隙度測試 41
2.4.2.2 膨潤度測試 42
2.4.2.3 機械強度測試 42
2.4.2.4 紅外線光譜分析 43
2.4.2.5 支架對細胞活性測試(XTT) 43
2.4.3.6細胞貼附效率測試 44
2.4.3.7支架降解率測試 45
2.4.3.8統計學 45

2.4.3 軟骨細胞取得及細胞植入支架培養 46
2.4.3.1 培養基的配製 46
2.4.3.2 軟骨細胞的分離與冷凍保存 46
2.4.3.3 支架消毒與軟骨細胞的植入 48
2.4.3.4 軟骨細胞的懸浮培養 48
2.4.4 軟骨細胞植入支架培養後之生化分析 49
2.4.4.1 細胞數的分析(DNA數定量) 49
2.4.4.2 聚葡萄醣胺定量分析(GAG定量分析) 50
2.4.4.3 膠原蛋白定量分析(collagen定量分析) 51
2.4.5 支架結構與孔洞分析(SEM) 53
2.4.6 組織切片製作 54
2.4.7 組織型態及胞外基質染色 55
2.4.7.1 蘇木紫-伊紅染色(Hematoxylin-eosin, H&E) 55
2.4.7.2 藏紅染色(safranin-O) 55
2.4.7.3 膠原蛋白第二型免疫螢光染色 56
2.4.8凋亡檢測 57
2.4.8.1 caspase-3檢測 57
2.4.8.2蛋白質濃度分析(BCA kit) 58
2.4.8.3 Mitochondria染色 59
第三章 結果與討論 61
3.1幾丁聚醣-聚麩胺酸支架機械性質 61
3.2幾丁聚醣-聚麩胺酸支架紅外線光譜分析 62
3.3幾丁聚醣-聚麩胺酸支架孔隙度與膨潤度測試 63
3.4 支架對細胞活性測試(XTT) 64
3.5 SEM分析 65
3.5.1幾丁聚醣-聚麩胺酸支架形態 65
3.5.2彈性蛋白、白蛋白、聚賴氨酸表面修飾之後幾丁聚醣-聚麩胺酸支架培養牛膝軟骨細胞形態4週後形態 65
3.6牛膝軟骨細胞植入幾丁聚醣-聚麩胺酸支架培養 66
3.6.1支架組成貼附的影響 66
3.6.2支架組成對細胞生長的影響 67
3.6.3支架降解率測試 68
3.6.4彈性蛋白、白蛋白、聚賴氨酸表面修飾支架對細胞生長的影響 69
3.7 組織切片染色分析 70
3.7.1蘇木紫-伊紅染色(H&E) 70
3.7.2藏紅染色(Safranin-O) 71
3.7.3膠原蛋白第二型免疫染色 71
3.8凋亡檢測分析 72
3.8.1 caspase-3分析 72
3.8.2 Mitochondria染色 73
第四章 結論與建議 75
4.1 結論 75
4.2 建議 76
參考文獻 77
附錄 113


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