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研究生:涂宣因
論文名稱:多醣類於膠原蛋白基質中之研究
論文名稱(外文):The study of polysaccharides in a collagen matrix
指導教授:黃玲惠
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:100
中文關鍵詞:傷口癒合膠原蛋白多醣類
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自膠原蛋白被應用為生物醫學材料以來,在組織工程的應用上一直被視為發展的主流之一,許多研究發現膠原蛋白基質具有幫助組織修復的功能,其中又以多孔狀海綿膠幫助傷口癒合的效果最佳,但結構脆弱導致膠原蛋白海綿膠在應用上頗受限制,如基質對水相的耐受性差,目前的解決方法主要以添加交聯劑或是物理方式來改善。本篇論文利用混合添加四種多醣類來製備膠原蛋白基質,分別為透明質酸(hyaluronan),硫酸軟骨素c(chondroitin sulfate c),水溶性幾丁聚醣(chitosan)及褐藻酸(alginate)等四種在傷口癒合上具有成效的多醣類,研究發現多醣類於基質中的分布為均勻相,不影響海綿基質的型態及孔洞範圍,而且可以增加膠原蛋白基質的熱穩定性。以體外的細胞培養方式比較四種多醣類的添加對於細胞遷移及增生的影響,結果發現透明質酸促進纖維母細胞遷移效果較其他多醣類為佳,而透明質酸、硫酸軟骨素c、褐藻酸於基質中對於細胞增生效果優於含幾丁聚醣之膠原蛋白基質。本研究並以天竺鼠動物進行背部全深度傷口的基質移植手術,評估傷口癒合後皮膚的組織結構差異,結果發現含透明質酸或硫酸軟骨素c之膠原蛋白基質對於傷口癒合效果較佳,且癒合後皮膚組織結構較接近正常組織,因此這類基質在皮膚替代物的發展上具有極大的潛力。
中文摘要..………………………………………………….………….… I
ABSTRACT…………………………………………………………….. II
目錄..……………………………………………………………………. III
表目錄..………………………………………………………………..… V
圖目錄..………………………………………………………………… VIII
符號說明…..………………………………………………………….... X
第一章 緒論..………………………………………………………...… 1
1.1 膠原蛋白……………………………………………………….... 1
1.1.1 膠原蛋白的來源與生合成…..………………………………... 2
1.1.2 膠原蛋白的型態及種類..……………………………………... 3
1.1.3 膠原蛋白應用的種類…..……………………………………... 3
1.1.4 膠原蛋白基質在組織工程上的應用情形..…………………... 4
1.2 添加多醣類的目的…..……………………………………….…. 4
1.2.1 多醣類的來源與種類…..……………………………………... 5
1.2.2 多醣類的功能……………………………………………….… 5
1.2.3 添加多醣類於膠原蛋白基質的研究……………………….… 7
1.2.4 多醣類於膠原蛋白結構的影響…..…………………………... 8
1.3 選擇添加的多醣類..………………………………………….…. 9
1.3.1 透明質酸(hyaluronan)..………………………………….… 9
1.3.2 硫酸軟骨素c(chondroitin sulfate c)……………………… 10
1.3.3 幾丁聚醣(chitosan)………………………………..…….… 11
1.3.4 褐藻酸(alginate)…………………………………………... 12
1.4 傷口癒合體外研究模式…..………………………………….… 12
1.4.1 細胞培養系統….……………………………………………... 13
1.4.2 細胞在基質上的行為….……………………………………... 13
1.4.3 動物實驗模式...…………………………………………….… 14
1.5 研究背景及研究動機….………………………………….….… 23
第二章 實驗藥品設備及方法……...……………….………………… 24
2.1 實驗藥品.……………………………………………………..… 24
2.2 實驗儀器………..……………………………………….…....… 26
2.3 實驗方法……………………………………………………...… 28
2.3.1 豬真皮膠原蛋白溶液之取得………………………………… 28
2.3.2 膠原蛋白的濃度測定………………………………………… 28
2.3.3 基質製備方式………………………………………………… 28
2.3.4 基質水相耐受性量測方式…………………………………… 29
2.3.5 基質的包埋與切片…………………………………………… 29
2.3.6 基值孔洞分析方法…………………………………………… 29
2.3.7 基質及組織切片之H&E染色....…….………………………. 30
2.3.8 基質切片之alcian blue染色……………....…………………. 30
2.3.9 DSC儀器操作………………………………………………... 30
2.3.10 CNBr活化多醣類…………………………………………….. 31
2.3.11 掃描式電子顯微鏡觀察…………………………………...….. 31
2.3.12 穿透式電子顯微鏡觀察………………………………………. 31
2.3.13 纖維母細胞遷移實驗……………………………………….… 32
2.3.14 纖維母細胞增生實驗…………………………………………. 32
2.3.15 基質吸水性試驗…………………………………………..…... 32
2.3.16 動物基質移植手術……………………………………………. 32
2.3.17 動物傷口閉合評估方式…………………………………….… 34
2.3.18 動物傷口皮膚厚度量測方式………………………………… 34
2.3.19 動物組織的包埋與切片……………………………………… 34
第三章 含多醣類的膠原蛋白基質與其性質分析…………………… 35
3.1 實驗目的…………………………………………………………. 35
3.2 實驗材料與方法…………………………………………………. 35
3.2.1 實驗材料……………………………………………………….. 35
3.2.2  水相耐受性試驗……………………………………………….. 36
3.2.3 基質中多醣類分佈試驗……………………………………….. 37
3.2.4 觀察基質中多醣類與膠原蛋白纖維型態…………………….. 37
3.2.5 基質熱穩定性探討…………………………………………….. 37
3.2.6 基質孔洞結構觀察…………………………………………….. 37
3.2.7 基質孔洞大小範圍分析……………………………………….. 37
3.3 實驗結果………………...……………………………………….. 38
3.3.1 含不同多醣類的膠原蛋白基質之水相耐受性……………….. 38
3.3.2 含經共價鍵結之多醣類的膠原蛋白基質之水相耐受性…….. 38
3.3.3 含活化之透明質酸的膠原蛋白基質之水相耐受性………….. 39
3.3.4 混合共價鍵結之多醣類及活化的透明質酸之膠原蛋白
基質之水相耐受性……………………………………..…….. 39
3.3.5 混合多醣類及活化之透明質酸的膠原蛋白基質之水相
耐受性...……………………………………………….….…... 39
3.3.6 含多醣類之膠原蛋白基質中多醣類分佈情況………….……. 40
3.3.7 多醣類對於膠原蛋白纖維形態的影響……………………….. 40
3.3.8 添加多醣類對基質熱穩定性的探討………………………….. 40
3.3.9  含多醣類之膠原蛋白基質的孔洞結構……………………….. 41
3.3.10 含多醣類之膠原蛋白基質孔洞大小範圍之評估…………….. 41
3.4  討論…………………………………………………….………… 42
第四章 含不同多醣類的膠原蛋白基質對於細胞及傷口癒合
之影響………………………………………………………… 65
4.1  實驗目的……………………………………………………….… 65
4.2  實驗材料與方法………………………………………….……… 65
4.2.1  實驗材料…………………………………………………..…… 65
4.2.2 多醣類影響細胞遷移能力之試驗………………..…………… 65
4.2.3 多醣類影響細胞增生能力之試驗………………..…………… 66
4.2.4 基質吸水性試驗…………………………………..…………… 66
4.2.5 動物傷口外觀閉合情形…………………………..…………… 66
4.2.6 動物傷口癒合後組織型態觀察…………………..…………… 66
4.2.7 動物傷口皮膚厚度量測…………………………..…………… 66
4.3  實驗結果………………………………………….....…………… 67
4.3.1  含不同多醣類之膠原蛋白基質對細胞遷移的影響………….. 67
4.3.2  含不同多醣類之膠原蛋白基質對細胞增生的影響………….. 67
4.3.3  含不同多醣類之膠原蛋白基質吸水性測試………………….. 67
4.3.4 含不同多醣類之膠原蛋白基質移植後傷口外觀閉合
之情形………………………………………………………… 68
4.3.5 含不同多醣類之膠原蛋白基質移植六個月後之
組織型態...…………………………………………………… 68
4.3.6 含不同多醣類之膠原蛋白基質移植六個月後之
皮膚厚度情形...……………………………………………… 68
4.4 討論……………………...………………………………….…… 69
第五章 結論…………………………...…………………………..…… 83
第六章 文獻參考……………………………...…………………..… 85
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