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研究生:林佩姿
研究生(外文):Pey-Tzy Lin
論文名稱:膠原蛋白立體基質中之血管新生研究
論文名稱(外文):The study of neovasculizationin a collagen scaffold
指導教授:黃玲惠
指導教授(外文):Ling-Hui HUANG
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2003
畢業學年度:91
語文別:中文
論文頁數:77
中文關鍵詞:血管新生基質膠原蛋白
外文關鍵詞:collagenscaffoldneovasculization
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隨著組織工程研究的蓬勃發展,對於幫助組織或器官之修復被視為重要課題之一,且新的組織生長與發育需要適當的血管用以傳送養分以及移除廢物,而目前許多研究發現許多血管新生生長因子具有幫助血管新生、組織修復的功能,因此,尋找適當的血管新生生長因子,以體外培養方式,來進行血管新生之實驗。本論文希望瞭解4種血管新生生長因子及其混合液的功能,進一步結合實驗室之前所研發之多孔狀膠原蛋白基質,進行血管新生之試驗。於實驗發現添加生長因子於二維培養下,對於細胞活性有顯著之影響,而於細胞爬行實驗上,會隨著不同生長因子、濃度及細胞而有所差異。因此進一步以三維立體培養方式進行共同培養內皮細胞及纖維母細胞於多孔狀膠原蛋白基質中,來評估並比較生長因子的添加對於細胞增生及血管新生之影響,於實驗發現添加生長因子於多孔狀膠原蛋白基質中的確可促進血管新生,且進一步利用組織免疫染色證實其管狀結構為細胞所構成,且其管狀結構周圍有基底膜生成。
In light of the rapid development of tissue engineering, angiogenesis has become one of the major issues in tissue and organ repair. Since the development of new tissues requires blood vessels to transport nutrient and remove wastes, it was found recently that, some angiogenic factors are capable of facilitating angiogenesis and tissue rehabilitation. Thus, this research aims at the screening of adequate angiogenic factors, and their ability to promote angiogenesis was investigated in an in vitro cell culture system. The functions of 4 various angiogenic factors was also examined. For a three-dimensional culture system, porous collagen matrices previously developed in our laboratory were applied.
It was found in this study that the adding of angiogenic factors under 2-D condition exhibited a significant effect on cell activities. Further, the concentration of angiogenic factors, the cell types, and various combinations of angiogenic factors were shown to affect the behavior of cell migration. Besides, a co-culture system of simultaneously cultivating endothelial cells and fibroblasts in porous collagen matrices were established to evaluate and compare the effect on cell proliferation and angiogenesis. It was found that the existence of angiogenic factors in porous collagen matrices did facilitate angiogenesis. The immunohistochemical staining was further carried out in this study to corroborate the tubular structure which was composed of cells, and basement membrane formation around these tubular structures.
目錄
中文摘要
英文摘要
目錄
表目錄
圖目錄
第一章 緒論…………………………………………………………………1
1.1 膠原蛋白………………………………………………………….……1
1.1.1 膠原蛋白的性質與結構………..…………………………………1
1.1.2 膠原蛋白在醫學上的應用………..………………………………2
1.2 血管新生………………………………………………………….……3
1.2.1 血管新生的過程……………………………………….…….……3
1.2.2 血管新生的影響因素………………………………….…….……5
1.2.3 血管新生的調控機轉………………………………….…….……8
1.2.4 血管新生對於組織工程之重要性…………….……….…….…..10
1.3 血管新生之培養模式……………………………………...…….……11
1.3.1 體外試驗模式…………….…………………………...…….……11
1.3.1.1 二維培養模式………….……………………….…...…….……12
1.3.1.2 三維培養模式………….……………………….…...…….……12
1.3.2 活體實驗模式………….……………………….…...…….…...…13
1.3.2.1 雞胚胎卵黃囊試驗法….…………………….…...……..…...…14
1.3.2.2 眼角膜上血管新生試驗.…………………….…...……..…...…14
1.3.2.3 The Matrigel plug assay.…………………….…...…..…..…...…15
1.3.2.4 Chamber assays.…………………………….…...…..…..…...…15
1.4 研究目的.………………………………………….…...…..…..…...…15
第二章 實驗儀器、藥品、與方法…………………….…...…..…..…...…17
2.1 實驗儀器………………………………….……….…...…..…..…...…17
2.2 實驗藥品………………………………….……….…...…..…..…...…19
2.3 實驗方法………………………………….……….…...…..…..…...…21
2.3.1 添加不同比例濃度之bFGF對於細胞活性之影響.…..…..….…21
2.3.2 添加不同比例濃度之bFGF對於細胞數目之影響.…..…..….…22
2.3.3 添加不同之生長因子對於細胞爬行之影響.……….....…..….…22
2.3.4 PI染色…………….………………….……….…...…..…..…...…23
2.3.5 三維細胞培養…….………………….……….…...…..…..…...…23
2.3.6 基質的包埋與切片.………………….……….…...…..…..…...…23
2.3.6.1 冷凍切片…….………………….……….……....…..…..…...…23
2.3.6.2 塑膠切片…….………………….……….……....…..…..…...…24
2.3.7 基質之組織切片H&E染色……….……….……....…..…..…...24
2.3.7.1 冷凍切片……….……….……………………....…..…..…...…24
2.3.7.2 塑膠切片……….……….……………………....…..…..…...…25
2.3.8 免疫組織化學染色法…….……………………....…..…..…...…25
2.3.9 Masson’s trichrome染色…….……………………....………...…25
第三章 生長因子對於細胞之影響…….……………………....……...…27
3.1 實驗目的…….……………………....……………………………...…27
3.2 實驗材料與方法…………………....……………………………....…27
3.2.1 實驗材料…………………....……..…………………………...…27
3.2.2 添加不同比例濃度之bFGF對於細胞活性之影響…………...…28
3.2.3 添加不同比例濃度之bFGF對於細胞數目之影響…………...…28
3.2.4 添加不同生長因子對於細胞爬行之影響………………..…...…29
3.4 實驗結果…………………....……..………...……………………...…29
3.4.1 添加不同比例濃度之bFGF對於細胞活性之影響…………...…29
3.4.2 添加不同比例濃度之bFGF對於細胞數目之影響…………...…29
3.4.3 添加不同生長因子對於細胞爬行之影響………….……………30
3.5 討論…………………....……..……….……..……………………...…31
第四章 三維立體基質培養.……..……….……..……………………...…32
4.1 實驗目的.……..……………………....……..……………………...…32
4.2 實驗材料與方法……………………....…....……….……………...…32
4.2.1 實驗材料……………………....…....……….……………...…….32
4.2.2 三維細胞培養………………....…....……….……………...…….33
4.2.3 生長因子對於刺激細胞增生之影響..…….………………...…...33
4.2.4 抗原表現情況………………....….....……………………...…….33
4.2.5 血管結構變化………………....….....……………………...…….33
4.2.6 血管管狀型態………………....….....……………………...…….33
4.3 實驗結果…………………………....…....……………………...…….33
4.3.1 生長因子對於刺激細胞增生及管狀生成之影響…………...…..34
4.3.2 生長因子對於抗原表現之情況……………………………...…..34
4.3.3 生長因子對於血管結構之變化……………………………...…..35
4.4 討論………………………………………...……………………...…..35
第五章 實驗結論……….……………………...……………………...…..36
第六章 文獻回顧……….……………………...……………………...…..38
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