臺灣博碩士論文加值系統

近幾年之內利用組織工程技術來進行角膜上皮細胞的重建與修補，是以羊膜為主要的治療工具，本研究主要是將角膜上皮細胞培養在幾丁聚醣薄膜上，期望相比於羊膜具治療的效能。我們進而將幾丁聚醣作為奈米粒子，並利用奈米粒子的特性去做些相關的研究。
我們利用了MTT、免疫細胞化學染色與器官培養來比較幾丁聚醣薄膜與羊膜培養角膜上皮細胞的差異，由結果可以發現角膜上皮細胞在幾丁聚醣薄膜上具有良好的活性，可以證明幾丁聚醣是ㄧ具有良好生物相容性的生醫材料。而在免疫細胞化學染色與器官培養的部份來看，我們可以發現幾丁聚醣薄膜與羊膜的結果具有ㄧ制性，這也可以證明幾丁聚醣薄膜具有生物方面的活性可讓角膜上皮細胞重建或修補。而在奈米粒子的部份，我們利用了不同的染劑去證實胞飲作用；並將幾丁聚醣奈米粒子當作基因轉殖的載體，去與微脂粒做比較；也做了不同大小奈米粒子下對角膜上皮細胞的MTT與LDH影響，發覺幾丁聚醣奈米粒子並不具有明顯的毒性且在130nm的奈米粒子影響下，角膜上皮細胞的數目有增加的趨勢。

In recent years, amniotic membranes transplantation is the most important advancement of treatment modality to treat ocular surface diseases. However, there are a lot of flaws for amniotic membranes transplantation. For example, the ethic issue and contamination of amniotic membrane make us to seek a new material to replace the usage of amniotic membrane. The purpose of this project is to evaluate the possibility of replacement of amnitoic membrane with chitosan membrane, which is a biocompatible materials used in many fields of biomedical science. We cultivated bovine corneal epithelial cells on chitosan membranes and evaluated their phenotypes.
MTT assay, indirect immunocytochemical staining and organ culture were used to compare the differences and evaluate the efficacy of treatment of corneal epithelial defects between amniotic membranes and chitosan membranes. According to the MTT assay, we found that the chitosan membrane was a good biomaterial for cultured corneal epithelial cells. In addition, the results of indirect immunocytochemical staining and organ culture, we found that chitosan membrane was compatible and as good as the use of amniotic membrane. Our results indicated that chitosan membrane is a good material for corneal tissue regeneration and repair.
For nanoparticle studies, we used different dyes to study endocytosis of chitosan nanoparticles in cultured cells. We found that chitosan nanoparticles could transfect cultured cells with a less effeciency than the liposome. From the results of MTT and LDH assays, we found that chitosan nanoparticles were low toxic and 130 nm of chitosan nanoparticles were helpful to increase cell numbers.

目錄............................................................................................................I
中文摘要..................................................................................................IV
英文摘要...................................................................................................V
圖表目錄.................................................................................................VII
第一章 背景..............................................................................................1
第二章 文獻回顧......................................................................................6
2-1高分子薄膜...................................................................................6
2-2 羊膜..............................................................................................7
2-3角膜上皮細胞的簡介...................................................................8
2-4幾丁聚醣奈米粒子…...................................................................8
第三章 材料與方法................................................................................10
3-1 實驗材料....................................................................................10
3-2 實驗儀器....................................................................................12
3-3 試劑配製....................................................................................16
3-4幾丁聚醣薄膜的製備.................................................................17
3-5分離純化角膜上皮細胞.............................................................18
3-6角膜上皮細胞的培養.................................................................19
3-6-1薄膜的前處理...................................................................19
3-6-2細胞的培養.......................................................................20
3-7細胞於材料上生長與死亡之評估.............................................20
3-7-1 MTT測試（評估細胞生長的情形）..................................20
3-7-2 LDH測試（評估細胞死亡的情形）..................................21
3-8間接免疫細胞化學染色.............................................................22
3-9器官培養.....................................................................................23
3-10奈米粒子粒徑大小與表面電量的測定...................................24
3-10-1製作幾丁聚醣奈米粒子.................................................24
3-10-2測定大小與電位.............................................................25
3-11奈米粒子的改質........................................................................26
3-11-1接枝螢光染劑.................................................................26
3-11-2接枝近紅外光染劑....….................................................27
3-11-3與脫氧核糖核酸結合...........................................……..27
3-12大小不同奈米粒子的MTT與LDH測試.................................29
第四章 結果與討論................................................................................30
4-1角膜上皮細胞在不同基材上培養情形：...................................30
4-2角膜上皮細胞對於不同大小奈米粒的活性與毒性測試.........31
4-3奈米粒子大小與濃度的關係.....................................................33
4-4角膜上皮細胞吞噬奈米粒子的情形.........................................34
4-5微脂粒與奈米粒子當作載體之轉殖.........................................36
4-6大小不同奈米粒子的MTT與LDH測試.................................37
4-7器官培養的情形………………………….................................40
第五章 結論............................................................................................43
參考文獻..................................................................................................44
附圖..........................................................................................................48

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