成人的角膜內皮細胞不具有再生能力。當角膜內皮細胞數量顯著減少會導致內皮層之排水功能失調，進而導致角膜水腫和不透明，嚴重影響視力。臨床上，角膜移植仍然是目前改善患者視力的唯一解決方法，但其往往受限於捐贈來源不足。此外，即使完成手術，移植的角膜可能因其本身品質不佳或受贈者免疫排斥等因素，導致移植角膜的內皮細胞密度下降，進而使移植體功能下降，甚至移植失敗。因此，若能開發一新型組織工程支架，用於培養角膜內皮細胞並支持其轉移到前房，或可做為捐贈角膜之替代品，進而有效解決捐贈角膜短缺或降低手術失敗風險。本論文擬利用明膠做為角膜內皮細胞培養與移植的支架，結合包覆有薑黃素之藥物緩釋系統，期望能藉由薑黃素之抗氧化及促進細胞增生等特性，改善移植細胞的存活率，並進一步提升移植成功率。實驗中，我們以單乳化方式製備出包覆有薑黃素的脂質-聚乳酸-乙醇酸複合微米粒子，並將其分散至明膠支架內部。實驗結果顯示，與未經載體包覆的薑黃素比較起來，包覆有薑黃素之複合微米粒子能夠更有效地提升角膜內皮細胞株B4G12細胞的增生速度，並增加其抗氧化能力。將此複合微米粒子與明膠薄膜結合成為支架後，可發現B4G12細胞可順利在支架表面貼附生長。以上實驗結果證實，本研究成功開發一新型組織工程支架，可應用於角膜內皮細胞的培養與移植，並降低氧化壓力對細胞的傷害，未來或有應用於角膜內皮細胞移植的潛能。

Human corneal endothelial cells (CECs) possess extremely limited regenerative potential. Dramatic loss of CECs results in endothelial dysfunction, thus leading to corneal edema and opacity and can be sight-threatening. Currently, corneal transplantation remains the only solution to improve patients’ visual acuity. However, worldwide shortage of donors continues. Additionally, the endothelial density of the harvested donor cornea can be reduced owing to various factors and thereby less than the threshold required for transplantation. Typically, a high pre-operative endothelial cell density is required in order to offset the postoperative cell loss and ensure graft survival. Biomaterial-based culture platform with enhanced structural support may provide a route for increasing the cell density of donor corneas or engineering artificial corneas. In this work, a curcumin loaded lipid-poly(lactic-co-glycolic acid) hybrid microparticles (MPs) was developed and embedded into a thin gelatin membrane, thus becoming a scaffold that can release curcumin continuously. Our in vitro results demonstrated that the corneal endothelial cell line B4G12 cells could attach and proliferate actively on the surface of the as-prepared gelatin scaffold. Additionally, the viability of cells under oxidative stress could be improved significantly, which could be attributable to the released curcumin. These experimental data indicated that the developed gelatin-based scaffold with sustained curcumin release capability might have great potential to be employed for the cultivation and transplantation of CECs.

摘要 I
ABSTRACT II
誌謝詞 III
目錄 IV
表目錄 VI
圖目錄 VII
第一章 緒論 1
1.1 角膜(CORNEA) 1
1.1.1 角膜上皮(Corneal Epithelium) 1
1.1.2 角膜基質(Corneal Stroma) 2
1.1.3 角膜內皮(Corneal Endothelium) 2
1.2 角膜移植手術 3
1.3 明膠(GELATIN) 5
1.4 薑黃素(CURCUMIN) 5
1.5 聚乳酸-甘醇酸(POLY LATIC-CO-GLYCOTIC ACID, PLGA) 7
1.6 脂質-聚合物複合型粒子載體(LIPID-POLYMER HYBRID PARTICLE CARRIER) 8
1.7 研究動機與實驗目的 9
第二章 材料與方法 12
2.1 薑黃素在水溶液中的穩定性測試 12
2.2 CUR@LIPID-PLGA HYBRID MP的製備 12
2.3 CUR@LIPID-PLGA HYBRID MP的特性分析 14
2.3.1. Cur@Lipid-PLGA Hybrid MP之大小與型態 14
2.3.2. Cur@Lipid-PLGA Hybrid MP包覆薑黃素之情形 14
2.3.3. Cur@Lipid-PLGA Hybrid MP於體外釋放薑黃素之情形 15
2.4 內含CUR@LIPID-PLGA HYBRID MP之明膠薄膜支架製備 15
2.5 CUR@MP/GELATIN支架特性分析 17
2.5.1. Cur@MP/Gelatin支架之型態 17
2.5.2. Cur@MP/Gelatin薄膜支架之光穿透度測試 17
2.6 細胞培養 17
2.7 細胞毒性分析 18
2.7.1. 未包覆之薑黃素 18
2.7.2. Cur@Lipid-PLGA Hybrid MP 18
2.8 薑黃素之抗氧化特性分析 18
2.9 建立裝載有角膜內皮細胞之CUR@MP/GELATIN支架 19
第三章 實驗結果與討論 20
3.1 薑黃素對角膜內皮細胞存活率之影響 20
3.2 薑黃素在水溶液中的穩定性測試 22
3.3 CUR@LIPID-PLGA HYBRID MP之大小與型態 22
3.4 CUR@LIPID-PLGA HYBRID MP之薑黃素包覆與釋放情形 24
3.5 CUR@LIPID-PLGA HYBRID MP對角膜內皮細胞存活率之影響 25
3.6 薑黃素之抗氧化性質分析 27
3.7 CUR@MP/GELATIN支架特性鑑定 28
3.8 建立裝載有角膜內皮細胞之CUR@MP/GELATIN支架 32
3.9 裝載有角膜內皮細胞之CUR@MP/GELATIN支架之抗氧化性質 34
第四章 結論 36
參考文獻 37

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