- 學位論文
以體外培養及體內刺激重建角膜內皮
Corneal endothelial regeneration through in vitro culture and in vivo stimulation approaches
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摘要
角膜內皮細胞對於調節角膜含水量及維持透明度十分重要，嚴重功能失調將造成角膜水腫，需要接受移植手術以恢復視力，雖然近年來手術技術持續進步，但是仍面臨器官來源不足及術後細胞數持續下降等問題，因此需要發展其它治療方式。 近年研究發現，對於不同程度及原因的角膜內皮疾病，可使用不同替代治療方式。早期的福斯氏角膜內皮失養症，由於病灶集中於角膜中央，因此可直接刮除中央病變區域，輔以促進細胞遷移藥物，使週邊功能相對健全的細胞往中央爬行。在嚴重角膜內皮疾病，則可使用體外培養角膜內皮細胞輔以細胞注射重建角膜內皮功能。然而，此兩種治療策略都有需要克服的問題。對於細胞注射治療而言，成功的關鍵在於培養出足夠數量及品質的細胞。人類角膜內皮細胞生長不易，雖可於培養過程中加入促進生長之藥物或激素，但此舉會導致角膜內皮細胞間質化，影響細胞產品的型態及功能。對於角膜內皮刮除治療而言，成功的關鍵在於促進細胞爬行，雖然近年發現Rho相關蛋白酶抑制劑有此作用，但其機轉不明，此外Rho相關蛋白酶抑制劑會影響細胞連結組成，破壞角膜內皮細胞屏障功能，因此尋找其它治療標的實屬必要。 本研究發現，角膜內皮細胞培養過程中會有間質化的現象，且基質金屬蛋白酶的表現有相對應的增加，若加入馬立馬司他此廣效型基質金屬蛋白酶抑制劑，可抑制角膜內皮細胞間質化，且神經鈣粘蛋白的切斷有相對應的減少，顯示兩者的關聯性。此效果另可與鹼性成纖維細胞生長因子結合，在促進角膜內皮細胞生長後，抑制其造成之細胞間質化的現象。在促進角膜內皮細胞遷移方面，本研究發現Rho相關蛋白酶及肌蛋白抑制劑都可促進角膜內皮細胞遷移及其方向性，機轉則是透過抑制細胞收縮，增加板狀偽足伸出持續時間，相較於Rho相關蛋白酶抑制劑，肌蛋白抑制劑能保留細胞連結份子及維持屏障功能完整，於動物模式中，肌蛋白抑制劑也較Rho相關蛋白酶抑制劑能消除角膜內皮傷害後的水腫。 基於上述研究成果，我們發現不同小分子藥物可分別應用於促進角膜內皮細胞培養品質，以應用於後續細胞注射治療，以及促進角膜內皮病灶刮除後的細胞遷移。此成果將有助於角膜內皮疾病的治療。
並列摘要
The corneal endothelium plays an important role in maintaining corneal clarity through regulating the hydration status of the corneal stroma. Severely dysfunctional corneal endothelium will lead to irreversible corneal edema that necessitates corneal transplantation to restore vision. Despite the advancement in surgical technique, there are still unmet needs, such as organ shortage and continuous corneal endothelial cells (CECs) loss postoperatively. Recently, different strategies have been developed to treat different corneal endothelial diseases. Early Fuchs dystrophy can be treated by simple descematorhexis to remove centrally-located lesions followed by migration-stimulating agents. For those afflicted with severe corneal endothelial diseases, in vitro culture of CECs followed by cell injection therapy has been shown to regenerate corneal endothelium. However, several issues remain to be addressed for these alternative treatments. Human CECs are known to have limited proliferative potential. Stimulating the growth of CECs often accompanies endothelial-mesenchymal transition (EnMT) and loss of endothelial function. On the other hand, rho-associated protein kinase (ROCK) inhibitor has been shown to promote CEC migration after descematorhexis, but the mechanism remains elusive. Furthermore, disruption of cell junction and thus barrier function are still concerns. In the first part of my study, I found that during in vitro culture, bovine CECs underwent EnMT and had an up-regulated expression and activity of matrix metalloproteinases (MMPs). Inhibition of MMP activity by marimastat, a broad-spectrum MMP inhibitor, in confluent bovine CECs suppressed the cleavage of N-cadherin and the EnMT process. Marimastat also suppressed EnMT triggered by basic fibroblast factor both in vitro and in vivo. In the second part of my study, I found that inhibition of either ROCK or non-muscle myosin II (NMII) promotes migration and directional persistence through increasing lamellipodial protrusion persistence, thus accelerated wound healing in vivo. Unlike ROCK inhibitor, directly targeting NMII preserves junctional integrity and barrier function upon reaching cellular confluence, which resulted in more rapid deturgescence in vivo. Based on these findings, we proposed that MMP inhibitor can be used to suppress EnMT, thereby enhancing the quality of the cell product designated for cell therapy. In addition, NMII inhibitor can be used to promote CEC migration following descematorhexis. These results will facilitate alternative treatment approaches for corneal endothelial diseases.
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