摘要：對人類而言，透明的眼角膜乃是擁有優良視力不可或缺的因子。若是角膜因各種原因包括角膜缺氧、鹼性物質灼傷、角膜輪部缺損或各種可能引起角膜發炎之疾病產生角膜新生血管，都有可能影響角膜的透明度。我們實驗室這幾年一直致力於角膜新生血管之研究，包括成功以結膜下注射bevacizumab 來治療兔子角膜新生血管，也嘗試使用另外兩個化學合成物來進行抑制角膜新生血管的研究，然而上述藥物治療僅能提供短效且限制性的臨床效用。MicroRNAs 就目前所知是一群non-coding 的RNA 分子，長度大約是十七至二十五個核甘酸，它們主要是藉由抑制細胞轉譯或是促使mRNA 分解來達成其調節基因的作用。因為microRNA 與基因是一對多的調控關係，所以抑制或投入相關的microRNA 較可以廣泛性治療複雜性的疾病，如血管新生。現今使用RNAi 來進行選擇性序列之基因抑制治療皆有初步成效，包括在癌症心肌梗塞及脈絡膜新生血管的治療。本研究的主要目的即是希望釐清各個血管形成控制因子與microRNAs 在角膜血管新生的狀況，以及測試以miRNA 為角膜新生血管的治療療效。第一年計畫主要是研究人類及兔子之結膜、輪部及角膜上皮細胞在不同時間之缺氧條件下，藉由流式細胞儀標的不同的血管形成因子，將性質不同的角膜上皮細胞分類後，分別研究其miRNA 表現，並分析培養液中之VEGF 濃度變化。再藉由knockdown 或ectopic expression 的方法來執行且確定所找出之miRNA 指標。第二年則執行兩種角膜新生血管動物模式(缺氧模式及化學灼傷模式)，以動物之組織研究前一年之miRNA 指標。第三年則是將結果執行於角膜新生血管動物模式，經角膜內或結膜下注射miRNA或其抑制物來應證體外實驗。我們相信此研究將能提供相當貢獻於角膜新生血管之標靶治療。
Abstract: Corneal transparency is essential for clear visual pathway. Corneal NV, a consequence ofanterior segment inflammation and injury, induce corneal opacity, affect visual acuity,increase the risk of graft rejection after penetrating keratoplasty, and cause complications ofthe ocular surface surgeries. For many years, our laboratory has successfully usedsubconjunctival injection of bevacizumab (human anti-VEGFmonoclonal antibody) to treatvarious rabbit models of corneal neovascularization. However, bevacizumab has followingdrawbacks: expensiveness, difficulty in storage, poor corneal penetration and the risk ofimmunological adverse reactions.MicroRNAs (miRNAs) are a class of 17-25 nucleotides non-coding RNA molecules thatregulate gene expression by either translational inhibition or mRNAs degradation. Becauseindividual miRNAs can regulate the expression of multiple target genes, manipulatingmiRNA expression can influence an entire gene network and thereby modify complex diseasepathology. Nowadays, application of sequence-selective gene inhibition by RNAi astherapeutic strategy becomes a promising alternative of treatment, such as in the treatment ofcancers, ischemic heart disease and choroidal neovascularization. Therefore, our proposedproject intends to elucidate the interplay between various angiogenesis regulatory moleculesand microRNAs (miRNAs) in the signal transduction pathway that are involved in cornealneovascularization (NV). The efficacy of miRNA-oriented therapeutic strategy for clinicalNV treatment will also be examined.In the first year, primarily cultured human and rabbit conjunctival, limbal and central cornealepithelial cells will be used to characterize miRNAs expression levels under 0, 2, 4, 24 and 72hrs of hypoxia. The responsiveness of hypoxia will be evaluated by FACS gating on theexpression of different angiogenesis-associated molecules. Differential miRNAs profile ofexperiment groups under various degree of hypoxia treatment will be determined by Q-PCR.The amount of secreted VEGF will be examined by ELISA of culture medium from eachgroup of cells. We will ectopicly express the miRNAs that are down-regualated, orknockdown the miRNAs that are up-regulated under hypoxia treatment to validate the roles ofmiRNAs in corneal angiogenesis. In the second year of the project, two in vivo corneal NVmodels, which are contact lens-inducing hypoxia and alkali burn-inducing corneal NV, will beconducted. After inducing corneal NV, the contribution of these miRNAs and associatedproteins will be evaluated by in situ hybridization, immunohistochemistry stain and real timePCR analysis. The result can verify our in vitro finding of year 1. Finally in the last year, thepotential of corneal NV treatment by miRNAs-oriented therapeutic strategy will be examined.We will compare the efficacy and efficiency of intrastromal or subconjunctival injection ofnaked miRNAs versus vector-based miRNA delivery system, including nanoparticle-based,lipid-based, polymer-based, and peg-liposome based vectors. The results will be determinedby biomicroscope observation of these experimental animals. We believe this study can opendoor for better targeted therapy of corneal NV.