高齡化社會來臨，許多與長壽有關的疾病孕育而生，加上3C產品普及，使得眼科疾病成為顯學。許多眼科疾病會導致眼盲，如青光眼及黃斑部病變至今無可完全治癒的方法，標準治療只是症狀控制，例如青光眼藥物是透過降眼壓，減少因高眼壓導致的視神經細胞缺損。現有的黃斑部病變透過減少黃斑部新生血管，達到延緩病程目的。非動脈性缺血性視神經病變發生的非常快速且無症狀，在發生的數天內即會導致失明，甚至沒有常規療法。這些疾病的真正臨床致病因子至今尚未被完全了解，主要原因是目前的研究工具有限制，使得醫生無法取得可靠的療法。
Extracellular Vesicles（EVs）是奈米尺寸的囊泡，當中含量最豐的exosomes作為細胞釋放訊號的傳遞工具，在細胞與細胞間溝通扮演決定性角色。Exosomes中傳遞的訊號同時扮演免疫調節、細胞外基質周轉、幹細胞分裂/分化、新生血管形成和細胞廢物清除等關鍵功能。目前關於exosomes在癌症研究上已有非常深入探討，但對眼睛exosomes功能的研究尚淺。主因是眼睛組織分化程度非常高，且可取得的樣品量很少，以目前exosomes的萃取方法，如超高速離心及梯度離心等，都需要大體積才能執行，因此以exosomes在細胞間傳遞的訊號研究多屬離體細胞等級，除非是可大量取得的樣本，如血液或細胞培養基等。
本實驗室於2014年開發出紙基平台晶片，透過以特定抗體，如anti-CD63抗體，直接進行眼房水的CD63 exosomes研究，突破微量體積樣品的研究限制，但尚缺可直接從紙基平台萃取exosomes的方法，故無法詳細調查exosomes的功能（functional assay）或是進行其內部訊號的完整圖譜分析（profiling）。本研究改良此法，利用一個在UV照射下不穩定鍵結（photolabile）取代原先的不可斷裂鍵結，以此紙基晶片萃取樣品後，僅需透過短暫紫外光照射，即可將exosomes從晶片上完整分離下來。
以大鼠非動脈性缺血性視神經病變模式（rNAION）收集的微量玻璃體，在不同的自然病程回復時間收集樣品，驗證本研究建立的光斷裂紙基晶片效能，並進行CD63 exosomes內微小核糖核酸的微陣列分析。本研究著重於分析與免疫反應相關的促進發炎（M1）及抗發炎（M2）的微小核糖核酸訊號分析，並以定量qPCR確認。研究發現CD63 exosomes中有5個與M1/M2巨噬細胞調控有關的微小核糖核酸有明顯變化，包含微小核糖核酸-31a-5p、微小核糖核酸-125a-5p、微小核糖核酸-182、微小核糖核酸-181a-5p、微小核糖核酸-124-3。經過文獻探討及動物實驗結果篩選出微小核糖核酸-124是當中最具有視神經保護潛力的微小核糖核酸。推測在非動脈性缺血性視神經病變發生時立即投藥，可能具有調控免疫保護視神經節細胞的功能。故設計試驗，在rNAION模式誘導後，立即注入人工合成的微小核糖核酸-124於大鼠玻璃體中，結果發現的確可以有效降低rNAION造成的視神經細胞死亡，並維持視神經訊號傳輸，達到視神經保護功用。
本研究證明以光斷裂紙基晶片收集微量樣品具實用性，利用此晶片可簡單的收集微量樣品中具特定生物標記的exosomes。未來透過本晶片可快速、簡易及有效的收集微量活體樣品中的exosomes、胞器、細胞，無論是從微量血液或是胚胎細胞的培養基等。透過本研究證明以此晶片萃取出的RNA，純度及數量足以完成如微小核糖核酸、基因體學、表觀遺傳學或蛋白質體學研究，加速研究速度，並直接以微量活體樣品分析生物標記與疾病關聯性，深入了解難解疾病的成因。

The aging society comes and aging associated diseases are commonly seen in older patients. The widespread use of 3C products making eye disease becomes an important issue in human life. Ophthalmic diseases such as glaucoma and age-related macular degeneration（AMD）are still incurable. Current treatment slows but cannot stop the progress of blindness. Standard treatments are mainly symptomatic control. Glaucoma drugs through lowering intraocular pressure（IOP）reduce high ocular pressure-induced cell damage of optic nerve. Current treatment of AMD delays retinopathy by decreasing retinal neovascularization. Non-arteritic anterior ischemic optic neuropathy（NAION）refers to loss of blood flow to the optic nerve. This condition typically causes sudden vision loss in one eye, without any pain. The main causes of the diseases have not been clarified because restricted research tool on ophthalmology which prevents the doctor from prescribing the disease.
Extracellular vesicles（EVs）are nano-sized cellular particles often bring informative molecules between cells. The most EVs, exosome, plays a key role in immune regulation, extracellular matrix turnover, stem cell division/ differentiation, neovascularization and cell waste remover. The role of exosomes in cancer has been explored in depth, but the functions of exosomes in eyes has just begun. The research hurdle is to obtain highly differentiated exosomes in vitreous humor. Current methods to collect exosomes by ultra-high-speed or gradient centrifugation etc. require large volume. Hence, many studies about exosomes are merely in vitro, except blood.
We developed a paper-based platform to obtain exosomes from clinical samples in 2014. It provided a tool to study small volume of aqueous humor in human, but the method cannot release exosomes from the paper platform. In this research, we used a photo-labile linker to replace the original stable linker as a bridge between antibody and paper. We are able to take the captured exosomes down from the paper through a short-term ultraviolet light exposure.
Through this designation, we study the miRNAs profile change from obtained CD63-exosomes in vitreous humor during a natural recovery process in nonarteritic anterior ischemic optic neuropathy rats（rNAION）. We focus on the miRNAs about macrophage differentiation, include M1 proinflammatory miRNAs and M2 anti-inflammatory miRNAs. Five M1/M2 related miRNAs（miRNA-31a-5p, miRNA-125a-5p, miRNA-182, miRNA-181a-5p, miR-124-3）are found dramatic change, and they are confirmed by quantitated PCR. MicroRNA-124 was hint its anti-inflammatory and optical neuroprotective effect in literatures and animal study. The artificial microRNA-124 was synthesized, and then we first proved its neuroprotective effect after intravitreous injection（IVT）in rNAION.
In this study, we develop a photolabile chip is simple and easy production and can capture specify antigen-exosomes in rare vitreous humor. In the future, the chip can be a useful tool to collect exosomes, organelles, cells, rare blood sample or embryonic culture medium. This tool can easily extract abundant information such as miRNAs, genomics, epigenetics, and proteomics in rare clinical samples. It will improve to understand more disease reasons in the future.

中文摘要 i
英文摘要 iii
致謝 v
圖目錄 vii
表目錄 ix
第一章 前言 1
第二章 文獻探討 6
第三章 理論模式推導 11
第四章 試驗設備與方法 12
第五章 結果與討論 18
第六章 結論 44
參考文獻 45
附錄 51

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