Signaling Pathway Controlling the Pathogenesis of Exposure Keratopathy (Evaporative Dry Eye, Desiccation Stress)---An in vivo and in vitro Study on Cell-Cell Junction, Cytokine Secretion and Inflammation

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Project title/計畫英文名

Project Number/計畫編號

MOST103-2314-B002-074-MY3

Translated Name/計畫中文名

探討暴露性(高揮發性)乾眼症在病理機轉上的重要訊息傳遞路線---針對「細胞-細胞間隙」「細胞激素的分泌」以及「發炎反應」的體外及體內實驗

Project Principal Investigator/計畫主持人

WEI-LI CHEN

Funding Organization

Ministry of Science and Technology

Co-Investigator(s)/共同執行人

胡芳蓉

Website

link

Start date/計畫起

01-08-2016

Expected Completion/計畫迄

12-07-2017

Description

Abstract

摘要：乾眼症為重要且常見的眼科疾病。其中「過度揮發性乾眼症」佔極大的比例。近年來台灣成立國家眼庫，角膜若在捐贈前過度暴露揮發將影響角膜的品質:加上現代人重度使用 3C產品，也導致「過度揮發性」乾眼症的風險。過去「過度揮發性乾眼症」的訊息傳遞與病理機轉未被充分研究。本實驗室做過前驅試驗，首度發現角膜在暴露一小時內，眼表面細胞就出現型態變化和發炎反應；如此早期顯的變化從甚少在文獻上報導過。由於本實驗室在眼表面研究極有經驗且有諸多論文發表，本計畫乃針對「過度揮發性乾眼症」進行研究；利用「動物實驗及細胞培養實驗模式」，模擬不同程度的「高度揮發性乾眼症」；逐年進行研究:(第一年)紐西蘭白兔經過 3, 6, 12, 24小時的角膜暴露後，研究眼表面上皮細胞型態、細胞間隙蛋白和細胞凋零行為變化。實驗中使用到免疫染色法(ZO-1、E-cadherin、beta-catenin, p120)、穿透式電子顯微鏡、活體共軛焦顯微鏡、RTPCR等方法。另探究 Notch 和Wnt-beta catenin這兩個候選訊息傳導路徑的角色。(第二年) 紐西蘭白兔經過 3, 6, 12, 24小時的角膜暴露後，對於淚液或細胞本身cytokine分泌，和發炎細胞浸潤的影響。實驗中收集眼淚和角膜上皮細胞，利用multplex bead analysis和 RTPCR偵測各種 cytokine的表現量(IL-1, IL-2, IL-4, IL-13, IFN-gamma和 TNF-alpha)；利用活體共軛顯微鏡偵測發炎細胞的浸潤，並用免疫螢光染色法鑑定發炎細胞類別。另外探究 NF-kB訊息傳導路徑是否扮演重要腳色。(第三年)角膜細胞培養環境下探討高揮發狀況(空氣暴露 1, 2, 3天)對於細胞的型態、細胞間隙蛋白和細胞發炎激素分泌的影響。實驗利用到免疫染色法(ZO-1、E-cadherin、beta-catenin, p120)、穿透式電子顯微鏡、RTPCR，multplex bead analysis和 RTPCR，偵測 cytokine(IL-1, IL-2, IL-4, IL-13, IFN-gamma和TNF-alph)隨著暴露時間的表現量變化,另外也設計在培養過程中加入了 NF-kB，Notch 和Wnt-beta catenin的促進或抑制藥品，藉此釐清這幾個訊息傳導路徑的腳色。本計畫設計明確，從各角度周延進行研究，相信對基礎醫學的知識，以及乾眼症治療，都有顯著的影響。
Abstract: Dry eye syndrome is a common and important diseases that bother many patients. Clinically, it can be classified into (1)aqueous tear deficiency (2) evaporative dry eyes (desiccating stress). Recently, The set up of National Taiwan Eye Bank points out the importance of protecting donor corneas from overexposure (desiccating stress) to decrease the damage of corneal surface before donation. The tendency that modern people over-read a lot of 3C screens may also lead to the decreased eye blinking, and cause evaporation dry eye. Although over-evaporative dry eye is so important, the underlying pathogenesis and signal transduction pathway has seldom been studied. Recently, our lab performed preliminary experiments on desiccating stress. We found that even cornea exposure was less than 1 hour, significant corneal epithelial morphological changes and inflammatory cell infiltration was found. To the best of our knowledge, such results has never been studied before. Since our lab have quite a lot of experience of performing in vivo or in vitro ocular surface studies, and published many papers on outstanding journals, we plan to continue the study, and use in vivo and in vitro experiment to mimic the evaporative dry eye (desiccating stress). In the first year, we will focus on the effects of desiccation stress (3,6,12,24 hours) on the morphology and cell-cell junction of corneal epithelial cells on New Zealand White rabbits. We will also evaluate the apoptosis behavior. Immunohistochemistry (ZO-1、E-cadherin、beta-catenin, p120), transmission electron microscopy, in vivo confocal microscopy and RTPCR will all be performed. We will also detect the possible role of Notch or Wnt-beta catenin signal transduction pathways. In the second year, we will focus on the effects of desiccation stress (3,6,12,24 hours) on the inflammation reaction on the ocular surface of New Zealand white rabbits. We will detect the cytokine expression (IL-1, IL-2, IL-4, IL-13, IFN-gamma and TNF-alpha) on tear film or the corneal epithelial cells by multplex bead analysis and RTPCR. We will also detect the time-sequential changes of inflammatory cells infiltration by in vivo confocal microscopy and IHC. The possible role of NF-kB signal transduction pathway will also be studied. In the third year, we will use in vitro corneal epithelial cell culture model to detect the effect of desiccation (airlift for 1, 2, and 3 days after confluency) on cellular morphology, cell-cell junctional proteins and cytokine expression. We will perform IHC (ZO-1 , E-cadherin, beta-catenin, p120) and transmission electron microscopy to detect cell morphology and cell-cell junction. RTPCR, multplex bead analysis and RTPCR will be performed to detect cytokine expression (IL-1, IL-2, IL-4, IL-13, IFN-gamma and TNF-alph) from cultivated corneal epithelial cells. To further identifying signal transduction pathways, we will inhibit or promote the signal transduction pathway of NF-kB, Notch or Wnt-beta catenin, and further define the roles of these three pathway under desiccating stress. Our study is well designed, and thorough. We anticipate the result will improve our limited knowledge about evaporative dry eye, and influence the clinical treatment of this important disease.