開發含白芨多醣體之人工淚液應用於乾眼症之治療

Abstract

乾眼症為一日漸興起的眼類疾病；起因於患者淚膜的不穩定，進而造成眼表面發炎。人工淚液與抗發炎藥劑為常見乾眼症治療方式；然而，多數人工淚液僅具保濕效果而不具實際治療效果；抗發炎藥劑雖然具實際治療效果，但會造成患者視力模糊與眼睛有異物感等情形。因此，本研究欲結合兩者之優點，並排除其缺點，開發含白芨多醣體之抗發炎特性人工淚液，應用於乾眼症之治療。 本研究以FTIR、NMR與TGA確認萃取出白芨多醣體的官能基、結構與熱分解溫度；WST-1與Live and Dead實驗確認細胞在不同濃度白芨多醣體之生物相容性；DCFDA與Gene expression確認抗氧化與抗發炎效果，並找出最佳治療濃度；溶於Buffer solution後測定物理性質。動物實驗分為兩部分: 以Ocular retention test確認人工淚液之保濕效果；以乾眼症動物模型藉由對兔子進行Schirmer test、角膜厚度測量與角膜上皮螢光染色確認乾眼症誘導與治療效果；最後將兔子犧牲並以H&E染色確認治療效果。 FTIR、NMR與TGA結果顯示，和先前文獻結果均相同，顯示本研究成功製備白芨多醣體；WST-1與Live and Dead實驗結果則顯示L929與人類角膜上皮細胞在不同白芨多醣體濃度下均具良好生物相容性；DCFDA與Gene expression結果顯示低濃度白芨多醣體具較佳抗氧化與抗發炎效果。本研究所製備之人工淚液在折射率、酸鹼值、黏度與滲透壓大小均與真實人眼淚液大小相近。Ocular retention test中由IVIS拍照結果顯示白芨多醣體能有效提升人工淚液在眼表滯留時間，顯示具良好保濕特性；乾眼症動物模型中，明視野、裂隙燈與螢光染色照片顯示本研究成功誘導兔子眼表發炎；與BAC誘導組相比，兩治療組別之螢光殘留量均下降，且含有白芨多醣體組螢光殘留量最低，顯示含藥物之人工淚液具一定治療效果。H&E染色角膜切片結果顯示，含有白芨多醣體之人工淚液組別之角膜上皮厚度與Control組相當，且基質層緻密，具有較佳治療效果。 由本研究材料分析、細胞實驗與動物實驗結果顯示，本研究成功開發具良好生物相容性，並具一定乾眼症治療效果之人工淚液。

Dry eye syndrome (DES) is now a very prevalent eye related disease, which is caused by the instability of tear film and results in the inflammation of ocular surface. Artificial tears and anti-inflammatory agents are two most comely used treatment methods for DES patients. However, most of the artificial tears can only moisturize but do not have substantial treatment effect, and anti-inflammatory agents may cause the patients have blurred vision and foreign body sensation although having substantial treatment effect. Hence, the purpose of this study is to combine the merits of artificial tears and anti-inflammatory agents, to develop artificial tears containing Bletilla Striata Polysaccharide (BSP) for DES treatment. In this study, FTIR, NMR and TGA tests are used to confirm the functional groups, structure and thermal decomposition temperature of BSP. WST-1 and Live and Dead tests are used to confirm the biocompatibilities of L929 and human corneal epithelial cells (HCEC) in different concentrations of BSP. DCFDA and Gene expression tests are used to confirm the anti-oxidant and anti-inflammatory effects of BSP, and to find the best treatment condition. Confirm the physical properties of artificial tears by testing refractive index, pH value, viscosity and osmolality. Use IVIS to confirm the moisturizing effect of BSP. For DES animal model, confirm the inducement effect and treatment effect by doing Schirmer test, fluorescence staining and testing cornea thickness. Finally, confirm the treatment effect by H&E staining. The results of FTIR, NMR and TGA in the study are all the same to the previous studies, which indicate that BSP is successfully extracted. WST-1 and Live and Dead results show that both L929 and HCEC have good cell viabilities in different concentration of BSP. DCFDA and Gene expression result show that lower concentrations of BSP have better treatment effect. The refractive index, pH value, viscosity and osmolality of artificial tears in this study are all very close to those of real human tears. Photos taken by IVIS show that BSP contained artificial tears have longer retention time than that of the buffer only one. The result of Schirmer test, cornea thickness and fluorescence staining show that we successfully induced inflammation of rabbit’s ocular surface in this study, and the artificial tears of this study has treatment effect to some extent. The result of H&E staining of the cornea shows that the BSP contained group has similar corneal epithelial cells thickness to that of control group, and has denser stromal layer than that of Buffer group, which indicate that the BSP contained artificial tears has better treatment effect. From the results of material analysis, in vitro study and in vivo study, we successfully develop artificial tears that have good biocompatibility and treatment effect for DES animal model.