幾個世紀以來，白內障一直是損害視力的全球性問題。據估計，在未來20年內，白內障患者的數量將增加約三分之一，並且白內障仍將是世界所有地區視力損害的主要原因。蛋白質聚集是白內障形成中最重要的因素;紫外線輻射是眾所周知的危險因素。基本上，蛋白質聚集的過程是不可逆轉的;但最近的研究表明，有一種化合物為羊毛甾醇能夠逆轉白內障的形成過程，這表明白內障是可以被治癒的。然而目前治療方式而以侵入式的方式治療，因此在本研究中我們的目的是使用新的治療方式治療白內障。首先我們使用成年純種斑馬魚，每天照射UVC每條魚右眼325 mJ / cm2。在平均照射7天後，他們所有的右眼都患上白內障，但左眼仍然正常。之後使用敷藥的方式將羊毛甾醇覆蓋在眼球上已達到類似於滴眼藥水的方式來做治療。而因為知道使用Lanosterol持續治療是有效果的，因此我們將做一組藥效實驗測試Lanosterol停止給藥後還能維持多久的治療效果。最後我們使用兩種方式觀察在未切片之前的白內障是否有被治療，從單純觀察的結果初步判定是有效果的，因此我們將用三種不同的染色方法做確認。本研究結果發現使用未侵入式的方式也是有治療的方式，這或許對於眼科會是一種全新的治療方式，從切片的結果我們可以清楚的看出治療前與後的明顯差異，這代表使用敷藥的方式是可以有效地進行治療。

For centuries, cataracts have been a global problem that undermines vision. It is estimated that the number of cataract patients will increase by about one-third in the next 20 years, and cataracts will remain the leading cause of visual impairment in all parts of the world. Protein aggregation is the most important factor in the formation of cataracts; ultraviolet radiation is a well-known risk factor. Basically, the process of protein aggregation is irreversible; but recent studies have shown that a compound called lanosterol can reverse the formation of cataracts, suggesting that cataracts can be cured. However, current treatments are treated in an invasive manner, so in our study we aim was to treat cataracts with new treatments. First we use an adult purebred zebrafish that illuminates the right eye of each fish with 325 mJ / cm2 per day. After 7 days of average exposure, all of their right eyes developed cataracts, but the left eye was still normal. After the application of the lanosterol on the eyeball by means of application, the treatment is similar to that of eye drops. And because we know that continuous treatment with lanosterol is effective, we will do a set of pharmacodynamic tests to test how long lanosterol can last after stopping the dosing. Finally, we used two methods to observe whether the cataract before the unslicing was treated. It is effective to judge from the results of the simple observation, so we will confirm it with three different staining methods. The results of this study found that the use of non-invasive methods is also a treatment, which may be a new treatment for ophthalmology. From the results of the test, we can clearly see the obvious difference between before and after treatment. The method of application is effective treatment.

謝誌------------------------------------------------------I

中文摘要--------------------------------------------------II

英文摘要-------------------------------------------------III

目錄------------------------------------------------------V

圖目錄--------------------------------------------------VII

表目錄-------------------------------------------------VIII

一.序論

1.眼球構造-------------------------------------------------2
2.晶狀體蛋白之功能與白內障產生------------------------------3
3.何謂紫外光-----------------------------------------------6
4.紫外光導致白內障原因--------------------------------------7
5.皮質性白內障等級-----------------------------------------8
6.白內障目前治療方式---------------------------------------9
7.Lanosterol的發現---------------------------------------10
8.Lanosterol治療白內障的分子機制---------------------------11
9.研究動機------------------------------------------------12
10.研究方法-----------------------------------------------14

二.實驗材料

1.藥品試劑------------------------------------------------15
2.儀器設備------------------------------------------------15
3.藥品溶液配置--------------------------------------------16
4.海綿製備------------------------------------------------16
5.切片抗染試劑--------------------------------------------17

三.材料方法

1.斑馬魚來源與養殖----------------------------------------18
2.麻醉藥劑量參考與配置-------------------------------------18
3.斑馬魚保護方式------------------------------------------19
4.拍攝斑馬魚眼球方式--------------------------------------19
5.UVC crosslinker使用方式---------------------------------20
6.敷藥治療方式--------------------------------------------20
7.拍照設備與參數------------------------------------------21
8.眼球用包埋與滲蠟之步驟-----------------------------------22
9.石蠟切片------------------------------------------------22
10.脫蠟--------------------------------------------------23
11.眼球切片染色-------------------------------------------23
12.免疫組織抗體染色---------------------------------------24

四.結果

1.使用UVC找出最佳照射強度與白內障分級----------------------26
2.誘發白內障產生時間--------------------------------------28
3.持續治療實驗與結果--------------------------------------29
4.四組實驗組織比較----------------------------------------32
5.藥效實驗與結果------------------------------------------35
6.藥效分析與結果------------------------------------------37
7. 切片與染色---------------------------------------------38

五.討論--------------------------------------------------41

六.參考文獻-----------------------------------------------45

七.附錄--------------------------------------------------49

圖目錄

附錄1. 眼球結構簡介----------------------------------------2
附錄2. 水晶體蛋白質之組成-----------------------------------3
附錄3. 三種晶狀體蛋白結構圖---------------------------------4
附錄4. 白內障產生之流程-------------------------------------5
附錄5. Lanosterol在人體內產生之步驟------------------------11
附錄6. Lanosterol抑制白內障產生之過程----------------------11
附錄7. 人類與斑馬魚眼球構造示意圖---------------------------12

圖1. 防止照射UV使用之海綿----------------------------------16
圖2. 敷藥用海綿-------------------------------------------17
圖3. 拍攝斑馬魚眼球側面圖----------------------------------19
圖4. 拍攝斑馬魚眼球俯視圖----------------------------------20
圖5. (A) 利用照射UV的方式誘發斑馬魚產生白內障---------------27
圖5. (B) 使用不同UV劑量測試白內障的產生---------------------27
圖5. (C) 斑馬魚白內障分級---------------------------------28
圖6. 斑馬魚出現白內障的時間表------------------------------29
圖7. (A) 持續治療實驗流程圖--------------------------------30
圖7. (B) 持續治療結果圖-----------------------------------31
圖7. (C) 持續治療方法結果圖與死亡隻數-----------------------32
圖8. 持續治療結果分析圖------------------------------------34
圖9. (A) 效實驗與結果圖 ----------------------------------35
圖9. (B) 藥效實驗結果圖-----------------------------------36
圖9. (C) 為藥效的死亡隻數---------------------------------36
圖10. 藥效結果分析圖--------------------------------------38
圖11. 切片與染色圖----------------------------------------40
圖12. 解剖水晶體說明--------------------------------------43
圖13. 水晶體內部的變化猜測---------------------------------44
圖14. 水晶體周圍深紅色物質---------------------------------44


表目錄

表1. 實驗所使用的藥品-------------------------------------15
表2. 實驗所使用到的儀器設備--------------------------------15

附表1.  不含Lanosterol成對樣本分析------------------------49
附表2.  含Lanosterol成對樣本分析--------------------------49
附表3.  No Treat和不包含Lanosterol藥物組別的獨立樣本分析----50
附表4.  No Treat和包含Lanosterol藥物組別的獨立樣本分析-----51
附表5. 包含Lanosterol與不包含Lanosterol藥物的獨立樣本分析---52
附表6  持續治療四組數據單因數變異數分析---------------------53

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