臺灣博碩士論文加值系統

當角膜遭受到局部性腫瘤、化學性傷害或是頑固性角膜潰瘍等疾病時，完全角膜移植手術是可以用作治療選擇之一，角膜保存之目標為保存延長角膜內皮細胞之活性。Kalevar-Majumdar (K-M) medium是個新合成用於保存角膜之保存液，而為了要在低溫保存時增長角膜內皮細胞的存活率，額外添加幫助細胞膜修復之物質於保存液中，應該是有助於延長細胞之活性，現今學者普遍認為經由鈣離子的傳遞路徑可以引發exocytosis，進而促進細胞膜之修復。本次實驗之目的為分析經 K-M medium保存兔子角膜之結果，並且比較於保存液中另外添加鈣離子之效果，我們利用五個月大的雌性紐西蘭大白兔之角膜，分別將其保存於不同天數的K-M medium或額外加了1.05 mM 鈣離子的K-M medium中。於4℃下保存了3、6、9、11與14天後，將角膜取出以電子顯微鏡( SEM )觀察其角膜內皮細胞型態並計算其密度，且以茜素紅染色來分析內皮細胞之存活率，另外，再以TUNEL染色，以了解於4℃下保存之角膜其細胞凋亡之情形。
結果以K-M medium分別保存3天與6天之角膜，其內皮細胞型態與正常內皮細胞相似，而SEM結果，分別保存6天於加鈣與不加鈣離子之K-M medium之角膜，其細胞缺損率為19.8% 以及20.4%。而以茜素紅染色之結果，其角膜內皮細胞存活率分別為92.1%及93.3%。因此，以K-M medium保存角膜之有效天數應為6天，而額外添加鈣離子於保存液中的效果與沒有加鈣的保存效果相同，沒有顯著差異；另外，比較SEM與茜素紅染色評估存活細胞之效果，SEM能夠準確呈現存活的角膜內皮細胞型態，而茜素紅須再搭配可以分辨死亡或活細胞之染色，如甲基藍，其染色結果才能更為精準。

Penetrating keratoplasty is generally the treatment of choice for corneas that have tumor, chemical damage or serious ulcer. The goal of corneal storage is to preserve endothelial integrity from procurement to transplantation. Kalevar-Majumdar ( K-M ) media is a new synthetic viscous medium for preservation of cornea. To improve corneal endothelial cell survival during cold preservation, the addition of a compound that enhances cell membrane repair can be beneficial. It is now accepted in animal cells that Ca2+-triggered exocytosis is necessary for membrane resealing. Our purpose is to analyze the outcome of using K-M medium for preservation of rabbit corneas, and compare the effect of addition calcium. We cut the corneas of 5-monthes-olds female New Zealand white rabbits and individually stored these corneas with 20ml K-M media or 20ml K-M media which contained 1.05 mM Calcium Chloride at 4℃ in different duration. After 3, 6, 9, 11 or 14 days at 4℃, the corneas were processed for scanning electron microscopy ( SEM ) or stained Alizarin red to analyze the morphology and cell density of cornea endothelium cells. Additionally TUNEL staining was performed to reveal the apoptosis of corneal endothelium stored in 4℃. The cornea preserved with K-M medium had good morphology at 3 and 6 days. The percentage of the lost endothelial cells stored in 6 days with or without calcium were 19.8% and 20.4% respectively in SEM. Furthermore, the proportion of the existence endothelium stained with Alizarin red were 92.1% and 93.3%.Therefore the better cornea storage time in K-M media was 6 day. The effect of additional Ca2+ in K-medium was the same as stored in K-M medium. Comparing the effects of SEM and Alizarin red, SEM can faithfully perform the morphology of survival corneal endothelial cells. Alizarin red has to cooperate to the stains that could differentiate dead cells or survival cells, for example methylene blue .

中文摘要 ……………………………………………………………i
英文摘要 ……………………………………………………………ii
目次……………………………………………………………………iv
圖次……………………………………………………………………vi
表次…………………………………………………………………viii
第一章 緒言…………………………………………………………1
第二章 文獻探討
第一節 角膜之構造與功能…………………………………2
第二節 角膜保存發展………………………………………5
第三節 低溫保存液與K-M保存液之比較 …………………8
第四節 促進細胞膜修復之物質……………………………13
第五節 保存後角膜細胞狀況………………………………16
第三章 材料與方法
3.1 掃描式電子顯微鏡 ……………………………………20
3.2 Alizarin red染色 ……………………………………21
3.3 TUNEL染色………………………………………………21
3.4 Hematoxylin and Eosin staining …………………22
3.5 微生物培養 ……………………………………………22
3.6 分析與統計 ……………………………………………22
第四章 結果……………………………………………………………23
4.1 掃描式電子顯微鏡……………………………………23
4.2 Alizarin red染色……………………………………32
4.3 TUNEL染色 ……………………………………………36
4.4 Hematoxylin and Eosin staining…………………40
4.5 微生物培養……………………………………………44
第五章 討論……………………………………………………………45
參考文獻………………………………………………………………52

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