臺灣博碩士論文加值系統

目前對於角膜潰瘍的治療方式大致可分為抗生素類、類固醇、隱形眼鏡、或外科手術等,但各有缺點,故試圖開發新藥劑,希望既能促進角膜的傷口癒合,又可避免以上缺點。本實驗室自1996年開始便有以真菌細胞壁來發展創傷敷材。1997年，台北醫學大學蘇慶華教授等人將松衫靈芝（Ganoderma tsμg ae）之殘渣作為創傷敷料之原料，將其命名為SACCHACHITIN。實驗證實，SACCHACHITIN具抗菌特性，且促進角質細胞增生，亦抑制傷口中過度活化之蛋白質(MMP)，進而減少組織細胞基質的過度分解，達到更有效的癒合，也幫助纖維母細胞的增生和位移與控制生長因子的釋放、能夠個別調控兩個以上的分子以及能讓特定的細胞增生。另雙紡錘孢子蟲草 (Cordyceps bifusispora, Eriksson) (Phytocordyceps ninchukispora, Su & Wang) 屬麥角菌科之真菌，係1984 年於台灣發現的新種。2005年，蘇慶華教授等人將雙紡錘孢子蟲草細胞壁經鹼處理，冷凍乾燥後，與甲基纖維素混合成為ASCOCHITIN。ASCOCHITIN 敷於傷口上，其結構可吸附過多的蛋白水解酶，並隔絕細菌感染，可以防止蛋白水解酶過度分解生長因子，進一步刺激傷口組織分泌生長因子，使傷口環境達到平衡，使傷口快速度過發炎期，進入增生重整期。又本實驗室於同年，將葡枝根黴菌(Rhizopus stolorifer)經UV光照射至突變後找到一株孢子形成較晚之菌株（ Rhizopus stolorifer F6），並用其製備出具有幾丁質特性的創傷敷料『RHIZOCHITIN』。使用RHIZOCHITIN能促進生長因子-TGF-β分泌，活化巨噬細胞與纖維母細胞及內皮細胞分泌更多細胞激素，吸引更多細胞修復組織並幫助血管新生作用，且能抑制MMPs避免細胞外間質及生長因子受過度破壞而延長傷口修補時間。
而本實驗希望藉由將上述三種材料經處理過後，選擇其中一種最適合的材料，製成加速眼角膜傷口癒合之藥劑。

SACCHACHITIN is a cell component containing polysaccharide and chitin derived from fruiting body of Ganoderma tsugae was previously proved to be a bio-compatible and biodegradable material for accelerating wound repair with properties in decreasing activity of MMP-9 and proteases from bacteria, increasing growth factors concentration, elevating type-1 collagen deposition in wound area tissue. In the present study, SACCHACHITIN was micronized into small particles in an average particle size of 2.0 ?慆 to meet the purpose as an eye drops for the healing of cornea from various damages especially those in the patients with laser eye surgery.
Statens Seruminstitut rabbit cornea cell line (SIRC) was employed as an in vitro model for the preliminary tests. Various plysaccharides including micronized SACCHACHITIN (MSC), SACCHACHITIOSAN (from G. tsugae) Rhizocchtosan (from Rhizopus stolonifer), Ascochitosan (from Cordyceps bifusispora), 1-3 b-glucan (from G. tsugae), polysaccharide (from Saccharomyces cereviseae) and commercial chitosan (Sigma) were compared to evaluate the toxicity or growth enhancement on SIRC. The prelimainary result indicated MSC was a choice for further study considering the growth enhancement effect on SIRC and the process in manufacturing.
New Zealand white rabbit was served as an animal model for corneal injuries created by attaching 1N NaOH or 75% alcohol dipped filter paper for 30 seconds on surface of cornea of rabbit and 0.02% MSC suspension was used as experimental group with normal saline served as control. The healing process of corneal epithelium was measured by fluorescent area, histo- logical section of cornea and MMP-9 activity in a time course of 6, 24, 48, 72 and 96 hours as well as on the 7 days after injury created. The result of 75% alcohol injury indicated that the healing in the group of MSC was accelerated by shortening the time in shrinking the area of wound up to 48 hr and the activity of MMP-9 inhibited since 24 hours after injuries. The healing of epithelial layer reached completely normal at 72 hours with the treatment of MSC when the control group was still in the initiative stage of healing. The repair process of epithelium layer in deep injury created by NaOH followed the same pattern of 75% alcohol injury, but the cells in stroma layer still remained irregular arrangement that light was not penetrable.
All the results indicate that MSC is a potential material for epithelium repair of the eyes but less helpful for the severe stroma layer injury.

【目錄】
第一章 緒論………………………………………………………………………………1
第二章 文獻探討…………………………………………………………………………3
第一節 角膜的解剖與生理功能 ……………………………………………………3
第二節 角膜的病理生理學 …………………………………………………………5
第三節 角膜缺損的疾病與診斷 ………………………………………………… 8
第四節 角膜缺損的治療方式…………………………………………………… 11
第五節、Matrix metalloproteinase（MMPs）在傷口癒合上的意義…………………15
第六節、本實驗室所開發之生物性材料……………………………………………17
第三章 研究動機 ………………………………………………………………………18
第四章 研究方法 ………………………………………………………………………20
第五章 實驗材料與方法 ………………………………………………………………21
第一節 實驗材料 (Materials) ……………………………………………………21第二節 實驗方法 (Methods) …………………………………………………… 24
第一部份、創傷敷料的製備流程………………………………………24
第二部份、細胞增生實驗………………………………………………28
第三部份、Micronized SACCHACHITIN之基本化學特性測定 ……31
第四部份、Micronized SACCHACHITIN之基本物理特性測定 ……33
第五部份、動物實驗……………………………………………………34
第六章 實驗結果與討論 ……………………………………………………………… 39
第七章 結論 …………………………………………………………………………… 45
參考文獻………………………………………………………………………………… 46
附錄……………………………………………………………………………………… 51

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