以透明質酸改質隱形眼鏡水膠及其促進眼部傷口癒合應用研究

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研究生：	施慶芳 Ching-Fang Shih
論文名稱：	以透明質酸改質隱形眼鏡水膠及其促進眼部傷口癒合應用研究 Semi-IPN modification of therapeutic soft contact lens with Hyaluronicacid for ocular wound healing applications
指導教授：	江安世 Ann-Shyn Chiang 薛敬和 Ging-Ho Hsiue
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	87
中文關鍵詞：	治療型隱形眼鏡、水膠、傷口癒合、角膜上皮細胞行為、透明質酸
外文關鍵詞：	therapeutic soft contact lens, hydrogel, wound healing, corneal epithelial cell behavior, hyaluronic acid, Semi-IPN
相關次數：	點閱：1 下載：0
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透明質酸(HA)在眼球中含量豐富並廣泛應用於眼科手術之高分子材料，在許多臨床研究發現定期給予HA能有效改善乾眼症症狀，增加淚液層的穩定度，同時也能促進眼表面傷口之癒合。本研究針對雞冠萃取之天然HA(5000kD)及細菌發酵產生之HA，以不同分子量(1500kD,810kD,50kD)材料塗佈表面對於人類角膜上皮細胞行為所造成之影響，如：細胞貼覆、細胞遷移、細胞增殖、細胞連結、細胞週期之影響，並以此實驗結果決定一最具療效之分子量進行下一步實驗。而後將HA以Semi-IPN方式與軟式隱形眼鏡進行物理性交聯，並元素分析儀進行含量分析。物性結構則除含水量分析外，更以SEM觀察改質前後之治療型隱形眼鏡表面形態，以萬能強力拉伸試驗機測量材料強力，以接觸角進行材料表面親疏水性分析，並以眼睛細胞株(SIRC)比較其促進傷口癒合之能力。
在以AFM觀察HA表面塗部形態，結果發現隨分子量增加，表面構造越顯粗糙。在HA含量相同(1mg/ml)之培養基進行人類角膜上皮細胞(HCEpiC)培養;低分子量(810kD,50kD)之HA相較於對照組，並未明顯促進細胞貼附及增殖，而天然之HA反而會抑制細胞貼附及增殖。但低分子量之HA卻能明顯促進細胞遷移，而天然之HA雖未有明顯促進細胞遷移卻也無抑制現象，而就人類角膜上皮細胞之貼附蛋白CD44之流式細胞儀分析結果發現，不同分子量之透明質酸，皆能有效促進CD44蛋白質之表現，此一結果可進一步推論HA本身的確可促進HCEpiC貼附；但因HA本身具有高黏稠性且分子量越大透明質酸之塗佈表面越粗糙，影響HCEpiC貼附於生長表面的穩定性，間接抑制人類角膜上皮細胞之生長。但一旦貼附於生長表面，透明質酸則能有效促進上皮細胞遷移。因此我們可發現在HA(50kD)這組之細胞貼附不受抑制，而細胞遷移能力也最好。
就隱形眼鏡材料添加透明質酸材料物性分析，可發現水膠材料在添加透明質酸後有較強的材料張力、更好的親水性，以SEM觀察改質前後之治療型隱形眼鏡表面形態發現材料表面皆有透明質酸的存在，且無細胞毒性。但其膨潤能力及含水率則有下降之現象，因此透氧率也會因此受到影響。就促進傷口細胞癒合的實驗顯示，此材料有些微促進傷口癒合之能力。由以上結果顯示將透明質酸添加於軟式隱形眼鏡材料中開發為治療型隱形眼鏡材料，的確具有相當潛力，但如何克服含水率、透氧率下降的問題，可能仍須改進其合成方式，以期得到具有更佳含水率及透氧率之材料。

Hyaluronic acid is a polymer that widely distributed in human coneectissues including the ocular tissues such as tears, cornea, aqueous humor and vitreous body. In ophthalmology, hyaluronic acid has been used as an main ingredient of artificial tear and visco-elastic agent during the intraocular surgeries to tent the anterior chamber in order to protect the corneal endothelium from the mechanical trauma during the operation. Htyaluronic havs been demonstrated to be able to promote the cell viability, cell migration, proliferation of corneal epithelial cells and the wound healing by several mechanism including CD 44 receptor binding, laminin/fibronectin secretion and water retentions, etc. In this project, we intend to modification the material of therapeutic soft contact lens ( hydrowyethyl methycrylate, HEMA) with hyaluronic acid by using simultaneous interpenetrating network to promote the effect of wound-healing of therapeutic contact lens.
Low molecular weight HA(MW=50kD,810kD) improves cell migration of human corneal epithelial (HCEpi) cell but cannot promote its adhesion and proliferation. High molecular weight HA(MW=5000kD, 1500kD) repel cell adhesion. This ability can be applied to HA eye drop and anti-adhesion membrane. Because high MW HA can decrease the friction between cornea and eyelid, and the damage of human cornea epithelial cell.
HA has several approaches to help cell migration, one of them is to increase the amount of CD44 receptor, regardless of the molecular weight. The increase in the amount of CD44 can explain how HA stimulates HCEpi cell migration. Though the result of HCEpi cell cycle with HA, we note that the S phase cell percentage of the low MW HA is higher than both the high MW HA and the control. But the resulted number of cell proliferation from the experiments is the same as the control. Further research is needed to demonstrate that HA can really improve cell proliferation.
The contact lens hydrogels containing HA, the surrounding of polymerized system must be maintained at pH 7.0 to increase the HA solubility and the transparency of hydrogels. The hydrogels were obtained as the water content below 50 % under photo free radical polymerization. If the water content was above 50 % in the polymerization, the hydrogels were not obtained. In SEM images, the HA was clearly observed on the surface of the hydrogels. The hydrogel containing HA has better tensile strength than that without containing HA. But the water content of hydrogel was decreased by increasing HA. The hydrogels that contained HA above 3 wt % exhibited well cell adhesion ability.
Through the current study, this novel therapeutic soft contact lens modified with hyaluronan or chitosan has great potential for its applications to corneal epithelial wound healing.

誌謝
目錄.......................................................i
圖目錄....................................................iv
表目錄....................................................vi
中文摘要.................................................vii
英文摘要..................................................ix
第一章 緒論................................................1
1-1 研究背景...............................................1
1-2 研究動機與目的.........................................2
第二章 文獻回顧............................................4
2-1 角膜潰瘍的成因.........................................4
2-1-1 角膜之基本構造.......................................4
2-1-2 角膜上皮細胞更新機制.................................4
2-1-3 造成角膜潰瘍、上皮輕微缺損之原因.....................5
2-1-4 目前角膜潰瘍之治療方法...............................7
2-2 透明質酸...............................................8
2-2-1 透明質酸於生物醫藥方面的應用.........................8
2-2-2 透明質酸與CD44生理作用機制...........................9
2-3 透明質酸促進角膜上皮損傷癒合作用之文獻回顧............11
2-3-1  HA對角膜缺損促進癒合作用的藥理和毒理學研究.........11
2-3-2  HA滴眼液治療角膜損傷的臨床研究.....................16
2-4 軟式隱形眼鏡..........................................18
2-4-1 隱形眼鏡材料的特殊性質..............................21
2-5 治療型隱形眼鏡........................................26
2-5-1 隱形眼鏡促進角膜潰瘍癒合之原理......................27
2-5-2 治療型隱形眼鏡之限制................................28
2-6 互穿型高分子網狀結構體(interpenetrating polymer  networks; IPNs)...........................................30
第三章 實驗大綱...........................................33
3-1 藥品與材料............................................34
3-1-1 實驗生物............................................34
3-1-2 藥品試劑............................................34
3-2 實驗設備..............................................35
3-3 不同分子量透明質酸材料人類角膜上皮細胞行為之研究......37
3-3-1 透明質酸表面製備....................................37
3-3-2 透明質酸表面形態分析(Atomic force microscopy; AFM)..37
3-3-3 表面電量測量(zeta potential)........................38
3-3-4 人類角膜細胞培養....................................38
3-3-5 細胞貼附分析........................................39
3-3-6 細胞生長能力分析....................................39
3-3-7 細胞移形(outgrowth migration)能力分析...............40
3-3-8 流式細胞儀(Flow cytometry)分析......................41
3-3-9 CD44 (cell adhesion molecule)含量分析...............41
3-3-10 細胞間物質運輸能力分析(cell-cell communication)....42
3-4 透明質酸改質隱形眼鏡..................................43
3-4-1 藥品純化............................................43
3-4-2 改質隱形眼鏡之製備..................................43
3-4-3 水膠元素分析(EA)....................................46
3-4-4 平衡含水量(equilibrium water content)的測定.........46
3-4-5 接觸角(contact angle)...............................46
3-4-6 水膠薄膜強力測試....................................47
3-4-7 透光率測定..........................................47
3-4-8 材料之細胞相容性測試................................48
3-4-9 表面形態觀察—掃描式電子顯微鏡(SEM).................48
3-4-10 蛋白質吸附實驗.....................................49
第四章 結果與討論.........................................50
4-1 透明質酸促進傷口癒合細胞實驗..........................50
4-1-1 透明質酸表面形態分析(AFM) ..........................51
4-1-2 表面電量測量(zeta potential) .......................52
4-1-3 接觸角分析(contact angle)...........................53
4-1-4 細胞貼附分析........................................54
4-1-5 細胞貼附細胞數量分析................................56
4-1-6 細胞生長能力分析....................................57
4-1-7 細胞移形(outgrowth migration)能力分析...............58
4-1-8 細胞週期(flow cytometry)分析........................59
4-1-9 CD44 (cell adhesion molecule)含量分析...............62
4-1-10 細胞間物質運輸能力分析(cell-cell communication)....65
4-2 透明質酸改質隱形眼鏡實驗..............................67
4-2-1 高分子共聚合水膠的合成及特性........................67
4-2-2 水膠元素分析(EA) ...................................68
4-2-3 平衡含水量(equilibrium water content)的測定.........69
4-2-4 水膠尺寸膨潤試驗....................................72
4-2-5 材料之細胞相容性測試................................73
4-2-6 表面形態觀察—掃描式電子顯微鏡(SEM).................75
4-2-7 蛋白質吸附實驗......................................77
第五章 結論...............................................80
第六章 參考資料...........................................82

                                

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