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研究生:黃士純
研究生(外文):Shih-Chun Huang
論文名稱:天然交聯劑Genipin交聯戴氏膜觀察對牛角膜內皮細胞之影響
論文名稱(外文):In-vitro Evaluation of Cultured Bovine Endothelial Cells on the Descemet’s Membrane Cross-linked by Genipin with Improved Physical Properties
指導教授:王賢達王賢達引用關係
指導教授(外文):Hsin-Ta Wang
口試委員:王一中葉龍坤
口試委員(外文):I-Jong WangLung-kun Yeh
口試日期:2012-11-01
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:有機高分子研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:55
中文關鍵詞:滬子素緻密層內皮細胞生物組織材料
外文關鍵詞:GenipinEndothelial cells
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眼角膜受損是常見意外之一,若嚴重傷害醫療上很難恢復原本的視力,尤其是內皮細胞嚴重受損之後,角膜易水腫甚或角膜混濁,導致無法維持角膜之清澈,因為內皮細胞無法自行更新必須接受全層角膜移植(Corneal transplantation)或部分內皮細胞移植(DSAEK彈力層撕去後角膜內皮細胞移植手術); 或DMEK (Descemet membrane endothelial keratoplasty)手術;也可以使用不同之載體(carrier)
來培養內皮細胞後移植。治療眼角膜受損的方式很多,眼科手術部分目前大多以羊膜為基材,但羊膜的取得不易,會有醫學倫理上的問題需人體試驗委員會同意並且有未知之感染風險,最重要的是其半透明材質而非透明常影響移植後之視力品質或形成新生血管而戴氏膜Descemet’s Membrane (DM)雖然是最自然且最適合之基底膜Basement membrane,然而自角膜上取下後容易捲曲碎裂不易運用操作,因此,尋找另一適當的基材作為載體來培養內皮細胞成為本研究之主要目的。
本實驗,藉由滬子果子萃取物Genipin交聯戴氏膜產生一種新的培養基材,並測試其對內皮細胞的影響。首先,從牛眼睛取下角膜之戴氏膜,以胰蛋白脢將內皮細胞去除掉,再利用Genipin交聯處理戴氏膜成為較為可塑性之薄膜並且測試交聯化薄膜之交聯指數、機械性質、含水率,接著使用牛角膜內皮細胞測試此交聯處理的生物組織薄膜之生物相容性,使用細胞存活率分析3- (4,5-
cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay)、掃描式電子顯微鏡Scanning Electron Microscope(SEM)、免疫螢光觀察牛角膜內皮細胞在交聯處理過後之生物組織薄膜上之生長情形及表現型態,由實驗中可發現製備出的薄膜表面平整適合其細胞的生長,並具有良好的生物相容性。
本研究實驗結果發現以Genipin備製之戴氏膜可進一步用來作為眼表層組織工程材料之一。


Genipin, a nature crosslinking material extracted from the fruits of Gardenia jasminoides, has been widely used in many biological applications. For in-vitro cell culture system, the Descemet’s membrane is most natural and suitable basement membrane provided for the cultured cells growth and migration during wound healing. However, the Descemet’s membrane is easy rolled and easy teared after peeling off from the corneal tissue. In this study, we try to develop a new culture system carrier by using genipin cross linker and observe the phenotypes of cultured endothelial cells on the genipin-crosslinker Descemet’s membrane.
The crosslinking process between Descemet’s membrane and genipin was performed before cell plating. The crosslinking characteristic of the genipin-fixed Descemet’s membrane was investigated. Furthermore, the mechanical strength and resistance were compared between the genipin-fixed Descemet’s membrane and non-genipin-fixed Descemet’s membrane. In addition, the proliferation rate of cultured bovine corneal endothelial cells was study by3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay). Furthermore, the immunohistochemistry staining and Scanning Electron Microscope (SEM) were performed to investigate the phenotypes of cultured bovine endothelial cells.
The mechanical strength and resistance were increased in the group of the genipin-fixed Descemet’s membrane as compared with the group of non-genipin-fixed Descemet’s membrane. The genipin-fixed Descemet’s membrane was easy handled, more smooth surface, and not easy teared. The Scanning Electron Microscope (SEM) analysis showed the normal endothelial cells phenotype between the two groups , and the cultured cells easily adhered to the new genipin-fixed Descemet’s membrane. The 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay) showed that the proliferation rate increased for the group of cells genipin-fixed Descemet’s membrane. The immunohistochemistry staining showed the ZO-1 positive and Na-K ATPase positive cultured cells on the genipin-fixed Descemet’s membrane. These results indicated that the phenotypes of cultured cells in genipin-fixed Descemet’s membrane were endothelial cells. Therefore, the phenotypes of cultured cells on genipin-fixed Descemet’s membrane were not significantly changed by treating with cross-linker genipin.
These results demonstrated that genipin can form stable crosslinked products and is an effective crosslinking agent for Descemet’s membrane fixation. Therefore, the non-toxic nature crosslinker genipin may be useful for the application on the reconstruction of the ocular surface.


目錄
中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
圖目錄 viii
表目錄 x
第一章前言 1
第二章文獻回顧 4
2.1生物組織材料 4
2.2膠原蛋白結構 5
2.3幾丁聚醣及應用 8
2.4交聯生物組織之目的 10
2.5交聯作用 10
2.5.1滬子素(Genipin) 10
第三章實驗材料與方法 13
3.1實驗材料 13
3.1.1實驗藥品 13
3.1.2實驗設備分析 16
3.2研究架構 17
3.3實驗方法 17
3.3.1牛角膜內皮細胞之取得 17
3.3.2生物薄膜的製備 20
3.3.3交聯指數(Fixation index)分析 21
3.3.4膨潤度測試(Swelling ratio) 22
3.3.5生物組織薄膜機械性質 22
3.3.6內皮細胞貼附及存活率分析 24
3.3.7生物組織薄膜之掃描式電子顯微鏡Scanning Electron Microscope (SEM)觀察 26
3.3.8免疫螢光染色 27
第四章實驗結果及討論 28
4.1組織材料的條件與外觀 28
4.2交聯指數分析 31
4.3膨潤度測試(Swelling ratio) 34
4.4機械性質分析(抗壓強度) 35
4.5組織斷裂面型態觀察 36
4.6細胞培養與型態觀察 38
4.7生物組織薄膜之細胞存活率分析 41
4.8免疫螢光染色 42
第五章結論 50
參考文獻 52


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