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研究生:傅振男
研究生(外文):Chen-Nan Fu
論文名稱:人類結膜杯狀細胞的培養及動物乾眼症模型的建立
論文名稱(外文):culture of human conjunctival goblet cells and establishment of an animal model to dry eye syndrome
指導教授:徐善慧徐善慧引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:56
中文關鍵詞:杯狀細胞結膜切片體外培養分化劑眼周手術PAS染色Rose bengal 試紙。
外文關鍵詞:conjunctival goblet cellsgrowth factorin vitroConjunctival biopsyPeriocular surgeryFGF1PAS reagentMUC5ACrose bengal strips.
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本研究利用正常人類含杯狀細胞的結膜切片,行體外培養,分析其特性,以分化劑增殖,於眼周手術同時,正常結膜切片取3 mm×3 mm置入PBS試管中之冰桶,於操作台將其切碎(1 mm × 1 mm)放入6公分培養盤,以DMEM+F12+10% FBS+1% PSA, FGF1,10ng/ml 之培養液加上蓋玻片,在37 ℃含5 % CO2,95 % O2培養箱中培養。
  經過七天培養後,將細胞以PAS染色,細胞頂端之黏液(mucin)可被染成紫色,而另一批細胞之RT-PCR MUC5AC在103bp位置亦有基因表現。另外細胞在6公分培養盤三日後,以0.05 % Trypsin+EDTA分離出細胞,去掉上清液後,於6well培養盤進行增殖,於一、三、七天分別計數細胞,比較細胞確有明顯增殖。
動物實驗方面完全遵守國立中興大學動物實驗管理小組的規定(Table 5)。我們選擇3.5kg雄性紐西蘭大白兔,依照人體模式培養出杯狀細胞,最後以不同PH值稀釋塩酸(HCL)破壞正常兔子的結膜,使Rose bengal 試紙呈陽性反應,建立人為乾眼症模型,已備將來結膜杯狀細胞移植使用。
The purpose of our study was to establish a system for culturing normal human conjunctival goblet cells and proliferating with growth factor in vitro.
During periocular surgery, a 3 mm×3 mm conjunctival biopsy was took into PBS containing tube in ice bottle, then it was cut into 1 mm×1 mm pieces. The cells were grown in a 1:1 mixture of DMEM/F12+10% FBS culture medium supplemented with 1% PSA+FGF1, 10ng/ml and characterized using morphology, histochemistry, molecular biology .
Goblet cells were cultured from differentiated epithelial cells, human goblet cells exhibited positive reactivity with periodic acid schift (PAS) reagent in MUC5AC mucin. The mRNA for MUC5AC was detected at 103 bp. The human epithelial cells including goblet cells can proliferate when fibroblast growth factor 1, 10ng/ml was added. Human goblet cells in vitro retain characteristics of goblet cells in vivo can be cultured.
In animal study we obey the rule of Chung Hsing University Council on Animal Care. We got NZW SPF, male rabbits (about 3.5 kg body weight) from VGH Taichung, the rabbit conjunctival epithelial cells were cultured by the way as human’s, we also set up the animal model of dry eye by destructed rabbit conjunctival epithelium with diluted HCL solution, Rose bengal strip staining showed positive reaction, the model will be used for rabbit conjunctival goblet cells transplantation through subconjunctival injection in the future.
摘要…………………………………………………………………I
Abstract…………………………………II
目錄……………………………………………………………………III
圖目錄…………………………………………………………………VII
表目錄…………………………………………………………………IX
第一章 前言…………………………………………………………1
1-1 研究背景及動機……………………………………………………1
1-2 研究目的…………………………………2
1-3 結膜簡介…………………………………………………………3
1-4 乾眼症簡介………………………………………………………5
1-4-1 乾眼症的症狀…………………………………………………5
1-4-2 淚液的組成及功用……………………………………………5
1-4-3 乾眼症的原因…………………………………………………7
1-4-4 乾眼症好發族群………………………………………………8
1-4-5 乾眼症之診斷…………………………………………………9
1-4-6 乾眼症之治療…………………………………………………11
1-4-7 如何預防乾眼症………………………………………………12
1-5 組織工程的定義與基本概念……………………………………13
1-6 文獻回顧……………………………………………………15
第二章 實驗藥品、器材與儀器……………………………………18
2-1 實驗藥品…………………………………………………………18
2-2 實驗器材與儀器…………………………………20
2-3 實驗流程與方法…………………………………………………23
2-3-1 實驗架構………………………………………………………23
2-3-2 實驗流程………………………………………………………25
2-3-2-1 實驗方法………………………………………25
2-3-3 Periodic Acid Schiff Reaction (PAS染色) ……………26
2-3-4 反轉錄聚合酶連鎖反應 (RT-PCR) …………………………31
2-3-4-1 RNA抽取……………………………………………………31
2-3-4-2 反轉錄反應 …………………………………………………32
2-3-4-3 聚合酶連鎖反應 ……………………………………………32
2-3-4-4 海藻膠DNA電泳分析……………………………………33
2-4 動物實驗………………………………………………………… 35
2-4-1 兔子結膜組織切片細胞培…………………………………35
2-4-2 乾眼症模型……………………………………………………35
第三章 結果………………………………………………………39
3-1 結果大意…………………………………………………………39
3-2 形態學特徵………………………………………………………39
3-3 人類結膜杯狀細胞PAS染色…………………………………39
3-4 人類結膜上皮細胞增生(含杯狀細胞增生)…………………40
3-5 RT-PCR證實MUC5AC之mRNA 基因表現……………………41
3-6 人類結膜組織切片………………………………………………41
3-7 動物實驗(兔子結膜培養細胞、組織切片PAS 染色,乾眼症模型之建立 )…………………………………………………………41
3-7-1 兔子結膜組織細胞培養…………………………………………42
3-7-2 兔子結膜組織切片……………………………………………43
第四章 討論…………………………………………………………44
4-1 人類結膜杯狀細胞體外可培養…………………………………44
4-2 染菌問題…………………………………………………………44
4-2 檢體不足問題………………………………………45
4-4 目前乾眼症治療的瓶頸………………………………45
4-5 黴菌感染探討……………………………………………46
4-6 如何避免染菌…………………………………………………47
4-7 本研究的貢獻……………………………………………………47
4-8 未來計劃…………………………………………………………48
第五章 結論…………………………………………………………49
第六章 參考文獻…………………………………………………50
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