臺灣博碩士論文加值系統

角膜為眼球表面上皮細胞所構成的無血管透明組織，由於角膜位於眼球的最外層，故角膜也扮演著防止外來之物理、化學或生物性傷害的角色。角膜上皮細胞是由角膜與結膜銜接處之輪部上皮組織基底層的幹細胞所分裂分化而來。隨著上皮細胞的汰舊換新，輪部基底層的角膜上皮幹細胞會被活化，藉由細胞增生分裂以維持角膜上皮的組織恆定性。
過去研究發現，在正常的人類角膜中，p63轉錄因子只在具備高分裂潛力的輪部基底層上皮細胞細胞核中表現，認為p63的表現為角膜上皮幹細胞的指標。然而卻有許多研究與眼科的臨床證據顯示，角膜上皮組織中亦發現p63的表現，此結果與前者理論相互矛盾。
角膜上皮組織汰舊換新或組織受創時，輪部上皮基底層的角膜上皮幹細胞會開始增生新的角膜上皮細胞並往角膜移動。我們的實驗發現角膜上皮於修復初期，新生的角膜上皮組織亦有p63的表現，與過去的理論不符。另外經由觀察同時期之角膜分化相關蛋白Keratin 3和 Keratin 14，發現修復初期的角膜上皮組織亦有Keratin 14的表現，與正常角膜上皮組織不同；而隨著角膜上皮組織修復完畢，Keratin 14的表現也漸漸消失，取而代之的則是Keratin 3的表現。說明角膜受創癒合後，新生的角膜上皮細胞會漸漸走向分化，成為成熟的角膜上皮細胞。
角膜組織ex vivo培養的實驗發現，修復初期的中央角膜上皮組織可以在人類羊膜上增生，與正常中央角膜上皮組織不同。配合免疫組織染色之結果，我們認為p63勢必參與修復初期角膜上皮細胞的分裂或分化。

Limbal epithelial stem cells (LSC) are the progenitors of corneal epithelium and are known to locate in the basal layer of the limbal epithelium. During corneal wound repair, limbal epithelial stem cells are stimulated to proliferate and generate new epithelial cells to fill the wound surface. Corneal epithelium is strongly positive with Keratin 3, a marker of the mature keratinocyte. In contrast, limbal epithelial basal cells are positive with p63, Keratin 14 and/or PCNA, the markers of either cell undifferentiation or proliferation. Here we show that in the epithelium of wound-healing cornea, the basal cells are positive with p63, Keratin 14 and PCNA, and are negative with Keratin 3, characteristics of that of the limbal basal cells. In contrast, Keratin 3 is expressed in cells located from suprabasal to superficial layers. Interestingly, immuno- fluorescent staining of serial sections shows that all p63 positive cells are also Keratin 14 and PCNA positive. Similar results were obtained with ex vivo cultured limbus. In vivo wounded corneas were allowed to undergo repair to complete the reepithlization. On day 7th post-wounding, central corneas were excised and explanted on AM to evaluate their growth potential. Central corneal epithelium taken from day 7th post-wounding grew vigorously to form an epithelial outgrowth with a rate that was faster than unwounded limbal tissue. In contrast, central corneas taken from day 30th post-wounding were unable to grow and no epithelial outgrowth was formed. The growth potential of the wound-repaired central cornea is positively correlated with the expressions of p63, Keratin 14 and PCNA. Our results suggest that the expression of p63 is an important indicator for the proliferation of corneal epithelial cells.

目次
中文摘要………………………………………………………………1
英文摘要………………………………………………………………3
1.序論…………………………………………………………………5
1-1.幹細胞……………………………………………………………5
1-2.成體幹細胞的存在………………………………………………5
1-3.成體幹細胞的特性………………………………………………6
1-4.成體幹細胞的鑑定………………………………………………6
1-5.角膜上皮組織……………………………………………………7
1-6.角膜上皮幹細胞…………………………………………………8
1-7.p63與上皮細胞…………………………………………………9
1-8.角膜上皮幹細胞的鑑定…………………………………………11
1-9.上皮組織受創修復………………………………………………11
1-10.角膜上皮受創修復與細胞激素的關係……………………….12
1-11.角膜上皮受創修復與p63的關係………………………………13
2.研究目的……………………………………………………………15
3.實驗材料與方法…………………………………………………16
3-1.實驗材料………………………………………………………16
3-1-1.實驗動物……………………………………………………16
3-1-2.人類羊膜製備………………………………………………16
3-1-3.實驗試劑……………………………………………………17
3-1-4.實驗儀器……………………………………………………18
3-2實驗方法…………………………………………………………19
3-2-1.全角膜破皮受創……………………………………………19
3-2-2.角膜受創癒合………………………………………………19
3-2-3.角膜組織培養………………………………………………20
3-2-4.角膜組織染色與影像呈現…………………………………21
3-2-5.Real-time quantitative reverse transcription PCR …………22
4.結果…………………………………………………………………24
4-1-1.輪部內的角膜上皮幹細胞並非平均分配……………………24
4-1-2.角膜破皮受創後，輪部產生新生細胞來修復角膜…………25
4-1-3.修復中的角膜組織具增生能力………………………………25
4-2.受創復原初期角膜上皮細胞中Keratin 3, Keratin 14及p63之表
現…………………………………………………………………26
4-3.以Real-time Q-PCR辨識受創角膜上皮的p63 ……………29
5.討論…………………………………………………………………30
6.Reference List…………………………………………………33

圖目錄
Fig.1…………………………………………………………………… 42
Fig.2.……………………………………………………………………43
Fig.3.……………………………………………………………………44
Fig.4.……………………………………………………………………45
Fig.5.……………………………………………………………………46
Fig.6.……………………………………………………………………47
Fig.7.……………………………………………………………………48
Fig.8…………………………………………………………………… 50
Fig.9.……………………………………………………………………51
Fig.10.…………………………..………………………………………52
Fig.11.…………………………..………………………………………53
Fig.12.…………………………..………………………………………54
Fig.13.…………………………..………………………………………55
Fig.14.…………………………..………………………………………56
Fig.15.…………………………..………………………………………57
Fig.16.…………………………..………………………………………58
Fig.17.…………………………..………………………………………59
Fig.18.…………………………..………………………………………60
Fig.19.…………………………..………………………………………61
Fig.20.…………………………..………………………………………62
Table 1 .…………….…………..………………………………………63
Fig.21.…………………………..………………………………………64

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