角膜幹細胞位於輪部。輪部幹細胞是角膜上皮細胞更新和修補的來源。近年來這方面的研究雖多，但輪部幹細胞分裂、及分化過程中的基因調控機制則仍不清楚。近年來發現p63，p53家族中的一個蛋白質，對於上皮組織的發育，上皮細胞的分裂和分化有著重要的影響。已知p63的isoform，DeltaNp63，可藉由抑制上皮細胞p21的表現來促進細胞分裂。在一些離體細胞實驗中也發現，快速進行分裂的上皮細胞會表現DeltaNp63 alpha，相反的，如果促使細胞老化，停止分裂或是開始分化，DeltaNp63 alpha的表現則下降。另外一個p63的isoform，TAp63，則可促進發育中的上皮組織開始進行多層化。因此，p63對於上皮組織，包含角膜組織在進行細胞更新，代謝應該扮演了相當重要的角色。另外，在一些上皮癌的組織中也發現DeltaNp63 alpha有過度表現的情形，顯現p63的功能確實跟細胞的分化和分裂有所關係。本實驗中利用Antisense oligonucleotides發現TAp63和輪部上皮細胞的分化有關，而TA和DeltaNp63都和細胞的分裂有關。另外，我們也利用組織免疫染色，檢定輪部及角膜上皮組織中EGFR，HGFR和KGFR的表現分佈。並測試EGF，HGF，以及KGF對於輪部上皮細胞在分裂以及分化上的影響。由實驗結果發現，HGF和KGF皆可促進輪部上皮細胞進行細胞分裂。KGF可以促進位於羊膜上的輪部組織生長出上皮層的能力，HGF則沒有影響。

Corneal stem cells are located in the limbus, and it’s responsible for the renewal and repair of the corneal epithelia. So far, the mechanism of gene regulation in proliferation and differentiation for limbal stem cells are not clear. p63, a membrane of the p53 family, plays important roles in the development of epidermis, proliferation and differentiation of epithelial cells. DeltaNp63 alpha, one of p63 isoform, promotes cell proliferation by suppressing the expression of p21. There is general agreement that the progenitor cells layers of skin, breast, and prostate express high levels of the nontransactivating DeltaNp63 isoforms. And, it is also found that the expression of DeltaNp63 is related to the proliferation of keratinocytes. TAp63, another p63 isoforms, initiate the stratification program of epidermis. So, it is possible that p63 participate in the cell renewal and repair in the epithelia, also in the cornea. Using of antisense oligonucleotides to blockade the expression of TAp63 and DeltaNp63, we find that the suppression of TAp63 and DeltaNp63 inhibit the epithelial outgrowth from the rabbit limbal explant on amniotic membrane. Suppression of TAp63 promote the differentiation of limbal keratinocytes, while DeltaNp63 has no effect. And, we also evaluate the functions of HGF and KGF to limbal epithelial in the cell proliferation and differentiation. We find that HGF and KGF promote the proliferation of limbal keratinocytes. KGF promote the epithelial outgrowth from rabbit limbal explant on amniotic membrane, but HGF doesn’t.

目 錄
指導教授推薦書………………………………………………………………… i
口試委員會審定書……………………………………………………………… ii
授權書……………………………………………………………………………. iii
簽署人須知………………………………………………………………………. iv
中文摘要…………………………………………………………………………. v
英文摘要………………………………………………………………………… vii
誌謝……………………………………………………………………………… viii
第一章 前言……………………………………………………………….. 1
第二章 材料與方法…………………………………………………………. 5
2. 1羊膜（Human Amniotic Membrane，AM）的製備…………………….. 5
2.2 輪部上皮細胞分離以及利用羊膜進行輪部組織培養………………… 5
2.3 使用Antisense oligonucleotides阻擋p63蛋白質的表現………………. 6
2.4 西方點墨法…………………………………………………………. 7
2.5 免疫染色…………………………………………………………………. 7
2.6 生長因子對輪部上皮細胞生長的影響…………………………………. 8
第三章 結果……………………………………………………………. 9
3.1離體培養輪部上皮細胞p63和keratin 3的表現............................…… 9
3.2 TAp63和Np63 antisense oligonucleotides對上皮細胞生長和分化的 影響.................................................................................…... 10
3.3 EGF，HGF和KGF受體在角膜組織中的分布........ ………………….12
第四章 討論…………………………………………………………………. 13
第五章 圖說……..…………………………………………………………. 18
第五章 圖表……..…………………………………………………………. 22
參考文獻…………………………………………………………………….……

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