Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model

博士 === 長庚大學 === 基礎醫學研究所 === 93 === Severe damage to the limbal epithelium due to chemical/thermal burns, virus infection and other pathological events may lead to loss of the limbal epithelial stem cells. Limbal epithelial stem cell deficiency lead to chronic inflammation and vascular invasion of t...

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Main Authors: DER-YUAN WANG, 王德原
Other Authors: JAN-KAN CHEN
Format: Others
Language:en_US
Published: 2005
Online Access:http://ndltd.ncl.edu.tw/handle/29736528650469859924
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spelling ndltd-TW-093CGU003250892015-10-13T11:42:57Z http://ndltd.ncl.edu.tw/handle/29736528650469859924 Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model 組織工程角膜兔子模式中輪部上皮幹細胞的特徵與鑑別 DER-YUAN WANG 王德原 博士 長庚大學 基礎醫學研究所 93 Severe damage to the limbal epithelium due to chemical/thermal burns, virus infection and other pathological events may lead to loss of the limbal epithelial stem cells. Limbal epithelial stem cell deficiency lead to chronic inflammation and vascular invasion of the cornea eventually and cause functional blindness. The autologous and allogenic transplantation of limbal epithelial cells expanded on human amniotic membrane have been introduced to improve the outcome of ocular surface reconstruction in limbal deficient eyes. However, the identification of limbal stem cells and the incorporation of sufficient number of these cells in the bio-engineered corneal tissue remain to be explored. In this study, we employ the cell biological and molecular biological means to identify the characteristics of rabbit limbal epithelial stem cells in normal limbus and in epithelial sheet outgrown from limbal explant cultured on amniotic membrane. The immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and integrin 6/4 and  subunits in corneal and limbal tissues, and in limbal explant and its epithelial outgrowth cultured for two weeks on amniotic membrane. Preliminary results showed that the epithelial cell sheet outgrown from limbal explant on amniotic membrane exhibits a phenotype similar to that of the limbus. These results suggested that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells (published in Invest. Ophthalmol. Visual Sci. 2003;44:4698-4704). The real-time Q-RT-PCR was employed to quantify the relative abundance of TAp63 and Np63 transcripts in limbal, peripheral corneal and central corneal epithelia. Antisense oligonucleotides were designed to specifically suppress the expression of TAp63 or Np63 in limbal keratinocytes and their effects on cell proliferation and differentiation were examined. The expressions of TAp63 and Np63 transcripts appeared to be site specific; TAp63 was expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Np63 was also expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promote differentiation. Suppression of Np63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. These suggested that TAp63 and Np63 affect the proliferation of limbal keratinocytes by a different mechanism (published in Invest. Ophthalmol. Visual Sci. 2005). We also compared the p63 expression pattern and differentiation levels in different rabbit limbal quadrates. We found that the superior limbal quadrate exhibited the highest level of p63 expression and was characterized by lowest differentiation level. In contrast, the inferior limbal quadrate exhibited very low or undetectable levels of p63 expression and was characterized with terminal differentiation. The ex vivo AM-based epithelial explant culture also showed that the explant from superior limbus has the greatest epithelial outgrowth activity than the cells from other limbal quadrates. Taken together, our results suggested that p63 is not a specific marker of corneal epithelial stem cells. However, different p63 isoforms still play specific roles in maintaining stem cell functions and the entrance to terminal differentiation lineage of corneal epithelial keratinocytes. JAN-KAN CHEN 陳君侃 2005 學位論文 ; thesis 94 en_US
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description 博士 === 長庚大學 === 基礎醫學研究所 === 93 === Severe damage to the limbal epithelium due to chemical/thermal burns, virus infection and other pathological events may lead to loss of the limbal epithelial stem cells. Limbal epithelial stem cell deficiency lead to chronic inflammation and vascular invasion of the cornea eventually and cause functional blindness. The autologous and allogenic transplantation of limbal epithelial cells expanded on human amniotic membrane have been introduced to improve the outcome of ocular surface reconstruction in limbal deficient eyes. However, the identification of limbal stem cells and the incorporation of sufficient number of these cells in the bio-engineered corneal tissue remain to be explored. In this study, we employ the cell biological and molecular biological means to identify the characteristics of rabbit limbal epithelial stem cells in normal limbus and in epithelial sheet outgrown from limbal explant cultured on amniotic membrane. The immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and integrin 6/4 and  subunits in corneal and limbal tissues, and in limbal explant and its epithelial outgrowth cultured for two weeks on amniotic membrane. Preliminary results showed that the epithelial cell sheet outgrown from limbal explant on amniotic membrane exhibits a phenotype similar to that of the limbus. These results suggested that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells (published in Invest. Ophthalmol. Visual Sci. 2003;44:4698-4704). The real-time Q-RT-PCR was employed to quantify the relative abundance of TAp63 and Np63 transcripts in limbal, peripheral corneal and central corneal epithelia. Antisense oligonucleotides were designed to specifically suppress the expression of TAp63 or Np63 in limbal keratinocytes and their effects on cell proliferation and differentiation were examined. The expressions of TAp63 and Np63 transcripts appeared to be site specific; TAp63 was expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Np63 was also expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promote differentiation. Suppression of Np63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. These suggested that TAp63 and Np63 affect the proliferation of limbal keratinocytes by a different mechanism (published in Invest. Ophthalmol. Visual Sci. 2005). We also compared the p63 expression pattern and differentiation levels in different rabbit limbal quadrates. We found that the superior limbal quadrate exhibited the highest level of p63 expression and was characterized by lowest differentiation level. In contrast, the inferior limbal quadrate exhibited very low or undetectable levels of p63 expression and was characterized with terminal differentiation. The ex vivo AM-based epithelial explant culture also showed that the explant from superior limbus has the greatest epithelial outgrowth activity than the cells from other limbal quadrates. Taken together, our results suggested that p63 is not a specific marker of corneal epithelial stem cells. However, different p63 isoforms still play specific roles in maintaining stem cell functions and the entrance to terminal differentiation lineage of corneal epithelial keratinocytes.
author2 JAN-KAN CHEN
author_facet JAN-KAN CHEN
DER-YUAN WANG
王德原
author DER-YUAN WANG
王德原
spellingShingle DER-YUAN WANG
王德原
Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
author_sort DER-YUAN WANG
title Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
title_short Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
title_full Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
title_fullStr Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
title_full_unstemmed Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model
title_sort characterization and identification of limbal epithelial stem cells in a tissue-engineered rabbit corneal model
publishDate 2005
url http://ndltd.ncl.edu.tw/handle/29736528650469859924
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