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目的探讨人角膜上皮干细胞的分子标记。方法对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白(ABCG2,BCRP1)、烯醇化酶α、整合素α6、α9及β1、表皮生长因子受体(EGFR)、细胞角蛋白19(CK19)、14(CK14)及转铁蛋白受体(CD71)的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应(RT-PCR)和原位杂交以检测其相关基因的表达。结果角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素α9蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT-PCR结果显示角膜缘表达p63、ABCG2、整合素α9蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记:p63表达于细胞核、ABCG2表达于胞质、整合素α9表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。(中华眼科杂志,2005,41:1014-1019)
Objective To investigate the molecular markers of human corneal epithelial stem cells. Methods Histological examination of corneal and limbal tissues was performed to analyze the limbal anatomy. Immunohistochemical staining of human corneal slices and whole corneal tissue was performed to detect undifferentiated markers of the central cornea and limbus, such as nuclear protein p63, breast cancer resistance protein (ABCG2, BCRP1), enolase α, integrin α6, α9 Β1, epidermal growth factor receptor (EGFR), cytokeratin 19 (CK19), 14 (CK14) and transferrin receptor (CD71) were detected by fluorescence microscopy and laser scanning confocal microscope. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization were performed on mRNAs of the corneal central and limbal epithelial cells to detect the expression of their related genes. Results Transverse sections of the limbus show that the limbal epithelial cells are arranged radially, corresponding to the Vogt fence environment. The undifferentiation markers integrin β1, EGFR, enolase α and CK19 in the corneal basal cell cytoplasm staining more than the surface cells; p63, ABCG2, integrin α9 protein found only in the limbal base epithelial cells. Laser scanning confocal microscopy and RT-PCR results showed that limbal epithelium expressed p63, ABCG2, integrin α9 protein and mRNA. In situ hybridization showed that p63 was only expressed in the basal cells of the limbus. Conclusion The corneal limbal epithelium is arranged radially, and the limbal stem cell population has multiple markers: p63 is expressed in the nucleus, ABCG2 is expressed in the cytoplasm, and integrin α9 is expressed in the cell membrane. Using these labeled complexes, the limbal stem cell population can be distinguished from other epithelial cells. (Chinese Journal of Ophthalmology, 2005,41: 1014-1019)