Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells
Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells
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DOI：
10.1634/stemcells.2005-0363
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发表时间：
2006-05-01
期刊：
影响因子：
5.2
通讯作者：
Li, De-Quan
中科院分区：
文献类型：
--
作者：
Chen, Zhuo;Evans, W. Howard;Li, De-Quan
本研究评估了差距连接蛋白（Cx）43是否可以作为人角膜上皮干细胞的阴性细胞表面标志物。在角膜缘组织和原代角膜缘上皮培养物中评价Cx43表达。免疫荧光染色和激光扫描共聚焦显微镜显示，Cx43在角膜和角膜缘基底上上皮细胞中呈强阳性表达，而角膜缘上皮基底细胞呈阴性表达。在原代角膜缘上皮培养物中，Cx43抗体主要染色大细胞，但不染色小细胞。通过半定量逆转录聚合酶链反应（PCR）和实时PCR测定，角膜中的Cx43 mRNA比角膜缘上皮中更丰富，并且在成熟的角膜缘上皮培养物中表达水平更高。使用GAP 11（一种针对Cx 32细胞外环2（151-187）的兔多克隆抗体，该序列与Cx43高度同源），通过荧光激活细胞分选从原代角膜缘上皮培养物中选择Cx43（暗）和Cx43（亮）细胞，并评估干细胞特性。这些Cx43（暗）和Cx43（亮）细胞通过其Cx43蛋白和mRNA的表达水平来确认。发现Cx43（dim）细胞含有较高百分比的慢循环溴脱氧尿苷（BrdU）标记保留细胞和干细胞相关标志物p63、ABCG 2和整联蛋白01阳性，分化标志物K3和外皮蛋白阴性的细胞。通过BrdU掺入、集落形成效率和生长能力评估，Cx43（暗淡）细胞具有比Cx43（明亮）细胞和未分级细胞更大的增殖潜力。我们的研究结果表明，人角膜缘基底细胞不表达连接蛋白43，这可以作为一个负的细胞表面标记的干细胞的人角膜缘上皮细胞的人口。
This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151-187), a sequence that is highly homologous in Cx43, the Cx43(dim) and Cx43(bright) cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43(dim) and Cx43(bright) cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43(dim) cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin 01 and negative for differentiation markers K3 and involucrin. The Cx43(dim) cells possessed a greater proliferative potential than Cx43(bright) cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells.