Differentiation Status of Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting
Differentiation Status of Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting
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DOI：
10.1089/ten.tea.2009.0711
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发表时间：
2010-09-01
影响因子：
4.1
通讯作者：
Connon, Che John
中科院分区：
文献类型：
--
作者：
Chen, Bo;Mi, Shengli;Connon, Che John
我们研究了牛角膜缘上皮细胞在完整的(剩余的羊膜上皮细胞和基底膜)和裸露的人羊膜(AM)上扩增时分化的差异。在添加的培养液中，上皮细胞的体外扩增持续2周，然后在空气提升条件下持续1周。采用免疫组织化学、Western blotting和定量聚合酶链式反应对培养前后的分化细胞(K3/K12阳性)和未分化细胞(K14阳性)进行定量检测。在AM上扩张的角膜缘上皮细胞在完整AM(IAM)和裸露AM(DAM)上形成4-6层。在DAM上，空气提升后分化细胞的比例保持不变。然而，在IAM上生长的细胞中，空气提升后分化的细胞数量显著增加。这些结果对基础和临床研究都有重要意义。首先，他们表明牛角膜缘上皮可以作为研究体外角膜缘干细胞扩增的基础研究的替代细胞来源。其次，这些发现是对临床医生的一个警告，即AM对可移植细胞的影响尚未完全了解；一旦细胞暴露在空气中，使用IAM或DAM可以在分化量方面产生不同的结果。
We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane (AM), a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under air-lifting conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. Limbal epithelial cells expanded upon AM formed 4-6 stratified layers, both on intact AM (iAM) and denuded AM (dAM). On dAM the proportion of differentiated cells remained unaltered after air-lifting. Within cells grown on iAM, however, the number of differentiated cells increased significantly after air-lifting. These results have important implications for both basic and clinical research. First, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Second, these findings serve as a warning to clinicians that the effect of AM on transplantable cells is not fully understood; the use of iAM or dAM can produce different results in terms of the amount of differentiation, once cells are exposed to the air.