Conjunctival epithelial cell differentiation on amniotic membrane.
Conjunctival epithelial cell differentiation on amniotic membrane.
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DOI：
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发表时间：
1999-04
影响因子：
4.4
通讯作者：
D. Meller;S. Tseng
中科院分区：
文献类型：
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作者：
D. Meller;S. Tseng
羊膜（AM）重建结膜表面恢复正常上皮表型，细胞密度明显高于对照组。本研究旨在探讨AM对兔结膜上皮细胞分化的调节作用。方法将兔结膜上皮细胞（RCEs）接种于经分散酶预处理的AM的基底膜侧，基质侧接种或不接种兔结膜成纤维细胞（RCFs）。在7至12天后，一半的培养物上升到空气-液体界面，其余的保持浸没。用视黄酸处理一小组含有RCF的气提培养物。在1、2和4周后，终止培养物并进行处理，用于用针对不同类型的粘蛋白（SMC和AM 3）、糖萼（AMEM 2）、角蛋白K3（AE 5）和K12（AK 2）的抗体进行免疫染色。此外，对K3角蛋白表达进行蛋白质印迹分析。透射电镜观察超微结构变化。结果在AM上生长的RCE一般较小，对AE 5和AK 2抗体呈阴性，对AMEM 2和ASPG 1抗体呈阳性。上皮分层和细胞极性与突出的微绒毛，紧密连接，和半桥粒在空气提升培养更明显。与RCFs共培养的RCEs显示散在的AM3阳性杯状细胞，维甲酸不增加。结论：在AM上培养的RCE主要表现为非杯状结膜上皮表型。上皮分层和细胞极性，上皮分化所必需的功能，促进了空气提升。该培养模型将有助于进一步研究结膜上皮细胞的生长和分化。
PURPOSE Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation. METHODS Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid. CONCLUSIONS RCEs cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further.