Department of Ophthalmology, Soonchunhyang University College of Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Korea.
Curr Eye Res. 2012 Jan;37(1):33-42. doi: 10.3109/02713683.2011.620728. Epub 2011 Oct 26.
Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model.
HCLECs were grown in RA-supplemented media at various concentrations (0, 10(-9) to 10(-6) M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis.
HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10(-8) to 10(-7) M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10(-6) M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations.
This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.
视黄酸（RA）对上皮细胞分化和维持粘液表型至关重要。本研究旨在通过原代人角膜缘上皮细胞（HCLEC）培养模型研究 RA 对角膜上皮分化和粘蛋白表达的影响。
将 HCLEC 在不同浓度（0、10（-9）至 10（-6）M）的 RA 补充培养基中培养。通过免疫组织化学或免疫荧光染色检测 p63、ABCG2、CK3、CK19，并用 Western blot 检测 ABCG2 和 CK12，以评估分化情况。采用扫描和透射电子显微镜观察超微结构形态。用玫瑰红染料孵育以检查屏障功能。通过免疫组织化学、实时定量 PCR 和 Western blot 分析研究 RA 对 MUC1、-4 和 -16 表达的影响。
未加 RA 的 HCLEC 表现为过度角化，而用 10（-8）至 10（-7）M RA 诱导的则是非角化的分层上皮，外观正常。在这些条件下，p63、ABCG2、CK3、CK19、MUC1、-4 和 -16 的染色模式与活体角膜缘上皮相似。较高浓度（10（-6）M）的 RA 导致异常分化。用 RA 培养的 HCLEC 紧密贴合，保持完整的屏障功能，防止染料渗透。此外，MUC1、-4 和 -16 的表达与 RA 浓度高度相关。
本研究表明，通过控制培养基中 RA 的浓度，培养的 HCLEC 可以模拟生理和功能表型。此外，我们的结果表明 RA 对角膜上皮分化和粘蛋白表达有调节作用。
