Yoshioka Hokoru, Ueta Mayumi, Yokoo Seiichi, Yokoi Norihiko, Mizushima Katsura, Naito Yuji, Kinoshita Shigeru, Sotozono Chie
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Department of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2025 May 1;66(5):6. doi: 10.1167/iovs.66.5.6.
Keratinization of the mucosal epithelia develops in severe ocular surface disorders, causing severe visual loss. This study elucidates the molecular mechanisms of keratinization in conjunctival epithelial cells (CjECs) and investigates the involvement of the vitamin A pathway.
Keratinized conjunctival epithelial sheets were generated by a closed system culture of human CjECs and confirmed by immunostaining. Comprehensive gene expression analysis and quantitative real-time PCR (qRT-PCR) were used to examine whether the cells could be used as an in vitro keratinization model. Moreover, immunostaining and qRT-PCR were used to examine alterations of vitamin A pathway-related genes in the cells and also the effect of adding all-trans retinoic acid (ATRA) and retinoic acid receptor alpha/beta (RARA/RARB) agonist Am80. Knockdown of RARA or RARB was also performed using transfection of small interfering RNA to identify receptors for retinoic acid.
Immunostaining revealed that CjECs cultured in a closed system had increased expression of keratinization markers. Comprehensive gene expression analysis and qRT-PCR revealed expression changes in vitamin A pathway genes, in addition to keratinization. In the closed system culture, immunostaining revealed that conjunctival epithelial keratinization was suppressed or partially ameliorated by ATRA or Am80, and qRT-PCR revealed that vitamin A pathway-related genes were significantly altered. Moreover, knockdown of RARA or RARB induced an increase in keratinization marker involucrin.
Keratinization of CjECs involves RARA or RARB-mediated pathways, and ATRA and Am80 alter the expression of vitamin A pathway gene and suppress keratinization.
在严重的眼表疾病中，黏膜上皮会发生角化，导致严重的视力丧失。本研究阐明了结膜上皮细胞（CjECs）角化的分子机制，并研究了维生素A途径的参与情况。
通过人CjECs的封闭系统培养生成角化的结膜上皮片，并通过免疫染色进行确认。采用综合基因表达分析和定量实时PCR（qRT-PCR）来检测这些细胞是否可用作体外角化模型。此外，使用免疫染色和qRT-PCR来检测细胞中维生素A途径相关基因的变化，以及添加全反式维甲酸（ATRA）和维甲酸受体α/β（RARA/RARB）激动剂Am80的效果。还通过转染小干扰RNA敲低RARA或RARB，以鉴定维甲酸的受体。
免疫染色显示，在封闭系统中培养的CjECs角化标志物的表达增加。综合基因表达分析和qRT-PCR显示，除角化外，维生素A途径基因的表达也发生了变化。在封闭系统培养中，免疫染色显示ATRA或Am80可抑制或部分改善结膜上皮角化，qRT-PCR显示维生素A途径相关基因发生了显著改变。此外，敲低RARA或RARB会导致角化标志物内披蛋白的表达增加。
CjECs的角化涉及RARA或RARB介导的途径，ATRA和Am80可改变维生素A途径基因的表达并抑制角化。
