Bardag-Gorce Fawzia, Diaz Alissa, Niihara Robert, Stark Jeremy, Cortez Daileen, Lee Alexander, Hoft Richard, Niihara Yutaka
Department of Medicine, The Lundquist Institute, Torrance, CA 90502, USA.
Emmaus Life Sciences Inc., Torrance, CA 90503, USA.
Int J Mol Sci. 2022 Apr 5;23(7):4032. doi: 10.3390/ijms23074032.
The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD.
New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs.
In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated.
These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.
本研究旨在调查醛脱氢酶（ALDHs）在兔角膜缘干细胞缺乏（LSCD）角膜以及用培养的自体口腔黏膜上皮细胞片（CAOMECS）治疗以重建LSCD眼表的角膜中的表达情况。
从新西兰白兔颊部活检组织中分离出自体口腔黏膜上皮细胞，并培养后移植回LSCD兔模型的角膜上。采用免疫荧光染色和蛋白质印迹分析比较健康角膜、LSCD患病角膜、CAOMECS治疗角膜中ALDH1A1和ALDH1A3的表达。培养人口腔黏膜和角膜上皮细胞（OMECS和CECs）并用视黄酸（RA）处理，以进一步研究ALDHs的表达。
在健康角膜中，ALDH1A1和ALDH1A3在角膜上皮基底细胞中显著表达。在LSCD患病角膜中，ALDH1A1和ALDH1A3在结膜化的顶端上皮细胞、杯状细胞和基质中显著表达。与LSCD患病角膜相比，CAOMECS移植的角膜中ALDHs表达降低。蛋白质印迹分析证实LSCD患病角膜上皮细胞中ALDH1A1和ALDH1A3表达上调。与患病CECs（D-CEC）相比，CAOMECS中ALDH1A1和ALDH1A3表达水平较低。当在OMECS中用视黄酸处理上调ALDH1A3时，Pax-6表达下调，这表明当ALDH酶上调时再生能力降低。
这些发现首次报道了LSCD兔角膜中ALDH1A1和ALDH1A3的上调，并证明用于重建角膜上皮的CAOMECS移植可能会降低ALDH酶的表达水平。
