Department of Ophthalmology, Taipei Tzu Chi Hospital, The Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan.
College of Medicine, Tzu-Chi University, Hualien, Taiwan.
Invest Ophthalmol Vis Sci. 2022 Feb 1;63(2):31. doi: 10.1167/iovs.63.2.31.
To determine the role of transmembrane mucins in blocking fluorescein ingress to the corneal epithelium and its deficiency in contributing to corneal fluorescein punctate staining.
A dry eye model was established by extirpating lacrimal and Harderian glands in rabbits to correlate the expression of mucins with fluorescein-stained areas on the corneal button using immunofluorescence. Expression of transmembrane mucins was promoted in human corneal epithelial cells (HCECs) by culturing with the mucin-promoting medium (MPM) or diquafosol treatment. Conversely, the expression of mucins was downregulated by knockdown with short hairpin RNA. The role of mucin1 extracellular domain in fluorescein ingress was further verified by overexpression of N-terminally truncated mucin1 in HCECs.
In the rabbit dry eye model, the expression level of mucin1 was significantly decreased in superficial corneal epithelial cells where fluorescein punctate staining was observed. Upregulation of mucin1 and mucin16 in HCECs promoted by MPM or by diquafosol treatment impeded intracellular fluorescein ingress. Downregulation of mucin1 and mucin16 enhanced fluorescence ingress in HCECs after fluorescein staining. Overexpression of truncated mucin1 did not alter the fluorescein intensity of fluorescein-stained HCECs, supporting the notion that the ability of mucin1 to block fluorescein ingress was primarily mediated by its extracellular domain. Minimal inherent expression of mucin16 in the rabbit cornea limited the validation of its role in blocking fluorescein ingress in vivo.
Transmembrane mucin1 blocks fluorescein ingress in the corneal epithelium, explaining how fluorescein staining is positive when the level of transmembrane mucins is disturbed in dry eyes.
确定跨膜粘蛋白在阻止荧光素进入角膜上皮以及其缺乏对角膜荧光素点状染色的贡献中的作用。
通过摘除兔的泪腺和哈迪腺建立干眼症模型，用免疫荧光法将粘蛋白的表达与角膜纽扣上荧光素染色区域相关联。通过用粘蛋白促进培养基（MPM）或地夸磷索处理培养，促进人角膜上皮细胞（HCEC）中转膜粘蛋白的表达。相反，通过短发夹 RNA 下调粘蛋白的表达。通过在 HCEC 中转染 N 端截断的粘蛋白 1 进一步验证粘蛋白 1 细胞外结构域在荧光素进入中的作用。
在兔干眼症模型中，观察到角膜浅层上皮细胞中粘蛋白 1 的表达水平明显降低，这些细胞出现荧光素点状染色。MPM 或地夸磷索处理后 HCEC 中转录上调的粘蛋白 1 和粘蛋白 16 阻碍了细胞内荧光素的进入。HCEC 中转录下调的粘蛋白 1 和粘蛋白 16 增强了荧光素染色后的荧光素进入。转染截断的粘蛋白 1 并没有改变荧光素染色的 HCEC 的荧光素强度，这支持了粘蛋白 1 阻断荧光素进入的能力主要通过其细胞外结构域介导的观点。兔角膜中粘蛋白 16 的固有表达较少，限制了其在体内阻断荧光素进入的作用的验证。
跨膜粘蛋白 1 阻断角膜上皮细胞中的荧光素进入，解释了为什么在干眼症中跨膜粘蛋白水平紊乱时荧光素染色呈阳性。
