Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye
Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye
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DOI：
10.1167/iovs.05-0735
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Gipson, IK
中科院分区：
文献类型：
--
作者：
Argüeso, P;Tisdale, A;Gipson, IK
目的。孟加拉玫瑰是一种有机阴离子染料，用于评估眼表疾病对眼表上皮的损害。有人提出，粘蛋白具有保护作用，防止正常眼表上皮细胞的玫瑰色染色。本研究旨在评价一种人角膜缘上皮(HCLE)细胞株的玫瑰红染色，该细胞系可产生和糖化膜相关粘液。方法：将人角膜缘上皮细胞在无血清培养中培养成融合细胞，并在含有10%血清的DMEM/F12细胞上进行诱导分化。分别用单抗HMFG-2、OC125和JAX-Lectin对膜相关粘蛋白MUC1、MUC16和T抗原碳水化合物表位进行免疫定位。为了评估染料的吸收，培养物与0.1%的孟加拉玫瑰孵育5分钟并拍照。为了确定排除负电荷的孟加拉玫瑰是否需要在细胞表面带负电荷，细胞与荧光阳离子铁蛋白孵育。结果：无血清培养的融合HCLE细胞胞浆和胞核呈玫瑰色，不表达MUC16，但不表达MUC1；HCLE细胞在含血清的培养液中培养，可形成不含孟加拉玫瑰的层积细胞岛。层状岛的顶端细胞在其顶端表面产生MUC16和T抗原碳水化合物表位。共定位实验表明，荧光阳离子铁蛋白不与这些层叠细胞结合，表明孟加拉玫瑰被排除在缺乏负电荷的细胞之外。结论：这些结果表明，角膜上皮细胞的层积和分化，通过在其顶端表面产生膜相关粘蛋白MUC16和粘蛋白相关T抗原碳水化合物的能力来衡量，在体外提供了对孟加拉玫瑰的保护作用，并提示了膜相关粘蛋白及其寡糖在保护眼表上皮细胞中的作用。
PURPOSE. Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins.METHODS. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration ( Ca+2 and Mg+2) in the rose bengal uptake assay.RESULTS. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake.CONCLUSIONS. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.