Immunohistochemical studies on the role of glycosaminoglycans in thepathogenesis of corneal disorders

为了明确d周期胶原原纤维的表面形貌，我们应用原子力显微镜（AFM）对小鼠角膜基质和巩膜的胶原原纤维进行了观察。AFM显示角膜原纤维的d周期凹槽深度较巩膜浅。钌红（RR）法显示角膜和巩膜胶原原纤维表面分布有蛋白聚糖（pg）或糖胺聚糖（GAGs）。角膜和巩膜超微结构的差异可以解释两种组织中分子数量或分布的差异。在另一项实验中，我们通过解剖获得的角膜样本，比较了用软骨素酶ABC或角化酶消化前后的人角膜胶原表面结构。经软骨素酶ABC消化后，表面结构没有发生变化，但经角化酶消化后，d -周期的凹槽变深了很多。由此可见，硫酸角蛋白相关pg抑制了人角膜胶原原纤维表面的凹槽，这些pg可能对胶原原纤维的规则分布起作用，从而可能有助于维持角膜的透明度。我们利用再移植获得的人角膜移植物，通过免疫组化研究进一步研究了PGs或GAGs在角膜创面愈合过程中的分布，发现硫酸软骨素A在创面愈合过程中具有重要作用。

To clarify the surface topography of D-periodic collagen fibrils, we applied atomic force microscopy (AFM) to the collagen fibrils of mouse corneal stroma and sclera. AFM showed that the depth of the groove in the D-periodicity of corneal fibrils was shallow compared with that of the sclera. Ruthenium red (RR) method showed that proteoglycans (PGs) or glycosaminoglycans (GAGs) distributed on the surface of collagen fibrils in the cornea and sclera. The difference in ultrastructure between cornea and sclera could explain the differences in the number or the distribution of molecule in both tissues.In the other experiment, we compared the surface structure of human corneal collagen before and after the digestion with chondroitinase ABC or kertanase by using corneal samples obtained from autopsy. The surface structure did not changed after the chondroitinase ABC digestion where as the groove of the D-periodicity changed to be much deeper after keratanase.This shows that keratan sulphate associate PGs should depress the groove of the surface of human corneal collagen fibrils, and these PGs may act for the regular distribution of collagen fibrils, and thus, may contribute for maintaining the transparency of the cornea. We further investigated the distribution of PGs or GAGs during corneal wound healing by the immunohistochemical study by using the human corneal graft obtained during regrafting, and showed that chondroitin sulphate A had an important role during wound healing.