Cintron C, Covington H I, Kublin C L
Eye Research Institute, Boston, Massachusetts 02114.
Invest Ophthalmol Vis Sci. 1990 Sep;31(9):1789-98.
Ultrastructural localization of proteoglycans (PGs) in 1-week- to 2-year-old scar was determined by staining with cuprolinic blue dye (CBD) after specific enzymatic digestion of keratan sulfate (KS) glycosaminoglycans (GAGs) or chondroitin sulfate glycosaminoglycans (CSs). High critical electrolyte conditions were maintained for CBD-staining, specific for high-sulfated GAGs. Although KS was detected in the 1-week-old wound, no CBD-stained KS was seen in the anterior stroma adjacent to the wound. The CS was present throughout the 1-week-old wound and adjacent stroma, and PGs were biosynthetically 35SO4-labeled in normal stroma. Subsequently, radioactivity from labeled PGs in normal stroma adjacent to the wound moved into scar tissue during healing. Marked sensitivity of PGs to Chondroitinase ABC indicated an abundance of CS in 2-week-old scars. Punctate CBD-staining and immunohistochemical evidence suggested chemically altered KS is present in the 2-week-old anterior scar. The pattern of CBD-staining in 1- and 2-week scars, after chondroitinase treatment, suggested KS in the younger scar is similar to adult high-sulfated GAG, whereas KS in the 2-week scar contains primarily newly synthesized low-sulfated KS. The latter is consistent with previous immunochemical and biochemical analyses. Cytochemical and immunohistochemical evidence indicated that KS is not present in the 2-week-old posterior scar. By the week 8 of healing, CBD-stained KS was present throughout most of the scar, except along the posterior margin, consistent with earlier stages of healing. The CBD-stained structures in the first 8 weeks of healing were reminiscent of stained GAGs in normal developing cornea. This fetal-like CBD-staining pattern seen in scar, however, changed to that of the normal adult by the 2nd year of healing. The significance of these observations relate to our contention that healing adult cornea recapitulates some ontogenetic events of the normal cornea, and that the nonuniform distribution and chemical properties of GAGs in scar tissue are a function of the movement of existing proteoglycans and de novo synthesis of altered macromolecules.
通过对硫酸角质素（KS）糖胺聚糖（GAGs）或硫酸软骨素糖胺聚糖（CSs）进行特定酶消化后，用铜蓝染料（CBD）染色来确定1周龄至2岁瘢痕中蛋白聚糖（PGs）的超微结构定位。维持高临界电解质条件用于CBD染色，这对高硫酸化GAGs具有特异性。尽管在1周龄伤口中检测到KS，但在伤口附近的前基质中未见CBD染色的KS。CS存在于整个1周龄伤口及相邻基质中，且PGs在正常基质中进行生物合成性35SO4标记。随后，伤口附近正常基质中标记PGs的放射性在愈合过程中移入瘢痕组织。PGs对软骨素酶ABC的显著敏感性表明2周龄瘢痕中CS含量丰富。点状CBD染色和免疫组化证据表明2周龄前瘢痕中存在化学改变的KS。软骨素酶处理后1周和2周瘢痕中CBD染色模式表明，较年轻瘢痕中的KS类似于成人高硫酸化GAG，而2周瘢痕中的KS主要包含新合成的低硫酸化KS。后者与先前的免疫化学和生化分析一致。细胞化学和免疫组化证据表明2周龄后瘢痕中不存在KS。到愈合第8周时，除后缘外，大部分瘢痕中均存在CBD染色的KS，这与愈合早期阶段一致。愈合前8周中CBD染色的结构让人联想到正常发育角膜中染色的GAGs。然而，瘢痕中这种类似胎儿的CBD染色模式在愈合第2年时转变为正常成人的模式。这些观察结果的意义与我们的观点相关，即成年角膜愈合重现了正常角膜的一些个体发生事件，且瘢痕组织中GAGs的不均匀分布和化学性质是现有蛋白聚糖移动和改变的大分子从头合成的结果。
