Brown C T, Applebaum E, Banwatt R, Trinkaus-Randall V
Boston University School of Medicine, Massachusetts 02118, USA.
J Cell Biochem. 1995 Sep;59(1):57-68. doi: 10.1002/jcb.240590108.
Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14, 42, and 84 days. The tissue and/or discs were removed and labeled with 35S-sulfate for 18 h; GAGs were extracted with 4 M guanidine-HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS-PAGE. The nonbound fractions from the chromatography were assayed for TGF-beta using Western blot analysis and for hyaluronic acid using an 125I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-beta that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-beta. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling.
我们的目标是研究将多孔盘插入基质层间袋所造成的伤口对角膜糖胺聚糖（GAGs）合成与沉积的影响。在第14、42和84天，对盘及其周围的基质组织进行检测，并与对侧对照基质和假手术角膜进行比较。取出组织和/或盘，用35S-硫酸盐标记18小时；用4M盐酸胍提取GAGs。提取物在Q-琼脂糖柱上进行色谱分析，结合的蛋白聚糖用线性盐梯度洗脱，并分析放射性组分。使用二甲基亚甲基蓝通过比色法测定总GAG含量。使用选择性多糖裂解酶进行酶消化来测定特定的GAGs，并使用SDS-PAGE检查蛋白核心。对色谱分析中的未结合组分进行Western印迹分析以检测TGF-β，并使用125I放射测定法检测透明质酸。在盘植入基质42天后对特定的GAGs进行定位。将盘植入基质导致GAG总量减少。然而，在周围组织和盘中，硫酸皮肤素-硫酸软骨素和硫酸乙酰肝素与硫酸角质素的比例增加。周围组织中的透明质酸在第14天升高，而盘中直到第84天才升高。对周围组织提取物的Western印迹分析显示，迁移的TGF-β形式的表观分子量为63和43 kDa。结果表明，将盘插入层间袋会导致周围组织和盘中糖胺聚糖的硫酸化和比例发生变化。这些变化与TGF-β的出现同时发生。84天后，盘中糖胺聚糖的群体开始类似于周围的基质。该模型将使我们能够进一步研究在广泛伤口后蛋白质的合成与沉积，在这种伤口中细胞必须迁移到伤口部位，然后进行广泛的重塑。
