Quantitative and qualitative control of hyaluronan which plays a role in the assembly of extracellular matrix as a backbone of proteoglycan aggregate

本项目的目的是：1)阐明影响高分子量透明质酸解聚酶的因素，该酶可将高分子量透明质酸降解为分子量为40000的片段；2)表征在透明质酸合成酶抑制剂4-甲基limbelliferone存在下培养t细胞形成的缺乏透明质酸的细胞外基质。首先，肝素酶处理培养成纤维细胞的细胞层导致外源性透明质酸解聚的刺激。肝素酶处理也抑制了高分子量透明质酸的合成。这些结果表明，细胞表面硫酸肝素可能影响透明质酸代谢。其次，在0.5 mM 4- methylumbellliferone的作用下培养形成的缺乏透明质酸的细胞外基质分析显示，细胞层CD44和纤维连接蛋白减少。细胞层中I型胶原的数量没有变化，而4-甲基伞形酮在培养基中观察到I型胶原分子的增加。这些现象可能是由于细胞外基质中缺乏透明质酸所致。进一步探讨了透明质酸与基质金属蛋白酶的关系。在外源透明质酸存在下培养细胞，用酶谱法和免疫印迹法比较基质金属蛋白酶与对照细胞的活性。结果发现，高浓度大于1 mg/ml的高分子量透明质酸能强烈抑制基质溶素-1的分泌。

The purposes of this project are 1) to clarify the factors that affect hyaluronan-depolymerizing enzyme which degrade high-molecular-weight hyaluronan into fragments with molecular weight of 40,000, and 2) to characterize the hyaluronan-deficient extracellular matrix which were formed by cultivating tcells in the presence of 4-methylimbelliferone, an inhibitor of hyaluronan synthase. First, heparitinase treatment of the cell layer of cultured fibroblasts resulted the stimulation of the depolymerization of exogenous hyaluronan. Heparitinase treatment also showed the inhibition of the synthesis of high-molecular-weight hyaluronan. These results suggest that the cell surface heparan sulfate may affect hyaluronan metabolisim. Second, the analysis of hyaluronan-deficient extracellular matrix, which can be formed by cultivation of the cells in the presence of 0.5 mM 4-methylumbelliferone, showed the decrease of CD44 and fibronectin in cell layer. The amount of type I collagen in cell layer was not changed, while an increase of the molecule was observed in the medium by 4-methylumbelliferone. These phenomena may be caused by the lack of hyaluronan in extracellular matrix. Furthermore, the relation between hyaluronan and matrix metalloproteinases were investigated. Cells were cultured in the presence of exogenous hyaluronan and the activities of matrix metalloproteinases were compared with that of control cells by zymography and western blotting. As a result, the excretion of stromelysin-1 was found to be strongly suppressed by high-molecular weight hyaluronan with high-concentration of more than 1 mg/ml.