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

透明质酸的定量和定性控制，透明质酸在作为蛋白聚糖聚集体骨架的细胞外基质的组装中发挥作用

基本信息

批准号：
10680577
负责人：
NAKAMURA Toshiya
金额：
$ 1.98万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

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.

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透明质酸合酶。首先，肝素酶对培养的成纤维细胞的细胞层的处理导致刺激外源透明质酸的解聚。肝素酶处理还显示出抑制高分子重量透明质酸的合成。这些结果表明，细胞表面硫酸乙酰肝素可能影响透明质酸的代谢。其次，对透明质酸缺陷的细胞外基质的分析，可以通过在0.5 mm 4-甲基邻不纤维酮的情况下通过细胞培养而形成，显示了细胞层中CD44和纤连蛋白的降低。细胞层中I型胶原蛋白的量没有改变，而在培养基中通过4-甲基固体观察到了分子的增加。这些现象可能是由于细胞外基质中缺乏透明质酸引起的。此外，研究了透明质酸和基质金属蛋白酶之间的关系。在存在外源透明质酸的情况下培养细胞，并将基质金属蛋白酶与对照细胞的活性通过Zymography和Western blotting进行了比较。结果，发现Stromelysin-1的排泄被高分子量透明质酸强烈抑制，高浓度超过1 mg/ml。