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.

该项目的目的是：1) 阐明影响透明质酸解聚酶的因素，该酶将高分子量透明质酸降解为分子量为 40,000 的片段，2) 表征通过培养形成的透明质酸缺乏的细胞外基质。 t细胞在 4-methylimbelliferone（透明质酸合酶抑制剂）存在的情况下。首先，肝素酶处理培养的成纤维细胞的细胞层导致外源透明质酸解聚的刺激。肝素酶处理还显示出对高分子量透明质酸合成的抑制。这些结果表明细胞表面硫酸乙酰肝素可能影响透明质酸代谢。其次，对缺乏乙酰透明质酸的细胞外基质（可以通过在 0.5 mM 4-甲基伞形酮存在下培养细胞而形成）的分析显示细胞层中 CD44 和纤连蛋白的减少。细胞层中 I 型胶原的量没有变化，但在 4-甲基伞形酮的培养基中观察到分子增加。这些现象可能是由于细胞外基质中缺乏透明质酸造成的。此外，还研究了透明质酸和基质金属蛋白酶之间的关系。在外源透明质酸存在下培养细胞，并通过酶谱法和蛋白质印迹将基质金属蛋白酶的活性与对照细胞的活性进行比较。结果发现，高浓度1mg/ml以上的高分子量透明质酸强烈抑制Stromelysin-1的排泄。