Change of calpain and effect of mechanical stress on chondrocyte tube culture

软骨细胞管培养中钙蛋白酶的变化及机械应力的影响

• 批准号: 10671357
• 负责人: NAKAGAWA Yasuaki
• 金额: $ 2.11万
• 依托单位: KYOTO 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 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671357/
• 关键词: tube culture; endochondral ossification; mechanical stress; centrifugation; 軟骨細胞; m-カルパイン

Rat chondrocyte cultures grown in centrifuge tubes with intermittent centrifugation differentiate into hypertrophic chondrocytes and form calcification. This culture system provides an extracellular matrix mineralization which is produced by chondrocytes per se, and we can assess this culture system as a model for the endochondral ossification in vivo. In our previous study, m-calpain was demonstrated immunohistichemically in epiphyseal chondrocytes, and immunoreactive m-calpain content in cells increased with terminal differentiation into hypertrophic cells. Using this culture system, we examined whether various degree of centrifugation changed the pattern of the endochondral ossification. Growth-plate chondrocytes were isolated from the rib cartilages of 5-week-old male Wister rats, and the cell suspension was transferred into a 15-ml plastic centrifuge tube at a density of 16X10ィイD14ィエD1 cells/ml/tube and centrifuged at 1, 500 rpm for 5 minutes. In the following, we divided three groups : group A was centrifuged at 3, 000 rpm for 30 minutes every day, group B was centrifuged at 1, 500 rpm for 5 minutes every other day, and group C was added no centrifugation. Group C also had the increase of alkaline phosphatase activity and terminal differentiation like group A and B. There were no significant differences in DNA content, proteoglycan content and alkaline phosphatase activity of all three groups. Calcium content of group A was significantly larger than that of group B at 3-week-culture, but at 4-week-culture, there was no significant difference between them.

在间歇离心的离心管中生长的大鼠软骨细胞培养物分化为肥大软骨细胞并形成钙化。该培养系统提供了由软骨细胞本身产生的细胞外基质矿化，我们可以将该培养系统评估为体内软骨内骨化的模型。在我们之前的研究中，免疫组织化学证明了骨骺软骨细胞中的m-钙蛋白酶，并且细胞中免疫反应性的m-钙蛋白酶含量随着终末分化为肥大细胞而增加。使用该培养系统，我们检查了不同程度的离心是否改变了软骨内骨化的模式。从5周龄雄性Wister大鼠的肋骨软骨中分离生长板软骨细胞，将细胞悬液以16X10ィイD14ィD1细胞/ml/管的密度转移至15ml塑料离心管中，并以1, 500rpm离心5分钟。下面我们分三组：A组每天3, 000 rpm离心30分钟，B组隔天1, 500 rpm离心5分钟，C组不加离心。 C组与A、B组一样也有碱性磷酸酶活性的增加和终末分化。三组DNA含量、蛋白多糖含量和碱性磷酸酶活性无显着差异。培养3周时A组的钙含量显着高于B组，但培养4周时，两者之间无显着性差异。