Effects of Mechanical Stress on Proliferation and Differentiation of Chondrocytes

机械应力对软骨细胞增殖和分化的影响

• 批准号: 14370469
• 负责人: OWAN Ichiro
• 金额: $ 2.82万
• 依托单位: University of the Ryukyus
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370469/
• 关键词: mechanical strain; chondrocyte; ATDC5 cells; type II collagen; type X collagen; aggrecan; histone H4; II型コラーゲン; X型コラーゲン; ALP; PTH; PTHrP receptor; ATDC5

Mechanical strain is an important factor to regulate chondrocyte proliferation and differentiation. Response of chondrocyte to mechanical strain could be differentiation dependent. Chondrogenic ATDC5 cells differentiate into hypertrophic chondrocytes in response to insulin, and regulates type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNA expression. In the present study, we examined whether ATDC5 cells respond to mechanical strain in differentiation specific manner.ATDC5 cells were cultured on plastic plates at 1×10^6 cell/plate in the medium of F12/DMEM containing 5% FBS and 10 μg/ml insulin. ATDC5 were stained with toluidine-blue to observe the morphologic change. We utilized an. in vitro cell loading system using four-point bending. The magnitude of strain was 4200 μstrain and the frequency was 0.5 Hz. To assess the effects of mechanical strain on ATDC5 cells, the expression of type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNAs were determined using northern blot analysis. Six to 24 hours after beginning of loading on day 4 through day 28, total RNA was extracted and hybridized with various cDNA probe.ATDC5 cells initiated chondrogenic differentiation as reported before. Type II collagen mRNA expression was firstly observed on day 4, type X collagen and aggrecan on day 14. The effects of mechanical strain were not observed on day 4 or 7, but mechanical strain up-regulates type II, X collagen, and aggrecan mRNA in a time-dependent manner thereafter. However, there were no increase in ALP and PTH/PTHrP receptor expression.These results indicate that differentiated, but not undifferentiated ATDC5 cells up-regulates type II, X collagen, and aggrecan transcripts in response to mechanical strain.

机械应变是调控软骨细胞增殖和分化的重要因素。软骨细胞对机械应变的反应可能依赖于分化。在胰岛素的作用下，软骨ATDC5细胞分化为肥大的软骨细胞，并调节II型、X型胶原、聚集素、碱性磷酸酶和PTH/PTHrP受体mRNA的表达。在本研究中，我们研究了ATDC5细胞是否以分化特异性的方式对机械应变做出反应。ATDC5细胞以1×106细胞/皿的塑料平板培养，培养液中含有5%胎牛血清和10μg/ml胰岛素的F12/DMEM。用甲苯胺蓝染色观察ATDC5的形态变化。我们利用了一个。体外细胞加载系统采用四点弯曲。应变值为4200μ应变，频率为0.5 Hz。为探讨机械应变对ATDC5细胞的影响，采用Northern印迹法检测ATDC5细胞II型、X型胶原、聚集素、碱性磷酸酶和甲状旁腺激素/甲状旁腺素rP受体的表达。在第4天至第28天开始加载后6~24小时，提取总RNA并与各种cDNA探针杂交，ATDC5细胞启动软骨分化。在培养的第4天观察到II型胶原的表达，在第14天观察到X型胶原和聚集素的表达。在第4天和第7天没有观察到机械应变的影响，但此后机械应变以时间依赖的方式上调了II型、X型胶原和聚集素的mRNA表达。然而，ALP和PTH/PTHrP受体的表达并没有增加。这些结果表明，分化的但不是未分化的ATDC5细胞在机械应变下上调了II型、X型胶原和聚集素扫描蛋白的转录。