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A cell-stretching system as a model system for the study of biomechanics

细胞拉伸系统作为生物力学研究的模型系统

基本信息

批准号：
08558078
负责人：
OBINATA Takashi
金额：
$ 4.22万
依托单位：
DEPARTMENT OF BIOLOGY,CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1.With an aim to clarify the effects of mechanical stress on cells in an in vitro system, we manufactured a culture system which permits unidirectional stretch of a cell culture substrate repeatedly over long periods. Thin elastic silicon membrane with fine parallel grooves as a substrate for cell culture and an appropriate culture medium were also devised.2.Since muscle cells are formed and maintained under a mechanically active environment, the effects of mechanical stress on cultured cells were examined with muscle cells as a model and observed two remarkable phenomena caused by stretching : 1) stretch-dependent cell arrangement and 2) hypertrophy of the muscle cells as follows :1) When rat aortic smooth muscle cells (A10) and mouse C2 myoblasts were cultured under a standard condition and then the substrate was stretched repetitively at a frequency of 1 Hz with an amplitude of 20%, the cells became oriented at an angle of about 70o to the direction of stretching. Actin filaments al … More so oriented in the same direction, but filament orientation preceded cell orientation. We found that this stretch-dependent cell orientation was completely blocked by 100 mM gadolinium, an inhibitor of a stretch-activated ion channel, and cycloheximide, but filament reorientation was not affected by these agents at all. From these results, it is concluded that orientation of filaments and the orientation of the cells are closely related but regulated differently.2) In the case of muscle cells (myotubes), stretching in a longitudinal cell axis is important from a physiological view point. Since rearrangement of cell orientation did not occur during stretching of myotubes aligned along the grooves, it was possible to stretch myotubes for more than a week in the direction of the longitudinal cell axis. We observed that myotube diameters increased significantly as compared with the control myotubes cultured without stretch. We further found that a protein of about 67 kDa accumulated in the stretched myotubes. We identified this protein with albumin based on antigenicity, size and pI.The albumin was the same as that in the culture medium and in addition, biotinized albumin added to the medium was also detected in the stretched myotubes. Therefor, we conclude that albumin in the medium was incorporated into myotubes in a stretching-dependent manner. Physiological implication of stretch-dependent albumin uptake remains for future investigation. Less

1.为了阐明体外系统中机械应力对细胞的影响，我们制造了一种允许细胞培养基质长期单向拉伸的培养系统。还设计了具有细平行凹槽的薄弹性硅膜作为细胞培养的基质和适当的培养基。2.由于肌肉细胞在机械活动环境下形成和维持，因此以肌肉细胞为模型检查机械应力对培养细胞的影响，并观察到拉伸引起的两个显着现象：1）拉伸依赖性细胞排列和2）肌肉细胞肥大如下：1）当大鼠主动脉平滑肌细胞(A10)和小鼠C2成肌细胞在标准条件下培养，然后以1 Hz的频率、20%的幅度重复拉伸基底，细胞定向成与拉伸方向成约70°的角度。肌动蛋白丝的方向相同，但丝的方向先于细胞的方向。我们发现这种拉伸依赖性细胞定向被 100 mM 钆（拉伸激活离子通道的抑制剂）和放线菌酮完全阻断，但细丝重新定向根本不受这些药物的影响。从这些结果可以得出结论，细丝的方向和细胞的方向密切相关，但调节方式不同。2）就肌肉细胞（肌管）而言，从生理角度来看，纵向细胞轴的拉伸很重要。由于在沿着凹槽排列的肌管拉伸过程中没有发生细胞方向的重新排列，因此可以在纵向细胞轴方向上拉伸肌管一周以上。我们观察到，与未经拉伸培养的对照肌管相比，肌管直径显着增加。我们进一步发现约 67 kDa 的蛋白质在拉伸的肌管中积累。我们根据抗原性、大小和等电点将这种蛋白质鉴定为白蛋白。白蛋白与培养基中的白蛋白相同，此外，在拉伸的肌管中也检测到添加到培养基中的生物素化白蛋白。因此，我们得出结论，培养基中的白蛋白以拉伸依赖性方式掺入肌管中。拉伸依赖性白蛋白摄取的生理意义仍有待未来研究。较少的