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

1.为了阐明机械应力对体外系统中细胞的影响，我们制造了一种培养系统，该系统允许细胞培养底物在长时间内重复单向拉伸。设计了具有细平行沟槽的硅弹性薄膜作为细胞培养基质和合适的培养基。由于肌肉细胞是在机械活动的环境下形成和维持的，我们以肌肉细胞为模型，考察了机械应力对培养细胞的影响，观察到拉伸引起的两个显著现象：1)拉伸依赖性细胞排列，2)肌细胞肥大：1)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后，以1 Hz的频率和20%的振幅反复拉伸底物，细胞取向与拉伸方向成约70度角。肌动蛋白丝的方向相同，但丝的方向先于细胞的方向。我们发现这种依赖拉伸的细胞取向被100mm钆（一种拉伸激活离子通道的抑制剂）和环己亚胺完全阻断，但丝的重新取向完全不受这些药物的影响。由此可见，丝的取向与细胞的取向密切相关，但调控方式不同。2)就肌细胞（肌管）而言，从生理学的角度来看，纵向细胞轴的拉伸是重要的。由于在沿着凹槽排列的肌管拉伸期间没有发生细胞方向的重排，因此有可能在纵向细胞轴方向拉伸肌管超过一周。我们观察到，与未拉伸培养的对照肌管相比，肌管直径显著增加。我们进一步发现，拉伸后的肌管中积累了约67 kDa的蛋白质。我们根据抗原性、大小和pI与白蛋白鉴定了该蛋白。白蛋白与培养基中的白蛋白相同，并且在拉伸的肌管中也检测到添加到培养基中的生物化白蛋白。因此，我们得出结论，培养基中的白蛋白以拉伸依赖的方式并入肌管。拉伸依赖性白蛋白摄取的生理意义有待进一步研究。少

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