Toyota Central R&D Labs., Inc., Nagakute, Aichi, Japan.
Biotechnol Bioeng. 2010 Jun 15;106(3):482-9. doi: 10.1002/bit.22705.
We have developed a novel method for measuring active tension generated by cultured myotubes using UV-crosslinked collagen film. Skeletal myoblasts cell line C2C12 or human primary skeletal myoblasts were seeded onto a thin (35 microm) collagen film strip, on which they proliferated and upon induction of differentiation they formed multinucleated myotubes. The collagen film-myotube complex contracted upon electric pulse stimulation which could be observed by light microscope. When collagen film-myotube complex were attached to force transducer, active tension generation was observed upon electric pulse stimulation. Measurement of active tension was possible for multiple times for more than 1 month with the same batch of collagen film-myotube complex. Active tension generation capability of C2C12 myotubes increased with progression of differentiation, reaching maximal value 6 days after induction of differentiation. Using this method, we measured the effect of artificial exercise induced by electric pulse on active tension generation capability of C2C12 myotubes. When the electric pulses of 1 Hz were continuously applied to induce artificial exercise, the active tension augmentation was observed. After 1 week of artificial exercise, the active tension reached approximately 10x of that before the exercise. The increased active tension is attributable to the formation of the sarcomere structure within the myotubes and an increased amount of myotubes on the collagen film. The increased amount of myotubes is possibly due to the suppressed atrophy of myotubes by enhanced expression of Bcl-2.
我们开发了一种新的方法，用于测量使用 UV 交联胶原膜产生的培养肌管的主动张力。骨骼肌成肌细胞系 C2C12 或人原代骨骼肌成肌细胞接种到薄（35 微米）胶原膜条上，在其上增殖，并在诱导分化后形成多核肌管。胶原膜-肌管复合物在电脉冲刺激下收缩，可通过光显微镜观察到。当胶原膜-肌管复合物附着于力传感器时，可观察到电脉冲刺激下的主动张力产生。使用同一批胶原膜-肌管复合物，可进行多次主动张力测量，持续超过 1 个月。C2C12 肌管的主动张力生成能力随着分化的进展而增加，在诱导分化后 6 天达到最大值。使用这种方法，我们测量了电脉冲诱导的人工运动对 C2C12 肌管主动张力生成能力的影响。当以 1 Hz 的电脉冲连续施加以诱导人工运动时，观察到主动张力增加。经过 1 周的人工运动后，主动张力达到运动前的约 10 倍。增加的主动张力归因于肌管内肌节结构的形成以及胶原膜上肌管数量的增加。肌管数量的增加可能是由于 Bcl-2 的表达增强抑制了肌管萎缩。
