Strain magnitude dependent intracellular calcium signaling response to uniaxial stretch in osteoblastic cells
Strain magnitude dependent intracellular calcium signaling response to uniaxial stretch in osteoblastic cells
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DOI：
10.1299/jbse.15-00242
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发表时间：
2015
影响因子：
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通讯作者：
Katsuya Sato;Yuki Ogawa;S. Ito;S. Fujisawa;K. Minami
中科院分区：
文献类型：
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作者：
Katsuya Sato;Yuki Ogawa;S. Ito;S. Fujisawa;K. Minami
在成骨细胞中，细胞改变其细胞内Ca浓度（[Ca]i）作为对机械刺激的响应。虽然已有研究表明成骨细胞对多种机械刺激有反应，包括基底拉伸、流体流动中的剪切应力、玻璃微针直接压入和静水压力等，细胞内钙信号对底物拉伸的响应特征的细节仍然不清楚，因为拉伸过程中的运动伪影导致焦平面和观察区域外，并使单细胞水平上[Ca]i变化的原位时间推移观察复杂化。在这项研究中，我们结合了我们最初开发的细胞拉伸MEMS器件与比率显微镜方法与两种可见波长的钙指示染料。细胞拉伸微装置和比率测量方法减少了拉伸过程中运动伪影的影响，使我们能够定量评估细胞对拉伸的钙信号响应特性。将MC 3 T3-E1成骨细胞接种到细胞拉伸微型装置上，并通过Ca指示剂Fluo 3和Fura Red进行荧光标记。在恒定应变率下对细胞施加5%、10%和15%三个应变量的单轴拉伸，并以高时间和空间分辨率原位延时观察细胞对拉伸的钙信号响应。我们成功地在拉伸应用过程中获得了延时荧光图像序列，而没有过多的失焦和空白时间。结果表明，MC 3 T3-E1细胞对牵张的钙信号反应强度随牵张应变的大小而变化。随着拉伸应变量的增加，拉伸细胞中Ca指示剂的荧光比值的变化量也增加。这一结果表明，成骨细胞可以感受到的力学信号转导途径的上游，如细胞外钙离子的流入的机械刺激的大小的可能性。
In osteoblast cells, cells change their intracellular Ca concentration ([Ca]i) as the response to mechanical stimuli. Although it has been reported that osteoblast cells responded to many kinds of mechanical stimuli including stretch of substrate, shear stress in fluid flow, direct indentation of glass microneedle and hydrostatic pressure etc., the detail of the characteristics of intracellular calcium signaling response to substrate stretch still remains unclear because motion artifact during stretch application causes out of focus plane and observation area, and complicates the in situ time lapse observation of change in [Ca]i in a single cell level. In this study, we combined our originally developed cell stretching MEMS device with the ratiometric microscopy method with two kinds of visible wavelength calcium indicator dyes. The cell stretching micro device and the ratiometric method reduce the influence of motion artifact during stretch application, and enable us to quantitatively evaluate the characteristics of cellular calcium signaling response to stretch. MC3T3-E1 osteoblastic cells were plated onto the cell stretching micro device and fluorescently labeled by Ca indicator Fluo 3 and Fura Red. A uniaxial stretch with three magnitudes of strain 5%, 10% and 15% with constant strain rate were applied to the cells, and in situ time lapse observation of cellular calcium signaling response to stretch was conducted with high temporal and spatial resolution. We succeeded in obtaining time lapse fluorescent image sequences during stretch application without excessive out of focus and blank time. The results revealed that MC3T3-E1 cells change the intensity of calcium signaling response to stretch according to the stretch strain magnitude. As stretch strain magnitude was increased, the amount of change in fluorescent ratio value of Ca indicators in stretched cells also increased. This result suggests the possibility that osteoblastic cells can sense the magnitude of mechanical stimuli at upstream of mechanotransduction pathway such as influx of extracellular Ca.