Investigation of sensor mechanism of mechanical stress and relationship of low molecular G protein for bone formation

首先，我们检测了MC3T3-E1和ST-2细胞在暴露于Rho激酶抑制剂Y27632后，是否通过在细胞延伸装置ST-140中伸展来改变信号传递路线。但低密度组ALP活性无明显变化，高密度组因细胞脱离小室而不能使细胞伸展。其次，我们在硅胶室中培养EL和ST-2，以确定拉伸是否对Rho的活性有贡献，并将其与对照组进行12小时的比较，但我们不能识别显著差异。首先，我们用ST-2和EL检测了BMP4的ALP活性，ST-2对BMP的反应是容量依赖的，而EL的反应很差。随后，人们认识到1%的拉伸是提高ST-2细胞ALP活性的合适条件，但如果在ST-2中加入5%的BMP4 50 ng/ml培养48小时，再拉伸1%24小时，则ALP活性反而下降，没有观察到协同作用。作为进一步的工作，我们开发了突变型低分子G蛋白(显性阴性)，并考察了与拉伸的相关性，因为一种使用Rho抑制剂的方法直接阻断了细胞的黏附，抑制了拉伸，此外，我们在实验室还显示了cAMP增强BMP的互补作用。因此，由于RhoA的信号抑制可能与MAPK的过程不同，所以我们用ELISA法测定细胞的cAMP密度。在这种情况下，我们将通过使用其他信号级联的抑制剂来评估它们与其他信号级联的相关性。

First of all, we examined MC3T3-E1 and a ST-2 cell whether they make a change on a signal transmission course by making them stretch in cell extension device ST-140 after exposing them to Rho kinase inhibitor Y27632. But there was no difference in ALP activity by the low density dosage and we could not make them stretch by the high density dosage because cells were detached from chamber. Secondly, we cultured El and ST-2 in a silicon chamber to confirm whether activity of Rho contributed by stretch, and made stretch for 12 hours and compared RhoA activity with control, but we could not recognize the significant difference.Next, we examined correlation of stretch and BMP signal. At first we examined ALP activity for BMP4 with ST-2 and El, and ST-2 reacted for capacity dependence for BMP, whereas the response of El was bad. Successively, it was recognized that stretch of 1% was a suitable to raise ALP activity for ST-2, however the ALP activity fell adversely if we cultured ST-2 with 5%BMP4 50ng/ml for 48 hours after stretching 1% for 24 hours, so that the synergistic effect was not observed.As future work, we develop mutant low molecular G protein (dominant negative) and examine correlation with stretching, because a method to use Rho inhibitor for directly obstructs the cell adhesion and inhibits stretching,In addition, we showed a data the cAMP reinforce action of BMP complementarily in our laboratory. Thus, we measure the cAMP density of a cell by ELISA because the signal suppression of RhoA is expected to be different from MAPK course. On that occasion, we will evaluate correlations with other signal cascades by using inhibitor of them.