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

机械应力传感机制及低分子G蛋白与骨形成关系的研究

基本信息

批准号：
17591588
负责人：
INORI Fumiaki
金额：
$ 2.3万
依托单位：
OSAKA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591588/
关键词：
mechanical stress osteoblast bone formation BMP low molecular G protein

项目摘要

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.

首先，我们检查了MC3T3-E1和ST-2细胞在暴露于Rho激酶抑制剂Y27632后是否通过使它们在细胞延伸装置ST-140中伸展来改变信号传输过程。但低密度剂量的ALP活性没有差异，并且由于细胞与室分离，我们不能通过高密度剂量使它们拉伸。其次，我们在硅室中培养El和ST-2以确认Rho的活性是否由拉伸引起，并拉伸12小时并将RhoA活性与对照进行比较，但我们无法识别出显着差异。接下来，我们检查拉伸和BMP信号的相关性。首先我们用ST-2和El检测了BMP4的ALP活性，ST-2对BMP有容量依赖性反应，而El的反应很差。随后，人们认识到1%的拉伸适合提高ST-2的ALP活性，但是如果我们在拉伸1%24小时后用5%BMP4 50ng/ml培养ST-2 48小时，则ALP活性会不利地下降，因此没有观察到协同效应。作为未来的工作，我们开发了突变型低分子G蛋白（显性失活）并检查与拉伸的相关性，因为一种使用Rho抑制剂的方法直接阻碍细胞粘附并抑制拉伸，此外，我们实验室还显示了cAMP增强BMP作用的数据。因此，我们通过 ELISA 测量细胞的 cAMP 密度，因为 RhoA 的信号抑制预计与 MAPK 过程不同。那时，我们将通过使用它们的抑制剂来评估与其他信号级联的相关性。