周期性牵张应力作用下成肌细胞ROS生成及其与JNK和NFkappaB的交互对话

目的探讨不同应力作用下成肌细胞内ROS产生的量J、NK和NFkappaB的活化程度以及它们间的交互对话。方法利用Flexercell细胞加力仪对体外培养C2C12成肌细胞施加不同频率、不同幅度的牵张应力。细胞计数、MTT实验检测应力条件下细胞的活性,通过荧光染料检测ROS的生成,荧光报告系统检测NFkappaB的活性以及Western Blot检测磷酸化和总体JNK1的水平,分析三者之间的交互对话关系。结果随着周期性应力的增加,细胞内ROS生成逐渐增加。随着应力的增大,细胞的凋亡逐渐增加,与此同时J,NK1活性逐渐增高,而JNK1表达水平并没有明显改变。与ROS生成量和JNK1活化逐渐增加不同,应力在10%以下时NFkappaB的活性随着应力的增大而增大,而当应力大于15%时,NFkappaB活性开始下降。结论当ROS的量积累到一定的时候J,NK开始激活,而ROS的量较低的时候,NFkappaB被激活。

The crosstalk among ROS,JNK and NFkappaB of C2C12 myoblast under cyclic stretch

Objective To investigate the crosstalk among ROS,JNK and NFkappaB of C2C12 myoblast under cyclic strentch.Methods By using Flexercell strain system,cultured C2C12 myoblasts could be stretched by different stretch magnitudes.Cell counting and MTT was used to estimate the viability of the cells.Fluorescent dye was introduced to test ROS level.Fluorescent reporter and Western Blot were applied to test NFkappaB activity and pJNK1 level and total JNK1 and the crosstalk of them.Results With the stretch magnitudes increased,consistent with the increased apoptosis,JNK1 were gradually activated,with no obvious changes of JNK1 expression.In contrast,NFkappaB activity increased with the extension rate increasing within the magnitudes no more than 10%,and it began to decline when the extension rate is over 15%.Conclusion JNK activation occurred only when ROS production accumulated sufficiently while NFkappaB was activated at a much lower threshold of ROS abundance.