周期性张应力下成肌细胞中半胱天冬氨酸蛋白酶9参与力学信号传导

摘要

背景：功能矫形使面颌部肌肉发生适应性改建，以纠正错颌畸形。成肌细胞是适应性改建的主要体现者，周期性牵张应力导致成肌细胞凋亡，而半胱天冬氨酸蛋白酶9是线粒体凋亡通路中的重要因子。　　目的：明确半胱天冬氨酸蛋白酶9在不同时间周期性张应力作用下的表达变化。　　方法：以大鼠L6成肌细胞为实验对象，在成功构建成肌细胞体外培养-力学刺激模型的基础上，通过多通道细胞牵张应力加载系统，分别对待测细胞持续施加1，6，12，24 h的周期性张应力，不加力组为对照组。倒置相差显微镜下观察成肌细胞的形态学改变和生长状况；运用RT-PCR和Western Blot技术检测周期性张应力作用下半胱天冬氨酸蛋白酶9的mRNA和蛋白含量变化。　　结果与结论：倒置相差显微镜观察结果显示成肌细胞施加周期性张应力后贴壁生长情况良好，细胞无变性并且脱落率极低，说明成功构建了成肌细胞体外培养-力学刺激模型。RT-PCR和Western Blot检测结果显示，随着加力时间的延长，半胱天冬氨酸蛋白酶9 mRNA含量和活化型半胱天冬氨酸蛋白酶9的蛋白含量均显著增加，而半胱天冬氨酸蛋白酶9前体蛋白含量随着加力时间的延长均显著降低。可见半胱天冬氨酸蛋白酶9参与了周期性张应力作用下的力学信号传导。%BACKGROUND:The adaptive reconstruction of maxil ofacial muscles would happen when functional orthopedic treatment is done to cure micromaxil ary deformity. The myoblast is the main responder in the process of adaptive reconstruction, and cyclic stretch can induce apoptosis of myoblasts. Caspase-9 is an important factor in the mitochondrial apoptosis pathway. OBJECTIVE:To investigate the expression of Caspase-9 in different cyclic stretch. METHODS:Based on myoblasts cultured in vitro-mechanical stimulation model, the rat L6 myoblasts were loaded stretch for 1, 6, 12 and 24 hours through multi-channel cellstress loading system, while the control group received no stretch. The morphological change and growth of myoblasts were observed under inverted phase contrast microscope;the expression of the mRNA and protein of Caspase-9 were detected by RT-PCR and western blot analysis, respectively. RESULTS AND CONCLUSION:Under inverted phase contrast microscope, the rat L6 myoblasts at cyclic stretch maintained a good growth state and biological characteristics;there was no celldegeneration;and the loss rate was extremely low, which could demonstrate that myoblast in vitro-mechanical stimulation model was established successful y. The results of RT-PCR and western blot analysis showed that, the expression of Caspase-9 mRNA and Cleaved Caspase-9 protein was significantly increased as the loading time prolonged, and the expression of Procaspase-9 protein was significantly decreased as the time. We can conclude that Caspase-9 is involved in the mechanical signal transduction of cyclic stretch.