频率对拉伸应变诱导成骨细胞凋亡的影响

目的 考察频率对拉伸应变诱导成骨细胞凋亡的影响。方法 采用Flexcell力学加载系统对小鼠前成骨细胞MC3T3-E1施加1%双轴拉伸应变刺激，频率分别为1、2、3、4、5 Hz，每天加载1 h，间歇性拉伸8 d;通过测定乳酸脱氢酶(LDH)活性检测细胞死亡率；采用Annexin V-FITC/ PI流式细胞术观察细胞凋亡情况；实时PCR检测细胞凋亡标志基因caspase-3、-9以及Bcl-2、Bax基因水平；Western blotting检测caspase-3、-9蛋白表达。结果 不同加载频率对成骨细胞LDH活性无影响；不同频率下流式细胞术总体凋亡率无显著性差异，但是2 Hz频率可诱导成骨细胞早期凋亡。2 Hz拉伸应变可明显上调caspase-3、-9基因和蛋白表达，上调Bax/Bcl2蛋白比值。结论 1%双轴拉伸应变刺激下1~5 Hz频率不能引起成骨细胞凋亡和死亡，但2 Hz频率可诱导成骨细胞早期凋亡，且通过上调Bax/Bcl-2表达实现的。
Objective To investigate the effect of frequency on osteoblast apoptosis induced by tensile strain. Methods MC3T3-E1 cells were applied with 1% biaxial tensile strain at the frequency of 1, 2, 3, 4, 5 Hz, respectively for 1 hour per day in 8 days. The survival rate of the cells was determined by activity of lactate dehydrogenase (LDH). Annexin V-FITC/ PI Flow cytometry was used to test cell apoptosis. Real-time RT-PCR was used to detect the gene level of apoptosis markers caspase-3, -9 as well as Bcl-2 and Bax, and Western blotting was used to test protein expressions of caspase-3, -9. Results Different loading frequencies had no effect on osteoblast activity of LDH. There was no significant difference in the total apoptosis rate of flow cytometry at different frequencies. However, the frequency of 2 Hz could induce early osteoblast apoptosis. Tensile strain at the frequency of 2 Hz could significantly increase the expression of caspase-3, -9 gene and protein, and induce cell apoptosis with the up-regulation of the Bax/Bcl-2. Conclusions Osteoblast apoptosis and death cannot be induced by 1% biaxial tensile strain at the frequency of 1-5 Hz, but the frequency of 2 Hz can induce the early apoptosis of osteoblasts by up-regulating the expression of Bax/BCl-2.