频率对拉伸应变诱导成骨细胞凋亡的影响

Effects of frequency on osteoblast apoptosis induced by tensile strain

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, re- spectively for 1 hour per day in 8 days. The survival rate of the cells was determined by activity of lactate dehydro- genase （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 os- teoblast activity of LDH. There was no significant difference in the total apoptosis rate of flow cytometry at differ- ent 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 in- duced by 1% biaxial tensile strain at the frequency of 1-5 Hz, but the frequency of 2 Hz can induce the early apop- tosis of osteoblasts by up-regulating the expression of Bax/BCI-2.