Effects of tensile forces on serum deprivation-induced osteoblast apoptosis: expression analysis of caspases, Bcl-2, and Bax.
Effects of tensile forces on serum deprivation-induced osteoblast apoptosis: expression analysis of caspases, Bcl-2, and Bax.
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发表时间：
2012-07
影响因子：
6.1
通讯作者：
Xuan Li;Xiao-Ling Zhang;Gang Shen;G. Tang
中科院分区：
文献类型：
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作者：
Xuan Li;Xiao-Ling Zhang;Gang Shen;G. Tang
背景细胞凋亡参与骨对机械负荷的适应性反应。本研究旨在通过检测成骨细胞凋亡相关蛋白caspase、Bcl-2和Bax的表达，探讨张力对成骨细胞凋亡的影响及其相关机制。方法从新生大鼠颅骨分离原代成骨细胞，在含10%胎牛血清（FCS）的最小必需培养基（MEM）中，采用Flexcell 4000应变单元，在无血清和无血清的条件下，对成骨细胞施加72 h的周期性拉力。Annexin V/PI染色流式细胞术检测细胞凋亡。通过Elisa分析Caspase-3活性。逆转录-聚合酶链反应（RT-PCR）检测caspase-8、caspase-9、Bcl-2和Bax基因表达。结果在10%FCS培养条件下，牵张成骨细胞的凋亡率与未牵张成骨细胞的凋亡率无明显差异。去血清组成骨细胞凋亡率明显升高，caspase-3活性增强，caspase-9和Bax表达增加。6%的牵张可减轻血清饥饿诱导的细胞凋亡，同时降低caspase-3活性，降低caspase-8表达，升高Bcl-2水平。相反，12%的拉伸伸长增加了caspase-3的活性，促进了细胞凋亡，并增加了caspase-8和Bax的表达。在施力后，没有发现caspase-9表达的显著变化。结论牵张力通过caspase-3和caspase-8信号通路调控成骨细胞凋亡。轻力通过升高Bcl-2表达从血清剥夺诱导的细胞凋亡中拯救细胞，而重力通过诱导Bax表达促进凋亡损伤。
BACKGROUND Apoptosis is involved in the adaptive responses of bone to mechanical loading. The purpose of this study was to investigate the effects of tensile forces on osteoblast apoptosis and the related mechanism by analyzing the expression of caspases, Bcl-2, and Bax. METHODS Primary osteoblasts were harvested from neonatal rat calvaria and were subjected to cyclic tensile forces for 72 hours using Flexcell 4000 strain unit in Minimum Essential Medium (MEM) with 10% fetal calf serum (FCS) or with serum deprivation. Apoptosis was tested by flow cytometry using annexin V/PI staining. Caspase-3 activity was analyzed via Elisa. The gene expression of caspase-8, -9, Bcl-2, and Bax was quantified by reverse transcription (RT)-PCR. RESULTS In 10% FCS condition, no significant difference in cell apoptosis was found between the stretched and non-stretched osteoblast cultures. Serum withdrawal resulted in higher apoptosis rate in the osteoblasts with increased caspase-3 activity, and elevated expression of caspase-9 and Bax. Six-percent elongation of stretch attenuated the cell apoptosis induced by serum starvation, concurrent with a decrease in caspase-3 activity, a decline of caspase-8 expression, and an elevation of Bcl-2 level. On the contrary, 12% elongation of stretch increased caspase-3 activity and promoted the apoptosis with an elevated expression of caspase-8 and Bax. No significant change of caspase-9 expression was identified upon force application. CONCLUSIONS These results suggested that tensile forces regulate cell apoptosis of primary rat osteoblasts through caspase-3 and caspase-8 signaling cascade. Light forces rescue the cells from serum deprivation-induced apoptosis by elevating Bcl-2 expression, while heavy forces promote the apoptotic insult by inducing Bax expression.