机械牵张力对血清饥饿诱导的成骨细胞凋亡的作用研究--caspases，bcl-2及bax的差异表达

目的：细胞凋亡参与了机械力作用下骨组织的适应性改建。本研究的目的是观察机械牵张力对体外培养成骨细胞凋亡的影响，并通过检测caspases，bcl-2和bax的表达来探讨其相关机制。 方法：使用Flexcell 4000TM细胞应变加载系统对新生SD大鼠颅顶骨成骨细胞施加不同大小的牵张力，细胞培养于含10%胎牛血清或无血清的MEM培养液中。运用流式细胞技术检测成骨细胞的凋亡，Elisa检测caspase-3活性，Real-time RT-PCR检测细胞caspase-8，caspase-9，bcl-2和bax的基因表达。 结果：在含10% 胎牛血清的培养液中，牵张力对成骨细胞的凋亡无影响。无血清培养时，成骨细胞凋亡率和caspase-3活性增加，同时caspase-9和 bax表达也增加。6%牵张力对无血清培养诱发的成骨细胞凋亡有抑制作用，同时caspase-3活性和caspase-8表达下降，并伴随bcl-2表达升高。而12%牵张力作用于无血清培养的成骨细胞时，细胞凋亡率增加，并伴随caspase-3活性、caspase-8和bax表达升高。Caspase-9的表达在张应变刺激作用下无明显变化。 结论：机械牵张力通过caspase-8引发的caspase级联反应调控成骨细胞的凋亡。轻力上调bcl-2 的表达来抑制无血清诱导的成骨细胞凋亡，而重力通过上调bax的表达促进细胞凋亡。

Effects of tensile forces on serum deprivation-induced osteoblast apoptosis --A differential expression of caspases, bcl-2 and bax

Objective: 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 72hrs 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 real-time RT-PCR. Results: In 10% FCS condition, no significant change of 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. 6% 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.