周期性张应变作用下成骨细胞凋亡的体外研究

Study on osteoblast apoptosis in response to mechanical stretch in vitro

目的研究周期性张应变对体外培养的成骨细胞凋亡作用及其与细胞增殖和细胞分化之间的关系。方法酶消化法分离培养新生SD大鼠颅顶骨成骨细胞,P2-P4代成骨细胞培养2d和4d后,用无血清培养法诱导成骨细胞凋亡,同时使用Flexcell4000TM细胞应变加载系统分别对细胞施加72h变形率为6%和13.6%的等轴周期性张应变。根据总培养时间分为5d和7d两组。运用流式细胞技术对成骨细胞凋亡水平进行检测,同时进行细胞记数及碱性磷酸酶(ALP)蛋白含量检测。结果与不加力的对照组相比,6%周期性张应变使成骨细胞凋亡率有不同程度的降低并伴随细胞数量增加;而在13.6%的周期性张应变下,成骨细胞凋亡率增加的同时,细胞数量有不同程度地降低;而相同培养时间下,5d组细胞对6%的周期性张应变更加敏感,而7d组细胞对13.6%的周期性张应变更加敏感。ALP活性在两种张应变刺激作用下均有所下降。结论周期性张应变对成骨细胞的凋亡具有影响,不同力值的周期性张应变可通过促进或抑制细胞凋亡的方式调控成骨细胞的活性。

Objective To investigate the effect of mechanical stretch force on the apoptosis, proliferation and differentiation of osteoblast cultured in vitro. Method Rat calverial osteoblasts passage 2 to 4 were used and cell apoptosis was induced by serum starvation aRer 2 or 4 days of static culture. The osteoblasts were then subjected to cyclic equibiaxial stretch forces for 72 hours using Flexcell 4000TM strain unit. Apoptotic levels of the osteoblasts were quantified by flowcytometer right after the stretch application. The cell number and alkaline phosphatase （ALP） activity were also evaluated. Result 6% elongation of stretch force led to a decrease of apoptosis rate with an increase of cell number, in which cells stretched after 2 days culture were more sensitive, showing a 45% decrease of apoptosis rate and 34% increase of cell number. 13.6% elongation of stretch force elicited an increase of osteoblast apoptosis while the cell population was decreased, in which cells stretched arter4 days culture were more significantly affected, showing a 192% increase of apoptosis rate and 64% decrease of cell population/MP activity was declined upon the two force loading magnitudes.Conclusions Stretching force can affect apoptosis in osteoblast culture. Different force loading magnitudes could control the osteoblast activity by accelerating or restraining the osteoblast apoptosis.