FoxO1在持续张应力促MC3T3-E1细胞成骨向分化中的表达变化

目的 研究体外持续张应力对MC3T3-E1 细胞成骨向分化过程中Forkhead 转录因子 1（Forkhead box protein O1, FoxO1）表达的影响，探讨FoxO1在持续张应力促进骨向分化机制中的作用。方法 MC3T3-E1细胞接种，应用FX-4000TTM细胞应力加载系统予以体外力学干预。施加频率1 Hz、幅度10%的张应力，按照加力时间长短分为对照和1、4、6、12、24、48、72 h组。运用酶化学染色、实时荧光定量PCR、蛋白免疫印迹、细胞免疫荧光等方法观察持续张应力对MC3T3-E1成骨能力变化的影响及FoxO1基因、蛋白表达和细胞定位情况。结果（1）持续张应力能够促进MC3T3-E1骨向分化。细胞碱性磷酸酶（alkaline phosphatase, ALP）表达量在24、48 h显著高于对照组，骨钙素（osteocalcin, OCN）表达水平于72 h达峰值并显著高于对照组，骨特异性转录因子（runt-related transcription factor-2, Runx2）表达量在4 h与对照组相比显著升高，其蛋白表达量也随加力时间而改变。加力组ALP染色结果与对照组有显著差异。（2）持续张应力促进FoxO1的基因和蛋白表达。FoxO1基因表达水平24 h增高最明显，其蛋白表达量于12 h显著上升。（3）加力6 h FoxO1胞核聚集，胞浆可见，但于24 h转位在胞浆大量表达。结论 10%持续张应力可以促进MC3T3-E1的成骨向分化，同时FoxO1基因和蛋白表达上调、蛋白定位改变。探寻FoxO1表达水平和定位情况在力学刺激下的变化规律，可为研究其在力学刺激中发挥的作用提供实验基础。
Objective To study the effect of continuous tensile stress on expression of Forkhead box protein O1 (FoxO1) in MC3T3-E1 cells in vitro during osteogenic differentiation, and explore the role of FoxO1 in the mechanism of continuous tensile stress induced-osteogenic differentiation. Methods MC3T3-E1 cells were seeded and applied with tensile stress at the frequency of 1 Hz and amplitude of 10% by FX-4000TTM mechanical loading system. MC3T3-E1 cells were divided into control, 1 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h group, respectively, according to the time subjected to the tensile stress. Alkaline phosphatase (ALP) staining, real-time PCR, Western blotting and immunofluorescence were applied to detect the effects of continuous tensile stress on osteogenesis ability of MC3T3-E1 cells, mRNA and protein expression of FoxO1, and allocation of FoxO1 in MC3T3-E1 cells. Results (1) Continuous tensile stress could promote the osteogenic differentiation of MC3T3-E1 cells. Compared with the control group, the mRNA expression of ALP increased significantly at 24 h, 48 h, and the mRNA expression level of osteocalcin (OCN) reached the peak value at 72 h, which was significantly higher than that in the control group. The mRNA expression of runt-related transcription factor-2 (Runx2) significantly increased at 4 h as compared to the control group, and Runx2 protein level changed accordingly. The ALP staining results of the stress group and control group were significantly different. (2) Continuous tensile stress could increase mRNA and protein expression of FoxO1. The mRNA expression of FoxO1 markedly increased at 24 h, and its protein expression significantly elevated at 12 h. (3) FoxO1 was expressed in the nucleus and cytoplasm at 6 h, and then significantly increased in the cytoplasmat at 24 h. Conclusions 10% continuous tensile stress can stimulate