细胞骨架在流体剪切力诱导成骨细胞COX-2基因和蛋白表达中的作用

Cytoskeleton integrity in the expression of COX-2 gene and protein in osteoblasts induced by fluid shear stress

目的:探讨细胞骨架在流体剪切力(FSS)诱导成骨细胞COX-2基因和蛋白表达中的作用。方法:将第3代小鼠原代成骨细胞分为4组:对照组、细胞松弛素D(CD)组、FSS组、FSS+CD组。FSS组和FSS+CD组分别施加12 dyne/cm2的FSS 60 min,FSS组只加FSS,而FSS+CD组在加入CD预处理60 min后施加FSS 60 min;对照组不做任何处理;CD组仅加入CD。应用实时荧光定量PCR、蛋白质印迹法和激光共聚焦显微镜检测COX-2 m RNA和蛋白的表达以及肌动蛋白细胞骨架的变化,并对结果进行双因素方差分析。结果:FSS组与其他3组相比,COX-2的m RNA和蛋白表达显著增加(P<0.05);CD组与对照组及FSS+CD组相比,COX-2的m RNA和蛋白的表达水平无显著差异(P>0.05);FSS+CD组与FSS组相比,COX-2的m RNA和蛋白的表达水平显著降低(P<0.05)。结论:应用FSS能使成骨细胞骨架发生重排、增多、增密且与流体方向保持一致。细胞骨架微丝的完整性与COX-2基因和蛋白表达密切相关,而CD对COX-2基因和蛋白的表达基本没有影响。

Objective To investigate the role of cytoskeleton integrity in the expression of osteoblasts COX-2 gene induced by fluid shear stress（FSS）. Methods The third generation of mouse primary osteoblasts was divided into 4 groups, namely control group, cytochalasin D（CD） group, FSS group, FSS＋CD group. The FSS of 12 dyne/cm2 was applied for 60 min in FSS＋CD group and FSS＋CD group, respectively. Only FSS was added in FSS group, while FSS was applied for 60 min in FSS group after pretreatment with CD for 60 min; no treatment for the control group; only CD was added in CD group. The real-time fluorescence quantitative PCR, western blot and confocal laser scanning microscopy were used to detect the expression of COX-2 m RNA and protein and to observe the change of F-actin cytoskeleton. The obtained results were analyzed with two-way analysis of variance. Results The expression levels of COX-2 m RNA and protein were significantly increased in FSS group, compared with those in the other 3 groups（P0.05）. No significant differences were found in the expression levels of COX-2 protein and m RNA between CD group and control group or FSS ＋ CD group（P0.05）. The expression levels of COX-2 m RNA and protein were significantly decreased in FSS＋CD group, compared with that in FSS group（P0.05）. Conclusion The application of FSS can rearrange and increase the cytoskeleton, and increase the density of cytoskeleton, keeping consistent with the direction of the fluid. The integrity of cytoskeleton microfilament is closely related to the expressions of COX-2 gene and protein, while CD had trivial effect on the expression of COX-2 gene and protein.