摘要目的 采用RNA干扰沉默LIMK2基因来抑制细胞骨架的改建,观察流体剪切力作用下其对成骨细胞c-fos基因力学敏感性的影响.方法 对小鼠颅骨分离的成骨细胞分别给予RNA干扰、阴性RNA干扰2种处理后,进行流体剪切力加载或不加载.分别应用逆转录(RT)-PCR和免疫荧光检测c-fos mRNA和蛋白的表达,并进行统计学分析.结果 在无流体剪切力加载条件下,单纯应用RNA干扰方法抑制细胞骨架改建,并不能使成骨细胞c-fos mRNA(0.0108±0.0074与0.0042±0.0018,t=-1.86,P>0.05)和蛋白(121±7与119±6,t=-1.272,P>0.05)的表达升高;流体剪切力能使成骨细胞c-fos mRNA(0.2203±0.1532比0.0042±0.0018,t=-707.35,P<0.05)和蛋白(178±12比119±6,t=-30.761,P<0.05)的表达水平显著升高;RNA干扰处理可显著提高流体剪切力诱导成骨细胞c-fos mRNA(0.5280±0.0879比0.2203±0.1532,t=-1007.00,P<0.05)和蛋白(224±46比178±12,t=-6.853,P<0.05)的表达水平;采用RNA干扰的方法抑制细胞骨架的改建,可以对流体剪切力诱导成骨细胞c-fos mRNA(F=84.388,P<0.05)和蛋白(F=42.409,P<0.05)的表达起协同作用.结论 采用RNA干扰沉默LIMK2基因抑制细胞骨架的改建,可以促进成骨细胞在流体剪切力作用下的c-fos基因表达.

abstractsObjective To study the effects of cytoskeleton reorganization inhibition with LIMK2 RNAi upon the mechanosensitivity of c-fos gene in osteoblast. Methods Mouse primary osteoblast was treated with LIMK2 specific siRNA (RNAi Group), negative control siRNA (NC Group), and then were loaded or unloaded by fluid shear stress. Real-time PCR and immunofluorescence were used to detect the c-fos expression levels and statistics analysis was performed. Results When the cytoskeleton reorganization was inhibited with RNAi only, the c-fos mRNA (0.0108±0.0074 and 0.0042±0.0018, t = -1.86, P > 0.05) and protein (121±7 and 119±6, t= -1.272, P > 0.05) expression levels of each unloaded group had no significant difference; Fluid shear stress could up-regulate the c-fos mRNA (0.2203±0.1532 vs 0.0042±0.0018, t=-707.35, P<0.05)and protein (178±12 vs 119±6, t=-30.761, P<0.05) expression; After the cytoskeleton reorganization was inhibited with RNAi, the c-fos mRNA(0.5280± 0.0879 vs 0.2203±0.1532, t=-1007.00, P < 0.05) and protein (224±46 vs 178±12, t=-6.853, P < 0.05)expression induced by fluid shear stress had significant difference. Cytoskeleton reorganization inhibition with RNAi had synergistic effect upon the expression of c-fos mRNA(F=84.388, P < 0.05) and protein (F=42.409, P<0.05) induced by fluid shear stress. Conclusion Using RNAi against LIMK2 to inhibit the cytoskeleton reorganization can promote the expression of c-fos gene and thus enhance the mechanosensitivity of c-fos gene in osteoblast.