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目的探讨唑来膦酸(zoledronate acid,ZOL)在共培养体系中对脾酪氨酸激酶(spleen tyrosine kinase,Syk)基因表达及破骨细胞(osteoclast,OC)生成的影响。方法体外将小鼠颅骨成骨细胞与单核巨噬细胞RAW264.7共培养,将细胞分为2组:对照组及5×10-7mol/L ZOL处理24 h组(ZOL组)。应用TRAP染色、牙本质吸收陷窝检测OC生成及骨吸收情况;Real-time PCR、Western blot、免疫荧光化学检测Syk基因表达。结果 2组细胞均有TRAP染色阳性多核OC生成,并在牙本质磨片上形成吸收陷窝;但OC生成及吸收陷窝的数目和体积ZOL组均显著少于对照组(P<0.01)。SykmRNA及蛋白水平在ZOL组也显著下降(P<0.01)。免疫荧光检测显示,Syk在对照组细胞质内强表达,并在细胞周缘形成肌动蛋白环样结构;而ZOL组Syk表达明显减弱,肌动蛋白环样结构消失。结论 ZOL可显著抑制共培养体系中OC生成和骨吸收功能,这可能与其对Syk基因表达的抑制有关。
Objective To investigate the effect of zoledronate acid (ZOL) on the expression of spleen tyrosine kinase (Syk) gene and osteoclast (OC) production in co-culture system. Methods Cultures of mouse skull osteoblast and macrophage RAW264.7 were co-cultured in vitro. The cells were divided into two groups: control group and ZOL group treated with 5 × 10-7mol / L ZOL for 24 hours. TRAP staining and dentin absorption lacuna were used to detect OC production and bone resorption. Syk gene expression was detected by Real-time PCR, Western blot and immunofluorescence staining. Results Both TRAP-positive multinucleated OCs were produced in both groups, and lacunar abscesses were formed on the dentine disk. However, the numbers and volume of lacunar-shaped lacunas in the OC group were significantly lower than those in the control group (P <0.01). SykRNA and protein levels also decreased significantly in ZOL group (P <0.01). Immunofluorescence showed that Syk was strongly expressed in the cytoplasm of the control group and formed an actin loop-like structure at the periphery of the cell. Syk expression in ZOL group was significantly weakened and actin ring-like structure disappeared. Conclusion ZOL can significantly inhibit OC production and bone resorption in co-culture system, which may be related to its inhibition of Syk gene expression.