中国修复重建外科杂志

中国修复重建外科杂志

FTY720-P 对破骨细胞 EphA2-EphrinA2双向信号通路作用的研究

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目的 探讨 FTY720-P 对破骨细胞 EphA2-EphrinA2 双向信号通路的影响。 方法 取小鼠巨噬细胞 RAW264.7,采用地塞米松及 1α,25-二羟维生素 D3 诱导分化为破骨细胞,并行抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色鉴定。将诱导培养的破骨细胞分为两组,实验组给予 400 ng/mL 的 FTY720-P 处理,对照组不给予 FTY720-P。培养 48 h 后,取细胞行实时荧光定量 PCR 检测、Western blot 检测以及免疫荧光染色观察,分析细胞中 EphA2、EphrinA2、RhoA,以及骨重建相关蛋白 BMP-2 及 TGF-β1 的表达。 结果 经 TRAP 染色鉴定,RAW264.7 细胞成功诱导成为破骨细胞。培养 48 h 后,与对照组相比,实验组 EphA2 及 EphrinA2 mRNA 及蛋白相对表达量均明显降低(P<0.05),RhoA 蛋白相对表达量也明显降低(P<0.05);BMP-2 及 TGF-β1 mRNA 相对表达量明显增高(P<0.05),蛋白表达增强。 结论 FTY720-P 能通过影响破骨细胞 EphA2-EphrinA2 双向信号通路之间的传导下调 RhoA,并促进 TGF-β1 和 BMP-2 表达,最终影响破骨细胞的破骨作用。

Objective To investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts. Methods Murine RAW 264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D3, and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins[bone morphogenetic protein (BMP-2) and transform growth factor β1 (TGF-β1)] were analyzed and compared. Results RAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours (P<0.05), and the relative expression of RhoA protein also significantly decreased (P<0.05). The relative expressions of BMP-2 and TGF-β1 mRNAs were significantly increased (P<0.05), and those protein expressions were enhanced. Conclusion FTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β1 and BMP-2 by affecting the transduction of EphrinA2-EphA2 bidirectional signaling in osteoclasts.

关键词: FTY720-P; EphA2; EphrinA2; RhoA; 破骨细胞

Key words: FTY720-P; EphA2; EphrinA2; RhoA; osteoclasts

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