雌激素通过调节EphB4/EphrinB2信号参与破骨细胞分化

Estrogen regulates osteoclast differentiation through EphB4 /EphrinB2 signal pathway

目的研究雌激素通过调节EphB4/EphrinB2信号通路对破骨细胞分化的影响。方法 (1)采用RANKL诱导法培养卵巢摘除骨质疏松模型(OVX)组和假手术(Sham)组小鼠的破骨细胞,于第10天提取两组细胞的RNA和蛋白质样品,通过RT-PCR和Western blot检测细胞EphrinB2表达的变化;(2)采用雌激素及雌激素拮抗剂分别诱导培养RAW264.7破骨细胞,培养第8天提取RNA和蛋白质样品,利用RT-PCR和Western blot检测细胞EphrinB2表达的变化;(3)在OVX模型组小鼠的破骨细胞培养中添加EphrinB2配体EphB4-Fc片段,通过RT-PCR检测破骨细胞标志物的表达和抗酒石酸酸性磷酸酶(TRAP)染色并计数,观察破骨细胞分化能力的变化。结果卵巢摘除骨质疏松模型组小鼠EphrinB2的表达量低于假手术组(P<0.01);雌激素拮抗剂组EphrinB2的表达量低于对照组(P<0.01),而雌激素组EphrinB2的表达量高于对照组(P<0.05);给予EphrinB2的配体EphB4-Fc片段后,OVX组小鼠的破骨细胞标志物表达量降低(P<0.01,P<0.001),TRAP染色阳性细胞数减少。结论雌激素可以通过调节破骨细胞EphrinB2的表达影响破骨细胞分化,采用EphB4-Fc片段处理后,OVX组小鼠增强的破骨细胞分化受到抑制。

Objective To investigate the effect of estrogen on osteoclast differentiation by regulating EphB4/EphrinB2 signaling pathway. Methods Osteoclasts of mice in the OVX group and sham group were cultured in the presence of RANKL. The extraction of mRNA and protein in both groups was performed after 10-day culture. The expression of EphrinB2 was detected using RT-PCR and Western boltting. RAW264.7 cells were cultured and induced with estrogen （ E2 group） and estrogen （ ICI group） antagonist, respectively. At the 8th day, the extraction of mRNA and protein was performed. The expression of EphrinB2 was also detected using RT-PCR and Western blotting. The osteoclasts of mice in the OVX group were cultured in the presence of IgG-Fc or EphB4-Fc fragment, a ligand of EphrinB2. The expression of osteoclast markers was detected using RT-PCR. Simultaneously, TRAP staining and counting was performed, in order to observe the osteoclast differentiation. Results The expression of EphrinB2 in OVX group was significantly lower than that in sham group （P 〈 0. 01 ）. Compared with that in control group, the expression of EphrinB2 increased in E2 group but decreased in ICI group （P 〈 0. 01 ; P 〈 0.05 ）. The intervention of EphB4-Fc down-regulated the expression of osteoclast markers in OVX grQup （P 〈 0. 01 ; P 〈 0. 001 ）, and the counts of TRAP positive cells also decreased. Conclusion Estrogen can influence osteoclast differentiation by regulating the expression of EphrinB2 in osteoclasts. After the intervention of EphB4-Fc, the enhanced osteoclasts differentiation in OVX mice can be suppressed.