白细胞介素-21对RAW264.7细胞向破骨细胞分化的影响  

Effect of interleukin-21 on osteoclastogenesis in RAW264.7 cells

作　　者：张颖健[1,2] 李常虹[1] 邢瑞[1] 孙琳[1] 赵金霞[1] 刘湘源[1] 

机构地区：[1]北京大学第三医院风湿免疫科,北京100191 [2]北京市公安局强制治疗管理处

出　　处：《中国骨质疏松杂志》2015年第3期253-258,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金青年基金项目(81102255)

摘　　要：目的了解白细胞介素-21（IL-21）诱导小鼠巨噬细胞系RAW264.7细胞向破骨细胞分化的作用,并探讨其可能的机制。方法 1、以不同浓度IL-21（0,1,10,20,40 ng/ml）处理RAW264.7细胞,培养5天后进行抗酒石酸酸性磷酸酶（TRAP）染色,Western Blot和免疫组化法检测降钙素受体（CTR）表达,采用Real time PCR法检测CTR和组织蛋白酶（Cathepsin）-K的mRNA表达水平。2、以信号通路抑制剂AG490、LY294002和PD98059作用30min后再加入IL-21,培养5天后观察破骨细胞形成情况,以Western Blot检测IL-21作用不同时间点时信号通路分子总蛋白和磷酸化蛋白水平,探讨IL-21直接诱导破骨细胞分化的作用机制。结果 1、在无RANKL作用的情况下,随着IL-21浓度增加TRAP阳性细胞数逐渐增多,20 ng/ml作用最强,40ng/ml时减弱。IL-21能够诱导破骨标志分子CTR和Cathepsin-K mRNA水平表达上调。Western Blot和免疫细胞化学证实,与阴性组比较,IL-21能促进CTR蛋白水平表达增高。2、PI3K-AKT通路抑制剂（LY294002）可以显著抑制IL-21诱导的RAW264.7细胞向破骨细胞分化,IL-21刺激5~15 min时p-AKT表达增强。结论 IL-21可不依赖RANKL直接诱导小鼠巨噬细胞系RAW264.7细胞向破骨细胞分化,该作用可能由PI3K-AKT通路介导。Objective To investigate the role of IL-21 in osteoclastogenesis of RAW264.7 cells and its possible mechanism. Methods 1 ） RAW264.7 cells were treated with different concentrations of IL-21 （0, 1, 10, 20, and 40 ng/ml）. After 5 days, TRAP staining was performed. The calcitonin receptor （CTR） expression was detected using Western blot and immunocytochemistry. The mRNA levels of CTR and Cathepsin-K were examined using real-time PCR. 2） Before adding IL-21 into the culture, various signaling pathway inhibitors （AG490, LY294002 and PD98059） were incubated with RAW264.7 for 30 min. Osteoclast formation was determined after 5 days culture. Phosphorylation of signaling proteins was determined using Western blot under the stimulation of IL-21. Results 1） Without the presence of RANKL, TRAP positive cells increased with the increase of IL-21 concentration. The strongest effect was at the 20 ng/ml concentration, and the effect decreased at the 40 ng/ml concentration. IL-21 promoted mRNA expression of CTR and Cathepsin-K. Western blot and immunocytochemistr）, showed that the expression of CTR in IL-21 group increased compared to that in control group. 2） The PI3K-AKT pathway inhibitor LY294002 significantly suppressed IL-21-induced osteoclastogenesis in RAW264. 7 cells. P-AKT expression increased after 5-15 rain stimulation of IL-21. Conclusion IL-21 can induce osteoclastogenesis of RAW264.7 cells independent of RANKL via PI3K-AKT pathway.

关 键 词：白细胞介素-21 破骨细胞 RAW264.7 

分 类 号：R593.23[医药卫生—内科学]