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| 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 |
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| DOI：10.3969/j.issn.1006.7108.2015.03.002 |
| 中文关键词: 白细胞介素-21 破骨细胞 RAW264. 7 |
| 英文关键词:Interleukin-21 Osteoclast RAW264.7 |
| 基金项目:国家自然科学基金青年基金项目（81102255 ) |
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| 中文摘要: |
| 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后再加人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。 |
| 英文摘要: |
| 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 immunocytochemistry 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 min stimulation of LL-21. Conclusion IL-21 can induce osteoclastogenesis of RAW264.7 cells independent of RANKL via PI3K-AKT pathway. |
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