糖皮质激素环境下骨重塑相关细胞外泌体miRNA变化

Effect of glucocorticoids on expression of bone remodeling related cells-derived exosomal miRNAs

目的探讨糖皮质激素刺激下骨重塑相关细胞外泌体miRNA的变化。方法体外培养小鼠成骨细胞、破骨细胞,提取上清液中外泌体进行透射电镜观察、粒径分析、CD9\CD81表面标记物检测;利用不同浓度(1μg/ml,10μg/ml,100μg/ml)地塞米松刺激48 h,观察两种细胞增殖情况;收集外泌体检测骨重塑相关miR-133、miR-214、miR-4697和miR-487的表达。结果不同浓度地塞米松均能够抑制两种细胞增殖,其中100μg/ml组与对照组相比抑制作用显著(P<0.05)。当地塞米松浓度为100μg/ml时,成骨细胞外泌体miR-133含量降低为对照组的45%,miR-214降为对照组的12%,miR-4697降为对照组的56%(P均<0.05)。当地塞米松浓度为1μg/ml时,破骨细胞外泌体内miR-133和miR-487上升为对照组的12倍,miR-214升高为对照组1.67倍,miR-487升高2.12倍(P均<0.05)。结论随着糖皮质激素浓度增加,成骨细胞外泌体内miR-133、miR-214、miR-4697含量依次递减,而miR-487含量随着糖皮质激素浓度先升高后降低。小剂量糖皮质激素刺激破骨细胞后,其外泌体内4种miRNA含量均增加,而高浓度糖皮质激素刺激后miRNA含量降低。

Objective To investigate the changes of mi RNAs in exosomes derived from bone remodeling-related cells stimulated by glucocorticoid. Methods Osteoblasts and osteoclasts were cultured in vitro. Exosomes from the supernatant were collected for transmission electron microscopy, particle size analysis and CD9/CD81 surface marker detection. The proliferation of two kinds of cells was observed using CCK-8 assay after stimulating by different concentrations of dexamethasone（1μg/ml, 10μg/ml,100μg/ml） for 48 hours. Exosomes were extracted to detect the expression of mi R-133, mi R-214 and mi R-4697 and mi R-487.Results The proliferation of both cells could be inhibited by different concentrations of dexamethasone, of which 100μg/ml of dexamethasone had the strongest inhibitory effect. When the concentration of dexamethasone was 100μg/ml, the level of mi R-133 in osteoblast exosomes reduced to 45% compared with the control group, meanwhile mi R-214 and mi R-4697 reduced to 12% and 56%, respectively（P〈0.05）. Compared with the control group, when the concentration of dexamethasone was 1μg/ml, the expression of both mi R-133 and mi R-487 increased 12-fold, and the expression of mi R-214 increased 1.67-fold and mi R-487 increased 2.12-fold. Conclusion With the increase of glucocorticoid concentration, the content of mi R-133, mi R-214 and mi R-4697 decrease gradually in osteoblast exosomes, while the content of mi R-487 increase first and then decrease. After stimulating osteoclasts with low dose glucocorticoid, the content of four mi RNAs in exosomes increases, while it decreases in high dose glucocorticoids group.