MLO-Y4来源外泌体对破骨细胞调控机制的研究

目的 探讨骨细胞系MLO-Y4来源的外泌体对破骨细胞的调控机制。方法 收集MLO-Y4细胞的培养基,通过超高速离心法获得MLO-Y4细胞外泌体（MLO-Y4-Exo）,Western blot和透射电镜鉴定其特征;MLO-Y4-Exo与骨髓巨噬细胞共培养,TRAP染色检测其对破骨细胞分化的作用;小鼠顶骨皮下注射MLO-Y4-Exo检测其对体内破骨细胞分化的影响;通过小干扰RNA检测MLO-Y4-Exo对促破骨分化的机制。结果 MLO-Y4外泌体大小形态满足外泌体特征,高表达CD63和Alix外泌体蛋白;MLO-Y4-Exo在体外和体内均促进破骨细胞分化（P<0.05）;RANKL小干扰RNA实验证实MLO-Y4-Exo通过向破骨前体细胞传递RANKL蛋白促进破骨细胞分化（P<0.05）。结论 骨细胞系MLO-Y4来源的外泌体通过向破骨前体细胞传递促破骨分化因子RANKL促进破骨细胞生成。

Mechanism of regulation of osteoclasts by MLO-Y4-derived exosomes

Objective To investigate the effect of osteocyte-like cell MLO-Y4-derived exosomes on osteoclast differentiation. Methods Osteocyte-like cell MLO-Y4-derived exosomes (MLO-Y4-Exo) were obtained by ultrahigh speed centrifugation. Their characteristics were identified using Western blotting and transmission electron microscopy. MLO-Y4-Exo were co-cultured with bone marrow macrophages. TRAP staining was conducted to detect the osteoclast differentiation. Mice were subjected to subcutaneous supra-calvarial injection of MLO-Y4-Exo for 5 consecutive days to detect the osteoclast differentiation in vivo. The mechanism of MLO-Y4-Exo promoting osteoclast differentiation was detected with small interfering RNA. Results The size and morphology of MLO-Y4-Exo met the exosome characteristics and highly expressed CD63 and Alix exosome proteins. MLO-Y4-Exo promoted osteoclast differentiation in vitro and in vivo (P<0.05). RANKL small interfering RNA assay confirmed that MLO-Y4-Exo promoted osteoclast differentiation by transmitting RANKL protein to osteoclast precursors (P<0.05). Conclusion MLO-Y4-derived exosomes promote osteoclast formation by transferring RANKL to osteoclast precursors.