目的 探讨成骨细胞来源的外泌体（Exo）中微小RNA(miR)-494对骨质疏松症（OP）大鼠骨代谢与骨重建平衡的影响及其机制。 方法 从MC3T3-E1成骨细胞系中分离鉴定Exo，并将miR-494电转至Exo，Real-time PCR测定miR-494表达水平。40只SD大鼠随机分为对照组、模型组、miR-494模拟剂（miR-494）组和miR-494抑制剂组，每组10只。除对照组外，其余3组大鼠切除卵巢构建OP模型。模型制作成功后，miR-494组与miR-494 抑制剂组分别接受含相应miRNA的Exo尾静脉注射，剂量为3×109个微粒。4周后，Micro-CT检测各组大鼠的骨参数，ELISA测定血清骨标志物，HE染色观察骨组织病理变化，抗酒石酸酸性磷酸酶(TRACP)染色检测破骨细胞数量，Western blotting测定骨重建相关蛋白及Toll样受体4(TLR4)相关通路蛋白表达水平。 结果 成功从MC3T3-E1细胞中分离得到典型杯状或圆形，直径约100 nm的Exo，其含有CD63、CD9、肿瘤易感性基因101(TSG101)、热休克蛋白70(HSP70)蛋白，且能被MC3T3-E1细胞摄取。经电转处理后，miR-494模拟剂和miR-494 抑制剂的Exo中miR-494的相对表达量明显升高和降低（P<0.05）。与模型组相比，miR-494组大鼠骨参数降低，血清骨标志物骨钙蛋白(BGP)、TRACP、Ⅰ型胶原C端肽(CTX-Ⅰ )升高，骨保护蛋白(OPG)、Ⅰ型前胶原N端肽( PⅠNP)降低，骨小梁结构紊乱，破骨细胞增加，骨组织内骨形态发生蛋白2(BMP-2)、Runt相关转录因子2(RUNX2)下调，核因子κB受体激活剂配体(RANKL)上调，TLR4、核因子κB (NF-κB) p65及髓样分化主要响应蛋白88(MyD88)上调（P<0.05）。而miR-494 抑制剂组情况则相反，骨参数和OPG、 PⅠNP 升高，BGP、TRACP、CTX-Ⅰ降低，骨结构恢复正常，破骨细胞减少，骨组织内BMP-2、RUNX2上调，RANKL下调，TLR4、NF-κB p65及MyD88下调（均P<0.05）。 结论 Exo传递miR-494可加重OP大鼠骨代谢异常，并抑制骨重建平衡，推测其作用机制可能与调控TLR4通路有关。

Objective To investigate the effect of osteoblastderived exosome (Exo) mediating microRNA (miR)-494 on bone metabolism and bone remodeling balance in osteoporosis (OP) rats and its mechanism. Methods   Exosomes was isolated and identified from MC3T3-E1 osteoblast cell line and transferred to Exo by electrical transfer. Forty SD rats were randomly divided into control, model, miR-494 mimic (miR-494), and miR-494 inhibitor group, 10 rats in each group. The ovaries were removed to construct the OP model except the control group. After modeling, the miR-494 group and miR-494 inhibitor group received tail vein injections of exosomes containing the corresponding miRNA, at a dose of 3×109 particles. Four weeks later, bone parameters were detected in each group of rats by Micro-CT, serum bone markers were measured by ELISA, pathological changes in bone tissue were observed by HE staining, osteoclast numbers were detected by tartrate-resistant acid phosphatase (TRACP) staining, and the expression levels of bone remodeling-related proteins and toll-like receptor 4(TLR4) pathway-related proteins were determined by Western blotting. Results  Typical cup-shaped or round exosomes were successfully isolated with a diameter of about 100 nm from MC3T3-E1 cells, which contained CD63, CD9, tumor susceptibility gene 101(TSG101), heat shock protein 70(HSP70) proteins and can be taken up by MC3T3-E1 cells. Compared with the model group, the bone parameters of the rats in the miR-494 mimic group decreased, serum bone markers bone Gla protein (BGP), TRACP, C-terminal telopeptide of type Ⅰ collagen (CTX-Ⅰ) increased, osteoprotegerin (OPG), procollagen type Ⅰ N-terminal propeptide (PⅠNP) decreased, bone trabeculae structure was disordered, osteoclasts increased, bone morphogenetic protein 2(BMP-2), Runt related transcription factor 2(RUNX2) in bone tissue downregulated, receptor activator of nuclear factor kappa-B ligand (RANKL) upregulated, TLR4, nuclear factor kappa-B p65(NF-κB p65) and myeloid differentiation primary response 88(MyD88) upregulated (all P<0.05). In contrast, the situation of the miR-494 inhibitor group was opposite, bone parameters and OPG,  PⅠNP increased, BGP, TRACP, CTX-Ⅰ decreased, bone structure returned to normal, osteoclasts decreased, BMP-2, RUNX2 in bone tissue upregulated, RANKL downregulated, TLR4, NF-κB p65 and MyD88 downregulated (all P<0.05). Conclusion  The transfer of miRNA-494 by Exo aggravates abnormal bone metabolism in OP rats and inhibits bone remodeling balance, suggesting that the mechanism of action may be related to the regulation of TLR4 pathway.