破骨细胞释放凋亡小体微小RNA-30a对成骨活性的影响

Effect of osteoclast-derived apoptotic body microRNA-30a on osteogenic activity

目的探讨破骨细胞凋亡释放凋亡小体介导成骨活性的作用。方法通过小鼠(n=10)骨髓单核细胞体外诱导破骨细胞,用抗酒石酸酸性磷酸酶(TRAP)染色和细胞骨架F-actin与DAPI双标免疫荧光鉴定破骨细胞,破骨细胞与小鼠成骨细胞MC-3T3E1共培养体系,DNA片段化ELISA分析破骨细胞凋亡,凋亡小体标志物检测,骨形成标志物Real-time PCR分析,凋亡小体微小RNA(miRNA)表达谱芯片筛查。结果100μmol/L阿伦膦酸钠(ALN)诱导成熟破骨细胞凋亡,并释放凋亡小体;Western blotting检测表明,ALN诱导凋亡小体特异性表面标志物蛋白表达增强;破骨细胞凋亡小体蛋白C3b表达增高与成骨细胞活性负相关;凋亡小体表达谱芯片筛查提示,miR-30a与骨形成标志物血清碱性磷酸酶(ALP)相关。结论破骨细胞凋亡释放的凋亡小体携带miR-30a抑制成骨细胞活性,凋亡小体可能参与破骨细胞与成骨细胞的对话。

Objective To explore that whether apoptotic bodies released by osteoclasts mediate osteogenic activity.Methods The osteoclasts were induced from mouse(n=10)bone marrow monocytes in vitro,and were identified by tartrate resistant acid phosphatase(TRAP)staining,F-actin,and DAPI double labeling immunofluorescence.The Coculture system of osteoclasts and mouse osteoblasts MC-3T3E1 was established.The apoptosis of osteoclasts was analyzed by DNA fragment ELISA.Immunoblotting of apoptotic body markers was investigated.Real-time PCR analysis of bone formation markers was tested.MiRNA expression profiling of apoptotic body was identisfied.Results Alendronate(ALN)100μmol/L induced osteoclast apoptosis and caused apoptotic body release from osteoclasts.The expression of C3b and annexinⅤprotein was enhanced by ALN;the expression of C3b in osteoclasts was negatively correlated with the activity of osteoblasts;the microarray screening of apoptotic body showed that miR-30a was correlated with bone formation markers and serum alkaline phosphatase(ALP).Conclusion Osteoclast-derived apoptotic body miR-30a can inhibit the activity of osteoblasts.Apoptotic body may participate in the dialogue between osteoclasts and osteoblasts.