目的 探讨活化Notch2信号对高糖条件下核因子κB受体活化因子配体(RANKL)诱导破骨细胞分化的影响,初步明确Notch2信号在高糖条件下破骨细胞分化中的作用.方法 体外培养小鼠破骨前体细胞,在高糖条件下采用RANKL刺激破骨前体细胞.实验分为葡萄糖和甘露醇等渗对照组,每组设5个浓度(0、5、10、20、40 mmol·L-1),通过实时定量聚合酶链反应检测Notch2基因的表达水平,抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞分化情况.构建含Notch2细胞内片段(ICN2)的病毒载体(pMX-ICN2),转染包装细胞,收获病毒上清液感染破骨前体细胞；将病毒载体分为ICN2-OE (ICN2过表达)和EMPTY(仅感染pMX载体)两组,通过蛋白质印迹法检测ICN2蛋白的表达水平；并在高糖条件下(20、40 mmol·L-1)用RANKL刺激ICN2-OE和EMPTY组,设甘露醇等渗对照组,用TRAP染色检测破骨细胞分化情况.结果 RANKL诱导破骨细胞分化过程中,葡萄糖浓度为20、40 mmol· L-1时,破骨细胞数分别为110.3±6.8和72.0±8.0,Notch2相对表达量分别为1.65±0.23和1.10±0.11; 20、40 mmol· L-1甘露醇组的破骨细胞数分别为152.7±7.0和157.0±12.5,Notch2相对表达量分别为2.82±0.28和2.42±0.27,组间差异有统计学意义(P＜0.05).活化Notch2信号后,葡萄糖浓度为20、40 mmol· L-1时,ICN2-OE组破骨细胞数分别为206.7±7.8和178.3±11.5,EMPTY组分别为102.3±8.7和76.0±10.1,组间差异有统计学意义(P＜0.05).结论 活化Notch2信号可促进高糖条件下破骨细胞的分化.%Objective To study the effect of high glucose and mannitol (osmotic control) on receptor activator of nuclear factor kappa B ligand (RANKL) -induced osteoclastogenesis and Notch2 expression using bone marrow macro-phages (BMMs) from mice. Furthermore, the effect of Notch2 activation on RANKL-induced osteoclastogenesis with high glucose concentration was explored. Methods Preosteoclasts were cultured and exposed to sustained high glucose (0, 5, 10, 20, 40mmol·L-1) levels to mimic diabetic conditions. Osteoclast formation was analyzed using tartrate resistant acid phosphatase (TRAP) assay. Expression of Notch2 gene was analyzed using real-time polymerase chain reaction. Constitutively over-expressed active Notch2, via stable transfection of exogenous ICN2 (intracellular fragment of Notch2) in preosteoclasts and the effect of Notch2 over expression on osteoclastogenesis was analyzed using Western blotting and TRAP staining. Results The osteoclast number with 20mmol·L-1 glucose (110.3±6.8) and 40mmol·L-1 glucose (72.0±8.0) was significantly less than the group with 20mmol·L-1 mannitol (152.7±7.0) and 40mmol·L-1 mannitol (157.0±12.5). The relative gene expression of Notch2 with 20 mmol·L-1 glucose(1.65±0.23) and 40mmol·L-1 glucose (l.l0±0.11) was significantly less than the group with 20mmol·L-1 mannitol (2.82±0.28) and 40 mmol·L-1 mannitol (2.42±0.27)(P〈0.05). The osteoclast number after Notch2 activation (ICN2-OE) with 20mmol·L-1 glucose (206.7±7.8) and 40 mmol·L-1 glucose (178.3±11.5) was significantly more than the control group (EMPTY) (102.3±8.7 and 76.0±10.1 respectively) (P〈0.05). Conclusion Notch2 signaling activation may promote osteoclastogenesis under high glucose concentration.
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