高铁环境对破骨细胞分化的影响及机制

Effects of high level of ferric icon on osteoclasts differentiation

目的 观察高铁环境对单核细胞RAW264.7向破骨细胞分化的影响及核转录因子二聚体p50-p65在其中的作用.方法 RAW264.7细胞以梯度莉量0- 400 μmol./L枸橼酸铁铵（FAC）干预,细胞计数试剂盒（CCK-8）检测细胞增殖能力,以核因子-κB受体活化因子配体（RANKL）诱导,行抗酒石酸酸性磷酸酶（TRAP）染色,根据染色结果选择效果最优组,与磷酸盐缓冲液（PBS）干预组对照.破骨细胞分化能力采用噬骨陷窝实验及荧光定量聚合酶链反应（FQ-PCR）检测;荧光探针检测活性氧（ROS）水平.Western blot检测胞质蛋白p50、p65及胞核蛋白p50、p65、磷酸化p50（p-p50）、磷酸化p65（p-p65）表达差异.结果 CCK-8结果表明,浓度范围在0-50 μmol/L的FAC对细胞增殖无明显影响（P＞0.05）.TRAP染色示50 mol/L FAC处理组阳性细胞数为（41.67±5.46）个,明显高于对照组的（20.00 ±4.00）个,噬骨陷窝数也高于对照组（P＜0.05）.ROS检测结果显示：FAC干预组荧光较对照组增强.FQ-PCR结果示：FAC组酸性磷酸酶5（Acp5）、基质金属蛋白酶-9（MMP-9）、降钙素受体（CTR）表达量分别为对照组的2.49、3.31、9.20倍（P＜0.05）.Western blot结果：两组核蛋白p-p50、胞质蛋白p65表达差异无统计学意义,FAC组核蛋白p65、p50、p-p65表达为对照组的14.38、15.32、3.88倍（P＜0.05）,胞质蛋白p50为对照组0.76倍（P＜0.05）.结论 铁离子在一定范围内升高ROS水平,进而促进p50-p65二聚体核易位,促进破骨细胞分化.

Objective To investigate the impacts of iron accumulation on differentiation of monocytes RAW264.7 cells into osteoclasts,as well as the effects of p50-p65 dimer on iron induced activation of osteoclast differentiation.Methods RAW264.7 cells were treated with different concentrations of Ferric ammonium citrate （FAC）.Ability of cell proliferation in each group was observed by cell counting kit （CCK-8） method.Tartrate-resistant acid phosphatase （TRAP） staining was conducted under the induction of RANKL,which can stimulate osteoclasts differentiation.Chose the most obvious effect of dose group,then carry out a resorption pits assay and 2＇,7＇-dichlorofluorescin diacetate （DCFH） analysis.Expressions of acid phosphatase 5 （Acp5）,matrix metalloproteinase 9 （MMP-9） and caltitonin receptor （CTR） were examined with real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）.Cytoplasmic and nuclear proteins were extracted for Western bolting.Results FAC at 0-50 μmol/L showed no significant difference on cell proliferation ability.The numbers of TRAP positive cells and osteoclasts resorption pits were significantly increased with the treatment of FAC （P 〈 0.05）.Compared to control group,the level of Reactive oxygen species （ROS） in the FAC group was elevated.FQ-PCR results showed that the expressions of osteoclasts-related gene （Acp5,MMP-9,CTR） with the treatment of FAC were much higher than the control group （2.49,3.31,9.20 fold,respectively,P 〈 0.05）.Western blotting analysis results：There was no significant difference of nuclear protein p-p50 and cytoplasmic protein p65 between two groups.The levels of nuclear proteins （p65,p50,p-p65） in FAC group were increased as compared with those in control group （14.38,15.32,3.88 fold,respectively,P 〈0.05）,while the level of cytoplasmic protein （p50） in FAC group was reduced as compared with that in control group （0.76 fold,P 〈 0.05）.Conclusion Ferric icon can increase the levels of ROS and stimulate nuclear translocation of p50-p65 dimer in a certain range of doses,then regulate the transcription and facilitate osteoclasts differentiation.