铁过载对成骨细胞铁稳态及生物活性的影响

Iron overload affect on iron homeostasis and biological activity of osteoblasts

目的了解铁过载对成骨细胞铁稳态及生物活性的影响。方法 34℃条件下体外培养人成骨细胞(hFOB1.19),以不同浓度(50、100、200μmol/L)枸橼酸铁铵(FAC)干预,用RT-PCR检测成骨细胞铁调节基因膜转铁蛋白(FPN1)、转铁蛋白受体(TfR)和二价金属转运蛋白1(DMT1)表达的变化;用激光共聚焦扫描显微镜(CLSM)观察成骨细胞铁离子荧光强度;流式细胞仪检测成骨细胞活性氧(ROS)水平;碱性磷酸酶活性试剂盒检测碱性磷酸酶活性;Vonkossa染色法行钙结节染色。结果与对照组相比,FAC干预48h后FPN1mRNA的表达随FAC干预浓度增加呈剂量依赖性上调,TfR、DMT1mRNA的表达呈剂量依赖性下调(P<0.05);FAC干预48h后成骨细胞铁离子荧光强度剂量依赖性减弱,与对照组相比差异有统计学意义(P<0.05);48h后成骨细胞ROS水平随FAC干预浓度增加呈剂量依赖性升高(P<0.05);FAC干预10d后各组成骨细胞碱性磷酸酶活性均随FAC浓度增高而降低,差异有统计学意义(P<0.05);与对照组相比,FAC干预17d后成骨细胞钙结节染色显示矿化面积和钙结节形成随FAC浓度增加而减少。结论铁过载对成骨细胞生物活性有明显抑制作用,其机制可能与细胞内铁离子浓度增加及活性氧水平升高有关。

Objective To investigate the effect of iron overload on iron homeostasis and biological activity of os- teoblasts. Methods Human osteoblast cells （hFOB1.19） were cultured at 34℃ in vitro. Cells were incubated in media supplemented with different concentrations （50 μmol/L, 100 μmol/L, 200 μmol/L） of ferric ammonium citrate （FAC）. The genes expression of （ transferring receptor, TfR）, （ divalent metal transporter 1, DMT1 ） and （ ferroportin, FPNI ） were detected by RT-PCR; Confocal laser scanning microscopy （CLSM） observed the fluorescence intensity of the iron ions in the osteoblast; Intracellular ROS was measured using the oxidation sensitive dye DCF-DA by flow cytometry; Alkaline phosphatase （ALP） activity was measured using ALP viability kit, and the calcium nodules was detected usingthe Von kossa staining. Results Compared with the controls, the expression of FPNI was up-regulated and TfR and DMTI were down-regulated significantly by FAC in a concentration dependently manner （ P 〈 O. 05 ） ; the fluorescence in- tensity of the iron ions in the osteoblast was significantly decreased and the levels of intracellular ROS was enhanced signif- icantly by FAC treatment （ P 〈 O. 05 ） ; the activity of ALP and the mineralized nodules were reduced by FAC significantly concentration-dependently （ P 〈 O. 05 ）. Conclusion Iron overload affecd on the biological activity of osteoblasts which may be related to the increase of intracellular iron ion concentration and the elevated of ROS.