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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 understand the effect of iron overload on iron homeostasis and biological activity of osteoblasts. Methods Human osteoblasts (hFOB1.19) were cultured in vitro at 34 ℃. Interferon (50, 100, 200 μmol / L) ferric ammonium citrate (FAC) was used to detect osteoblast iron-regulated genes (FPN1), transferrin receptor (TfR) and divalent metal transporter 1 (DMT1) were detected by flow cytometry. The intensity of iron ion in osteoblasts was observed by laser confocal scanning microscopy (CLSM) The level of reactive oxygen species (ROS) in osteoblasts was detected by cytometry; alkaline phosphatase activity was detected by alkaline phosphatase activity kit; calcium staining was performed by Vonkossa staining. Results Compared with the control group, the expression of FPN1 mRNA was up-regulated in a dose-dependent manner with the increase of FAC concentration and the expression of TfR and DMT1 mRNA was decreased in a dose-dependent manner (P <0.05) 48 h after FAC intervention; (P <0.05). After 48h, the level of ROS in osteoblasts increased in a dose-dependent manner (P <0.05), while the level of ROS in osteoblasts increased in a dose-dependent manner (P <0.05) Alkaline phosphatase activity of osteoblasts in each group decreased with the increase of FAC concentration (P <0.05). Compared with the control group, calcified nodules of osteoblasts were observed 17 days after FAC intervention And calcium nodule formation decreased with increasing FAC concentration. Conclusions Iron overload significantly inhibits osteoblasts biological activity. The mechanism may be related to the increase of intracellular iron concentration and the increase of reactive oxygen species.