雌二醇下调铁对成骨细胞活性的抑制作用

目的了解雌二醇与铁对小鼠原代成骨细胞活性的影响及活性氧(ROS)的作用。方法提取新生小鼠颅骨原代成骨细胞,经差速贴壁法纯化后传代,选取3~4代进行实验,随机分为对照组,枸橼酸铁铵(FAC)组和FAC+17β-雌二醇(FAC+E2)组,分别用2.5μmol/L FAC和10 nmol/L E2干预7 d后收集细胞,用CCK-8法检测各组细胞增殖情况;用碱性磷酸酶(ALP)活性试剂盒检测各组细胞碱性磷酸酶活性;荧光定量PCR(Q-PCR)检测成骨相关基因(Runx2、osterix、Bglap、IBSP)表达;荧光酶标仪检测各组ROS水平并在荧光显微镜下观察。结果细胞活性氧水平检测结果显示,FAC组活性氧水平明显高于其余各组,FAC+E2组与FAC组相比ROS水平降低(P0.05);FAC干预后细胞增殖能力及ALP活性明显下降(P0.05),FAC+E2组细胞增殖能力较FAC组上升(P0.05),ALP活性上升,但差异无统计学意义(P0.05);Q-PCR结果显示,与对照组相比,FAC组Runx2、osteorix表达水平明显下降(P0.05),FAC+E2组与FAC组相比,各基因表达水平均显著升高(均P0.05)。结论 FAC可能通过升高ROS水平抑制成骨细胞生物活性;雌二醇可能通过清除FAC诱导生成的ROS下调该抑制作用。

Estradiol alleviate the inhibition of osteoblasts activity caused by iron accumulation

Objective To explore the effects of iron and estradiol on the biological activity of osteoblasts and the influence of reactive oxidative stress （ ROS） on this process.Methods Primary osteoblasts, extracted from suckling mice＆#39;s cranium and purified with differential adhesion method , were divided into 3 groups： control group, FAC group, FAC＋E2 group.Cells were intervened by FAC and estradiol .Harvested cells after 7d and then detected cell proliferative capacity using CCK-8 kit.Runx2, osterix, Bglap, and IBSP mRNA expression were detected by q-PCR.Alkaline phosphatase （ ALP） activity was measured using ALP kit .ROS level was detected using a fluorescence microplate reader .Results The level of ROS in FAC group was significantly higher than that in control group （ P〈0.05 ） , and level in FAC＋E2 group were lower than that in FAC group （P〈0.05）.Cell proliferative capacity and alkaline phosphatase activity were reduced with the treatment of FAC （ P〈0.05 ） , while estradiol improved the inhibition of cell proliferative capacity and alkaline phosphatase activity induced by iron accumulation .Q-PCR results showed that FAC decreased Runx 2 and osteorix mRNA expression （P〈0.05）.Runx2, osterix, IBSP, and Bglap mRNA expression were increased in FAC ＋E2 group, comparing to those in FAC group （ P〈0.05 ） .Conclusion Iron accumulation might increase ROS level and then reduce the biological activity of osteoblasts .This process might be inhibited by estradiol .