铁蓄积对小鼠骨量、骨内血管及内皮细胞影响的实验研究

Experimental study on the effect of iron accumulation on bonemass, intraosseous vessels and vascular endothelial cells in mice

目的探讨小鼠内源性铁蓄积对骨量、骨内血管的影响及外源性铁剂对血管内皮细胞活性的影响。方法根据是否敲除铁调素将小鼠分为正常组(未敲除铁调素,C57/BL6小鼠)和铁调素敲除组,每组各10只,均为8周龄,体重约为22 g的雄性小鼠。两组小鼠均培养至16周龄时处死,进行实验。酶联免疫吸附实验检测两组小鼠血清铁蛋白水平;肝组织石蜡切片普鲁士蓝染色检测肝脏铁蓄积程度;微型计算机断层扫描(Micro-CT)检测小鼠股骨微结构等参数;骨内H型血管免疫荧光染色检测骨内H型血管数量。细胞实验分为血管内皮细胞正常培养组(细胞正常组)和使用200 μmol/L枸橼酸铁铵干预培养的血管内皮细胞组(Fe组)。划痕实验检测血管内皮细胞的迁移能力;管型形成实验检测血管内皮细胞的成管功能。免疫荧光检测血管内皮细胞的内皮活性。结果铁调素敲除组血清铁蛋白水平[(318.30±12.53) ng/ml]较正常组[(109.60±4.66) ng/ml]明显升高,铁调素敲除组肝脏普鲁士肝铁染色蓝色面积百分比(80.80%±3.156%)较正常组(20.94%±2.813%)显著增加,铁调素敲除组小鼠骨密度(0.044±0.002 mg/m^3)较正常组(0.131±0.008 mg/m^3)低,铁调素敲除小鼠骨内血管数量(17.06%±1.060%)较正常组(38.76%±4.576%)显著减少;两组各指标比较差异均有统计学意义(t=-49.367、-13.788、35.293、6.165;均P< 0.05)。Fe组血管内皮细胞培养24 h后划痕缩减了24.300%±1.849%,较细胞正常组(39.060%±3.211%)显著减少,Fe组血管内皮细胞管型区域面积[(0.035±0.003) mm^2]较细胞正常组[(0.330±0.018) mm^2]显著降低,Fe组血管内皮黏蛋白阳性细胞数量百分比(12.000%±3.462%)较细胞正常组(0.035%±0.003%)明显降低;两组细胞各指标比较差异均有统计学意义(t=9.790、18.929、13.922;均P< 0.05)。结论内源性铁蓄积会导致小鼠骨量丢失,同时骨内血管的数量明显减少;血管内皮细胞在铁剂干预后,其迁移能力、管型形成能力及内皮活性均受到抑制。

Objective To investigate the effect of endogenous iron accumulation onbone mass, intraosseous vessels and the effect of exogenous iron on endothelial cell activity. Methods The mice were divided into control group (C57/BL6 mice without hepcidin knockout) and hepcidin-knockout group (10 mice in each group, 8 weeks old and weighing about 22 g). The mice in both groups were killed at the age of 16 weeks. Serum ferritin levels were measured by Enzyme-linked immunosorbent assay (ELISA), and iron accumulation in liver tissue was measured by Prussian blue staining, while femoral micro-structure was measured by micro-CT, and H-type vessel immunofluorescence staining was used to detect the number of H-vessels in bone. Cell experiments were divided into normal culture group (normal cell group) and intervention group (Fe group) with 200 μmol/L ammonium ferric citrate. Scratch test was used to detect the migration ability of vascular endothelial cells, and tube formation test was used to detect the function of vascular endothelial cells. The endothelial activity of vascular endothelial cells was detected by immunofluorescence. Results The level of serum ferritin (318.30±12.53 ng/ml) in the hepcidin-knockout group was significantly higher than that in control group (109.60±4.66 ng/ml). The percentage of blue area of Prussian liver iron staining in the hepcidin-knockout group (80.80%±3.156%) was significantly higher than that in control group (20.94%±2.813%). Bone mineral density in the hepcidin-knockout group (0.044±0.002 mg/m^3) was significantly higher than that in control group (0.131±0.008 mg/m^3). The number of intraosseous blood vessels in the hepcidin-depleted mice (17.06%±1.060%) was significantly lower than that in control group (38.76%±4.576%). There were significant differences between the two groups in each index (t=-49.367,-13.788, 35.293, 6.165;all P < 0.05). The scratches of vascular endothelial cells in Fe group were reduced by 24.300%±1.849% after 24 hours of culture, which was significantly lower than that in normal group (39.060%±3.211%). The area of vascular endothelial cells in Fe group (0.035±0.003 mm2) was significantly lower than that in normal group (0.330±0.018 mm^2). The EMCN positive cells of vascular endothelial cells in Fe group were significantly lower than those in normal group. The percentage of cell number (12.000%±3.462%) was significantly lower than that of normal cell group (0.035%±0.003%). There were significant differences in cell indices between the two groups (t=9.790, 18.929, 13.922;all P< 0.05). Conclusion Endogenous iron accumulation aggravated bone loss in mice, and the number of intraosseous blood vessels decreased significantly. Vascular endothelial cells were inhibited by iron intervention in migration, tube-formation and endothelial ability.