高铁环境对成骨细胞生物活性的影响  

The effects of excess iron on osteoblastic activity index

作　　者：赵理平[1] 徐又佳[1] 林华[2] 张伟[1] 马勇[1] 都本才[1] 王昌超[1] 张鹏[1] 

机构地区：[1]苏州大学附属第二医院骨科,苏州215004 [2]南京大学附属鼓楼医院骨病中心,南京210009

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2010年第4期256-262,共7页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：江苏省自然科学基金(BK2008165);教育部博士点基金(20103201110020)

摘　　要：目的探讨高铁环境对人成骨细胞(hFOB1.19)活性指标的影响。方法采用细胞贴壁法培养成骨细胞(hFOB1.19)后,将不同浓度枸橼酸铁铵(50、100、200μmol/L)加入细胞培养基,用MTT法检测成骨细胞增生活性;碱性磷酸酶活性试剂盒检测碱性磷酸酶活性;流式细胞仪检测成骨细胞凋亡情况;Vonkossa染色法行钙结节染色;用RT-PCR和Western blotting分别检测Ⅰ型胶原(COL1)的基因及蛋白表达变化。结果用不同浓度枸橼酸铁铵干预成骨细胞后,与对照组相比,48 h与72 h组成骨细胞增生活性呈剂量依赖性降低,差异有统计学意义(P<0.05);48 h组成骨细胞凋亡率呈浓度依赖性升高,与对照组比较差异有统计学意义(P<0.05);不同浓度枸橼酸铁铵干预10 d和14 d时各组成骨细胞碱性磷酸酶活性均随枸橼酸铁铵浓度增高而降低,差异有统计学意义(P<0.05);与对照组相比,枸橼酸铁铵干预17 d后钙结节染色结果显示,矿化面积和钙结节形成随铁离子浓度增加而减少;枸橼酸铁铵干预3 d后呈剂量依赖性下调Ⅰ型胶原基因和蛋白的表达。结论高铁环境使成骨细胞成骨活性指标均受到抑制。Objective To investigate the effects of Ferric Ammonium Citrate on osteoblastic activity index in human osteoblasts in vitro. Methods Human osteoblast cells （hFOB1.19） were cultured in a 5% CO2 atmosphere at 34℃. Cells were incubated in media supplemented with 0 -200 μmol/L of Ferric Ammonium Citrate （FAC）. Proliferation viability of osteoblasts were evaluated by MTT assay at 48 h and 72 h and capacity of cultures to form mineralized bone nodules at 15 d were used to evaluate the proliferation and function of osteoblasts by von-kossa staining assay. Counting the number of calcium nodular was used to reflect the effect of FAC on cell mineralization. Apoptotic hallmarks were detected by flow cytometer 48h after the treatment of FAC by using Annexin intervention V/PI staining. Alkaline phosphatase （ALP） activity was measured using ALP viability kit at 10 d and 14 d. The gene and protein expression of Type I collagen was detected by RT-PCR and western blot at 72 h after treatment with FAC. Results Compared with the controis, cell proliferation viability had obvious difference in FAC groups （P 〈 0. 01 ）. The proliferation and ALP activity of osteoblasts were significantly suppressed by iron overload concentration-dependently （P 〈 0. 05）. The number of mineralized nodules was reduced by FAC significantly concentration-dependently. The numbers of mineralized nodules and mineralized surface area were decreased significantly by FAC treatment （ P 〈 0. 05 ）. 200μmol/L of FAC decreased calcium nodules most obviously. Apoptotic events were induced by iron overload remarkably （P 〈 0. 05 ）. The expression of type I collagen was inhibited significantly by FAC （ 100 - 200μmol/L） （P 〈 0. 05 ）. Conclusion The activity indexes of osteoblasts are all inhibited in excess iron environment.

关 键 词：枸橼酸铁铵 hFOB1.19 细胞增生 凋亡 钙结节 

分 类 号：R681[医药卫生—骨科学]