铁离子对人成骨细胞(hFOB1.19)生物活性影响

摘要

目的 探讨铁离子对成骨细胞株(hFOB1.19)生物活性,包括碱性磷酸酶活性(alkaline phosphatase activity,APA)、细胞凋亡、钙结节、Ⅰ型胶原(type 1 collagen,COL 1)和骨钙素(osteocalcin,OC)的影响.方法 成骨细胞(hFOB1.19)于5% CO2 34 ℃培养箱内培养,将不同浓度枸橼酸铁铵(ferric ammonium citrate,FAC)50、100、200 μmol/L和去铁胺(deferoxamine,DFO)5、10、20 μmol/L分别加入细胞培养基中,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;流式细胞仪检测细胞凋亡;Von kossa染色法行钙结节染色;RT-PCR和Western blotting法分别检测 COL1和骨钙素(bone gla protein,BGP)的基因及蛋白表达.结果 (1)48 h后增生活性:FAC组细胞增生活性随FAC干预浓度的增加呈剂量依赖性降低(P<0.05),DFO组在5 μmol/L浓度组时促进成骨细胞增生,在10、20 μmol/L浓度组时抑制成骨细胞增生.(2)10 d时碱性磷酸酶活性:FAC组碱性磷酸酶活性随FAC干预浓度的增加而降低,DFO组碱性磷酸酶活性随DFO干预浓度的增加而升高(P<0.05);(3)48 h时凋亡:FAC组细胞凋亡率呈剂量依赖性升高(P<0.05),DFO组在5、10 μmol/L浓度时抑制细胞凋亡(P<0.05),在20 μmol/L则促进成骨细胞凋亡(P<0.05);(4)21 d时钙结节染色:FAC组随铁离子干预浓度增加,成骨细胞矿化面积、钙结节形成数量均减少,DFO组在5、10 μmol/L浓度时促进成骨细胞矿化功能,而20 μmol/L浓度时对成骨细胞矿化有抑制效应;(5)3 d时基因及蛋白表达:FAC组COL1、BGP基因和蛋白的表达呈剂量依赖性下调(P<0.05);DFO组COLⅠ、BGP基因的表达呈剂量依赖性上调(P<0.05),DFO在5、10 μmol/L浓度时促进COL1、BGP蛋白表达,在20 μmol/L浓度时抑制COL1、BGP蛋白表达.结论 不同浓度枸橼酸铁铵均抑制成骨细胞功能活性,提示高铁环境不利于成骨细胞的成骨功能.低铁环境对成骨细胞有双重效应,低剂量去铁胺对成骨细胞活性有促进作用,而高剂量去铁胺则对成骨细胞活性有抑制效应.%Objective To investigate the effects of ferri ion on the proliferation and activity of human osteoblast line hFOB1.19 cells in vitro.Methods Human osteoblasts (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) and 0-20 μmol/L of deferoxamine(DFO).Proliferation ability of osteoblasts were evaluated by MTT assay and capacity of forming mineralized nodules were used to evaluate the proliferation and function of osteoblasts by von-kossa staining assay.Counting the number of calcium nodular and mineral area was used to reflect the effect of FAC on cell mineralization.Apoptotic hallmarks were detected by flow cytometer after 48 h of the treatment of FAC and DFO by Annexin intervention Ⅴ/PI staining.Alkaline phosphatase (ALP) activity was measured using ALP viability kit.The gene and protein expression of type Ⅰ collagen(COL1) and osteocalcin(BGP) was detected by RT-PCR and Western blotting at 72 h after treatment with FAC and DFO.Results Compared to controls,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 in dose dependent manner(P<0.05).The number of mineralized nodules was reduced by FAC significantly in dose dependent manner.The number of mineralized nodules and mineralized surface area were decreased significantly by FAC treatment (P<0.05).200 μmol/L of FAC decreased calcium nodules most obviously.Apoptotic events were induced by iron overload remarkably (P<0.05).The expression of COL1 was inhibited significantly by FAC(100-200 μmol/L) (P<0.05).Conclusion Different concentrations of FAC had negative effect on the osteoblastic activity,while DFO could promote the activity of hFOB1.19 cells at high levels and decrease activity of cells at low levels.