摘要
目的探讨铁调素对小鼠单核细胞株RAW264.7向破骨样细胞分化过程的影响及活性氧(reactive oxygen species,ROS)在其中的作用。方法常规培养两组RAW264.7,一组作为对照,另一组以1.6μmol/L铁调素(hepcidin,Hepc)干预,以巨噬细胞集落刺激因子、核因子-κB受体活化因子配体诱导向破骨样细胞分化,分化能力采用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色检测。采用免疫荧光(immunofluorescence,IF)检测膜铁转运蛋白(ferroportin,FPN)表达水平,同时检测细胞内铁离子水平差异。二氯二氢荧光素(dichlorodihydrofluorescein,DCFH)荧光探针标记并检测细胞ROS水平差异。抗氧化剂吡咯烷二硫代甲酸铵(pyrrolidine dithiocarbamate ammonium,PDTC)干预后采用TRAP染色检测破骨样细胞分化水平。结果铁调素干预后小鼠破骨样细胞分化能力增强,TRAP阳性染色细胞增多(P<0.001)。IF结果示铁调素干预后,破骨样细胞FPN表达下调。铁含量检测试剂盒检测结果表明铁调素可升高破骨样细胞内铁水平。进一步检测DCFH荧光示铁调素使细胞ROS水平升高。进一步使用抗氧化剂PDTC干预可抑制破骨样细胞分化(P<0.001),降低铁调素对破骨样细胞分化的促进作用。结论铁调素可封闭破骨样细胞胞膜FPN,造成胞内高铁状态,上调ROS水平,最终促进破骨样细胞分化。
Objective To investigate the effects of hepcidin on the differentiation of RAW264.7 into osteoclast-like cells and the roles of reactive oxygen species(ROS).Methods Mouse-derived RAW264.7 were extracted and at incubated with 1.6μmol/L hepcidin(Hepc).Differentiation into osteoclast-like cells was induced by the addition of macrophage colony stimulating factor(M-CSF)and nuclear factor-κB receptor activating factor ligand(RANKL).The differentiation ability was detected by tartrate resistant acid phosphatase(TRAP)staining.Immunofluorescence(IF)was used to detect the expression level of ferroportin(FPN).The difference of intracellular iron level was also detected.Dichlorodihydrofluorescein(DCFH)fluorescent probe was used to label and detect the difference of cellular ROS.After the intervention of antioxidant pyrrolidine dithiocarbamate ammonium(PDTC),the level of osteoclast-like cells differentiation was detected by TRAP staining.Results After Hepc intervention,the differentiation ability of osteoclast-like cells was enhanced and TRAP positive cells increased(P<0.001).IF results showed that the expression of FPN in osteoclast-like cells was down-regulated after Hepc intervention.The results of iron assay kit showed that Hepc could increase the iron level in osteoclast-like cells.Further detection of DCFH fluorescence showed that Hepc increased the ROS level.Antioxidant PDTC could inhibit osteoclast-like cells differentiation(P<0.001)and reduce the promoting effect of Hepc on osteoclast-like cells differentiation.Conclusion Hepc can block the membrane FPN of osteoclast-like cells,cause intracellular high iron level,subcequently increase ROS level,and finally stimulate the differentiation of osteoclast-like cells.
作者
王啸
孙婧悦
钱晨月
王爱飞
华俊
徐又佳
WANG Xiao;SUN Jing-yue;QIAN Chen-yue;WANG Ai-fei;HUA Jun;XU You-jia(Department of Orthopedics,the Second Affiliated Hospital of Soochow University,Suzhou 215004,Jiangsu,China;Department of Oncology,the First Affiliated Hospital of Soochow University,Suzhou 215006,Jiangsu,China;Department of Pharmacy,the Second Affiliated Hospital of Soochow University,Suzhou 215004,Jiangsu,China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2022年第3期252-256,共5页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
国家自然科学基金(82003473)
江苏省药学会药学科研项目(A201913)
院科研预研基金项目(SDFEYBS1810)。
关键词
骨质疏松症
活性氧
铁调素
破骨样细胞
osteoporosis
reactive oxygen species
hepcidin
osteoclast-like cell
