铁调素对RAW264.7向破骨细胞早期分化及晚期分化的影响.docVIP

  铁调素对 RAW264.7 向破骨细胞早期分化 及晚期分化的影响# 王啸，张鹏，汪升，沈光思，徐又佳** 5 10 15 20 25 30 35 40 （苏州大学附属第二医院骨科） 摘要：目的 探索梯度剂量铁调素干预小鼠单核细胞系 RAW264.7 后，向破骨细胞前期分化 及后期分化能力的差异。方法 铁调素以梯度浓度干预 RAW264.7，24h 后以 CCK8 法检测 各组细胞增殖能力。各组细胞均匀接种至噬骨馅窝板中，在含核因子 kB 受体活化因子配体 （RANKL）培养基的环境下，梯度浓度铁调素干预 RAW264.7，早期分化组细胞干预 3d 后 行抗酒石酸酸性磷酸酶（TRAP）染色；晚期分化组 RANKL 诱导 3d，再以梯度浓度铁调素 干预 3d，6d 后行 TRAP 染色。各组在干预终点时以 Trizol 消化细胞并冲洗晾干噬骨馅窝 板，镜下观察噬骨馅窝吸收情况；消化下的细胞行 q-PCR 检测破骨相关基因表达差异。结 果 铁调素在 0.4~1.6μM 范围内能促进破骨细胞分化，早期分化各浓度组 TRAP 阳性细胞数 及噬骨馅窝数无统计学差异，而晚期分化各组细胞分化有统计学差异。q-PCR 结果与 TRAP 染色结果一致。结论 铁调素对 RAW264.7 向破骨细胞晚期分化有促进作用，且呈剂量依赖 性，而对早期分化影响甚微。 关键词：实验外科学；铁调素；破骨细胞；骨质疏松 中图分类号：R-681 The influence of hepcidin on early and terminal differentiation of RAW264.7 monocytes into osteoclasts WANG Xiao, ZHANG Peng, WANG Sheng, SHEN Guangsi, XU Youjia (Department of Orthopaedics,The Second Affiliated Hospital of Soochow University,SuZhou 215004) Abstract: Objective To explore the influence of graded doses of hepcidin on differentiation of mouse RAW264.7 cells into osteoclasts,especially on the early or ternimal differentiation. Methods RAW264.7 cells were intervered with graded doses of hepcidin in the presence of receptor activator of NF-kB ligand(RANKL).Cell viability was tested with CCK8 after 24h,and then replated into osteo assay plates.To study the effect on the early differentiation,RAW264.7 cells were treated with indicated concentrations of hepcidin for 3d.On the ternimal differentiation,cells were cultured in the presence of RANKL for 3d and then treated with hepcidin for another 3d.,then stained with tartrate-resistant acid phosphatase(TRAP).Numbers of pits were measured.Gene expressions were examined with q-PCR. Results Hepcidin at 0.4~1.6μM might stimulate osteoclasts terminal differentiation,not the early differentiation,which was indicated with the results of TRAP staining and resorption pit assay.The gene expressions of TRAP,CTK and CTR were in correspondence with the consequences of TRAP staining and resorption pit assay. Conclut