机械刺激/铁调控成骨细胞活性促进骨愈合的实验研究

Basic Research of Mechanical Stimulation/Iron on the Regulation of Osteoblast Activity to Promote Bone Healing

目的:探讨机械刺激/铁通过体内外COX2/PGE2通路介导成骨细胞活性、促进骨愈合的潜在机制。方法:将18只10周龄雄性Balb/c小鼠随机分成正常组、骨折组、机械刺激/铁组3组,制作小鼠胫骨骨折动物模型,酶联免疫吸附试验(ELISA)检测Ⅰ型胶原c端交联末端肽(CTx)、骨钙素(OC)浓度;取骨折组成骨细胞培养并用Western Blot法检测成骨细胞相关蛋白表达,免疫荧光检测成骨细胞核膜上COX2总蛋白量,用流式细胞仪分析铁对成骨细胞COX2/PGE2通路和细胞凋亡的影响。结果:机械刺激/铁组较骨折组骨折线模糊,血清中CTx含量降低、骨钙素含量升高,血清骨钙素(BGP)、碱性磷酸酶(ALP)和COI1水平均升高;铁能使成骨细胞CTx含量升高,骨钙素含量降低;骨折组BGP、ALP和COI1蛋白上升较正常组和高/低铁组明显;铁激活成骨细胞COX2/PGE2通路,且高铁组可显著提高COX2、PGEX和PGE2蛋白水平,成骨细胞核膜上COX2荧光强度较低铁组和正常组明显增强;高铁组上调多腺苷二磷酸核糖聚合酶(PARP)和Bax水平、下调Bcl2水平,更能显著降低成骨细胞凋亡率,同时高铁组COX2、PGE2、ALP和COI1水平显著提高。结论:机械刺激/铁通过体内外COX2/PGE2通路介导成骨细胞活性,促进骨愈合,拓展了机械刺激/铁在骨愈合中的应用。

Objective:To investigate the potential mechanism of mechanical stimulation/iron in promoting bone healing by mediating osteoblast activity through the COX2/PGE2pathway in vitro and in vivo.Methods:18male Balb/c mice with 10-week-old were randomly divided into normal group,model group and MS+iron group.The animal model of tibial fracture was prepared.The concentrations of C-terminal cross-linked peptide(CTx)and osteocalcin(OC)of typeⅠcollagen were detected by enzyme-linked immunosorbent assay(ELISA).Osteoblasts in model group were cultured,and Western Blot was used to detect the expression of related proteins in osteoblasts.The total amount of COX2protein in the nuclear membrane of osteoblasts was detected by immunofluorescence.The effects of iron on COX2/PGE2pathway and apoptosis of osteoblasts were analyzed by flow cytometry.Results:Compared with model group,the fracture line of MS+iron group was blurred,the content of CTx in serum was decreased and the content of OC was increased,and the levels of bone glaprotein(BGP),alkaline phosphatase(ALP)and COI1were increased.Iron can increase the content of CTx and decrease the content of OC in osteoblasts.The protein levels of BGP,ALP and COI1in model group were significantly higher than those in normal group and high/low iron group.Iron activated the COX2/PGE2pathway of osteoblasts,and the high iron group can significantly increase the protein levels of COX2,PGEX and PGE2.The fluorescence intensity of COX2on the nuclear membrane of osteoblasts was significantly increased compared with that in the low iron group and the control group.The apoptosis rate of osteoblast could be significantly decreased by up-regulating poly(ADP-ribose)polymerase(PARP)and Bax levels and down-regulating Bcl2levels in high iron group,while the levels of COX2,PGE2,ALP and COI1in high iron group were significantly increased.Conclusion:Mechanical stimulation/iron can promote bone healing through osteoblast activity mediated by COX2/PGE2pathway in vitro and in vivo,which expands the application of mechanical stimulation/iron in bone healing.