自噬铁环镜变化对成骨细胞(hFOB1.19)及其骨代谢异常的影响  

Effect of change of iron environment on osteoblasts(hFOB1.19)autophagy and abnormal bone metabolism

作　　者：赵理平[1,2] 陈国兆 吴耀刚[1] 沙卫平[1] 徐又佳[2] ZHAO Li-ping;CHEN Guo-zhao;WU Yao-gang;SHA Wei-ping;XU You-jia(Department of Orthopedics, the Affiliated Zhangjiagang Hospital of Soochow University, Zhangjiagang 215600,Jiangsu,China;Department of Orthopedics, the Second Affiliated Hospital of Soochow University/Osteoporosis Institute of Soochow University, Suzhou 215600, Jiangsu,China)

机构地区：[1]苏州大学附属张家港医院骨科,江苏张家港215600 [2]苏州大学附属第二医院骨科、苏州大学骨质疏松诊疗技术研究所,江苏苏州215000

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2021年第3期267-275,共9页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：国家自然科学基金(81874018);苏州市科技发展计划(民生科技-医疗卫生应用基础研究)(SYSD2020007);张家港市卫计系统青年科技项目(ZJGQNKJ201701)。

摘　　要：目的探讨成骨细胞自噬在铁环境变化情况下致骨代谢异常的作用。方法实验分为对照组、枸橼酸铁铵(ferric ammonium citrate,FAC)组、枸橼酸铁铵+3-甲基腺嘌呤(3-methyladenine,3-MA)组、去铁胺(deferoxamine,DFO)+FAC+3-MA组、DFO+FAC组。用Western blot法检测成骨细胞自噬(LC3、P62、Beclin1)和成骨(collagen typeⅠ,COLⅠ)和骨钙素(bone gla-protein,BGP)标志蛋白的表达;共聚焦显微镜观察成骨细胞COLⅠ、BGP的荧光蛋白;流式细胞仪检测成骨细胞凋亡;Von kossa染色法行钙结节染色;碱性磷酸酶活性试剂盒检测碱性磷酸酶活性。结果与对照组比较,FAC组LC3Ⅱ、Beclin1蛋白表达增加、LC3Ⅱ/Ⅰ比值增大,可溶性P62、COLⅠ、BGP蛋白表达减少,细胞凋亡增加,细胞矿化能力(0.68±1.63)及碱性磷酸酶活性(0.68±1.84)下降(P<0.05),不可溶性P62蛋白表达差异无统计学意义(P>0.05)。加入3-MA抑制剂后,与对照组、FAC组比较,LC3Ⅱ、可溶性P62、Beclin1蛋白表达减少、LC3Ⅱ/Ⅰ比值减小,COLⅠ、BGP蛋白表达进一步减少,细胞矿化能力(0.44±1.63)及碱性磷酸酶活性(0.53±0.97)下降,细胞凋亡、不可溶性P62蛋白表达增加(P<0.05);但FAC+3-MA组与DFO+FAC+3-MA组结果比较,差异无统计学意义(P>0.05)。与对照组比较,DFO+FAC组LC3Ⅱ/Ⅰ比值变小,LC3Ⅱ、Beclin1、可溶性P62、COLⅠ、BGP蛋白表达减少,细胞凋亡增加,细胞矿化能力(0.86±0.39)及碱性磷酸酶活性(0.76±0.52)下降(P<0.05),不可溶性P62蛋白表达差异无统计学意义(P>0.05)。与FAC组比较,DFO+FAC组LC3Ⅱ/Ⅰ比值变小,LC3Ⅱ、Beclin1蛋白表达减少(P<0.05),可溶性P62、COLⅠ、BGP蛋白表达增加,细胞凋亡减少,细胞矿化能力及碱性磷酸酶活性升高(P<0.05),不可溶性P62蛋白表达无差异(P>0.05)。结论成骨细胞自噬在骨代谢过程中起着重要作用,过度激活或抑制自噬均会对细胞产生损害,适宜的自噬水平对成骨细胞的功能活性有促进作用。降铁�Objective To investigate the roles of osteoblast autophagy in abnormal bone metabolism caused by changes in iron environment.Methods The experiment set control group,ferric ammonium citrate(FAC)group,3-methyladenine(3-MA)group,deferoxamine(DFO)+FAC+3-MA group and DFO+FAC group.The autophagy protein(LC3,P62,Beclin1)and osteoblast protein was detected by Western blot.The fluorescent proteins of collagen typeⅠ(COLⅠ)and bone gla-protein(BGP)was observed by confocal laser scanning microscope.The apoptosis of osteoblasts was detected by flow cytometry.Von Kossa staining was used for calcium nodule staining.Alkaline phosphatase activity was detected by alkaline phosphatase activity kit.Results Compared with the control group,the expression of LC3Ⅱ,Beclin1 protein,LC3Ⅱ/Ⅰratio and apoptosis were increased,and the expression of soluble P62,COLⅠ,BGP protein,mineralization ability(0.68±1.63),and alkaline phosphatase activity(0.68±1.84)decreased in FAC group(P<0.05),but the expression of insoluble P62 protein had no difference(P>0.05).Compared with control and FAC group,the expression of LC3Ⅱ,Beclin1,COLⅠ,BGP,soluble P62 protein,LC3Ⅱ/Ⅰratio,cell mineralization ability(0.44±1.63)and alkaline phosphatase activity(0.53±0.97)decreased and the insoluble P62 protein expression and apoptosis increased after adding 3-MA inhibitor(P<0.05),but without statistical difference between FAC+3-MA and DFO+FAC+3-MA group(P>0.05).Compared with the control group,the protein expressions of LC3Ⅱ,Beclin1,soluble P62,COLⅠ,BGP,the ratio of LC3Ⅱ/Ⅰ,cell mineralization ability,and alkaline phosphatase activity decreased(P<0.05)and the cell apoptosis increased,but the protein expression of insoluble P62 had no difference in DFO+FAC group.Compared with the FAC group,the protein expressions of LC3Ⅱ,Beclin1,LC3Ⅱ/Ⅰratio,and cell apoptosis decreased and the protein expressions of soluble P62,COLⅠ,BGP,cell mineralization ability(0.86±0.39),and alkaline phosphatase activity(0.76±0.52)inecreased(P<0.05),but the protein e

关 键 词：枸橼酸铁铵 去铁胺 成骨细胞自噬 骨代谢 3-甲基腺嘌呤 细胞凋亡 

分 类 号：R681[医药卫生—骨科学]