摘要
目的:探究衰老对小鼠颌骨骨再生能力及小鼠胚胎成骨细胞前体细胞(mouse embryo osteoblast precursor cells,MC3T3-E1 cells)成骨分化能力的影响。方法:采用衰老加速小鼠(SAMP8小鼠)构建上颌骨骨缺损模型,通过Micro-CT分析年轻组(2月龄)与衰老组(8月龄)小鼠上颌骨缺损区新生骨的骨微结构参数。在体外实验中,采用过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导MC3T3-E1细胞衰老,通过实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和蛋白质印迹法检测衰老相关基因的表达。通过衰老相关β-半乳糖苷酶(senescence-associated-β-galactosidase,SA-β-Gal)染色比较细胞阳性面积;利用流式细胞术分析细胞周期分布;通过碱性磷酸酶(alkaline phosphatase,ALP)染色及茜素红(alizarin red S,ARS)染色评估细胞成骨分化能力,采用蛋白质印迹法检测成骨相关蛋白的表达水平。结果:骨缺损术后14 d,衰老组小鼠新生骨量低于年轻组。体外实验表明,经H_(2)O_(2)处理后,MC3T3-E1细胞的衰老相关基因与蛋白表达水平均显著上升,SA-β-Gal染色阳性面积显著增加;细胞周期分析显示,S期+G2期的细胞比例显著下降;同时,ALP与ARS染色显示细胞成骨分化能力减弱;成骨相关蛋白骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)和Runt相关转录因子2(Runt related transcription factor 2,Runx2)的表达水平也显著下降。结论:衰老可抑制小鼠颌骨骨再生能力,部分机制源于衰老状态下成骨前体细胞的分化能力下降。
Objective:To investigate the effects of aging on the bone regeneration capacity of mouse jaw and the osteogenic differentiation ability of mouse embryo osteoblast precursor cells(MC3T3-E1 cells).Methods:A maxillary bone defect model was established using senescence-accelerated mice(SAMP8 mice).Micro-CT was used to analyze the bone microstructural parameters of new bone in the maxillary bone defect area of young(2-month-old)and aged(8-month-old)mice.In vitro,hydrogen peroxide(H_(2)O_(2))was used to induce senescence in MC3T3-E1 cells.The expression of senescence-related genes was detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Senescence-associatedβ-galactosidase(SA-β-Gal)staining was used to compare the positive area;flow cytometry was employed to analyze cell cycle distribution;alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining were used to evaluate osteogenic differentiation ability;and Western blotting was applied to detect the expression levels of osteogenesis-related proteins.Results:On the 14th day after bone defect surgery,the amount of new bone in the aged group was lower than that in the young group.In vitro experiments showed that after H_(2)O_(2) treatment,the expression levels of senescence-related genes and proteins in MC3T3-E1 cells were significantly increased,and the SA-β-Gal staining positive area was significantly enlarged.Cell cycle analysis revealed a significant decrease in the proportion of cells in the S and G2 phases.Meanwhile ALP and ARS staining indicated weakened osteogenic differentiation,and the expression levels of osteogenesis-related proteins,bone morphogenetic protein-2(BMP-2)and Runt-related transcription factor 2(Runx2)were also significantly decreased.Conclusion:Aging can inhibit bone regeneration in the mouse jaw,partly due to the decreased differentiation capacity of osteoblast precursor cells under aging conditions.
作者
徐亦凡
王佐林
XU Yifan;WANG Zuolin(Shanghai Engineering Research Center of Tooth Restoration and Regeneration&Tongji Research Institute of Stomatology&Department of Oral Implantology,Shanghai Tongji Stomatological Hospital and Dental School,Tongji University,Shanghai 200072,China)
出处
《口腔颌面外科杂志》
2025年第6期448-455,共8页
Journal of Oral and Maxillofacial Surgery
基金
国家重点研发计划(2018YFE0202200)
中央高校基本业务费专项资金(22120180196)
国家自然科学基金(81670962)。
