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，H₂O₂）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H₂O₂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。

关键词: 骨缺损, 衰老, 骨再生, SAMP8小鼠

Abstract:

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 (H₂O₂) 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-associated β-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 H₂O₂ 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.

Key words: bone defect, aging, bone regeneration, SAMP8 mice

中图分类号: 

• R782