骨形态发生蛋白2在小鼠模型中诱导破骨细胞活化的机制

陈翔; 汪萍萍

doi:10.13898/j.cnki.issn.1000-2200.2020.01.005

骨形态发生蛋白2在小鼠模型中诱导破骨细胞活化的机制

陈翔,
汪萍萍

Mechanism of the bone morphogenetic protein 2 inducing osteoclast activation in the mouse model

摘要

摘要:
目的探讨骨形态发生蛋白2（bone morphogenetic protein 2，BMP-2）在诱导小鼠破骨细胞活化的作用以及相关的机制。
方法选取20只小鼠，按照随机数字法分为对照组和实验组，每组10只，均接受小鼠脊柱后外侧入路的横突间融合手术，实验组植入骨片和BMP-2，对照组仅植入骨片，评估术后骨骼变化情况。在小鼠胫骨内提取骨髓来源的巨噬细胞（bone marrow-derived macrophages，BMMs），应用RANKL及M-CSF试剂诱导为破骨细胞，并在其中加入不同浓度的BMP-2，应用TRAP染色法检测破骨细胞情况，荧光定量聚合酶链反应（polymerase chain reaction，PCR）方法测定Smad1及p65基因，Western blotting法测定Smad1及p65蛋白表达情况。
结果微型CT仪影像检查显示，实验组小鼠术后的脊柱融合情况强于对照组。2组小鼠术后1、2、4周骨小梁体积和骨小梁厚度均呈增高趋势，骨小梁间隙呈降低趋势，差异均具有统计学意义（P < 0.01）。实验组术后1、2、4周骨小梁体积高于对照组，实验组术后2、4周骨小梁间隙低于对照组（P < 0.01）。在BMP-2浓度为60、90及120 ng/mL时破骨细胞总数和总面积均高于BMP-2浓度为0、30 ng/mL时（P < 0.01）。Smad1及p65 mRNA表达在不同浓度BMP-2诱导破骨细胞比较差异均具有统计学意义（P < 0.01），Smad1和p65 mRNA表达呈线性相关（P < 0.05）。
结论BMP-2能够促进小鼠脊柱手术模型的融合，BMP-2参与了破骨细胞活化的过程，可能通过Smad1/p65信号通路介导破骨细胞活化。

Abstract:
ObjectiveTo investigate the role of bone morphogenetic protein 2 (BMP-2) in inducing the osteoclast activation in mice, and its related mechanisms.
MethodsTwenty mice were randomly divided into the control group and experimental group (10 mice in each group), and treated with intertransverse fusion surgery through the posterolateral approach of the spine.The experimental group was implanted with bone fragments combined with BMP-2, the control group was only implanted with bone fragments, and the postoperative bone changes in two groups were evaluated.The bone marrow-derived macrophages (BMMs) were extracted from the tibia of the mice, induced into osteoclasts using RANKL and M-CSF reagents, and co-cultured with different concentrations of BMP-2.The osteoclasts, mRNA levels of Smad1 and p65 and protein levels of Smad1 and p65 were detected using TRAP staining, RT-PCR and Western blot, respectively.
ResultsThe results of image examination of micro-CT showed that the spinal fusion in experimental group was stronger than that in control group.The trabecular bone volume and trabecular thickness in two groups showed an increasing trend after 1, 2 and 4 weeks of operation, and the trabecular bone gap showed a decreasing trend, and the difference of which was statistically significant (P < 0.05).The volume of trabecular bone in experimental group was higher than that in control group at 1, 2, and 4 weeks after operation, and the trabecular space in experimental group was lower than that in control group at 2 and 4 weeks after operation (P < 0.01).The total number and total area of osteoclasts at the 60 ng/mL, 90 ng/mL and 120 ng/mL of BMP-2 were higher than those at 0 ng/mL and 30 ng/mL of BMP-2 (P < 0.01).The differences of the expression levels of Smad1 and p65 mRNA in osteoclasts induced by different concentrations of BMP-2 were statistically significant (P < 0.01), and the expression level of Smad1 mRNA was linearly correlated with p65 (P < 0.01).The differences of the expression levels of Smad1 and p65 protein in osteoclasts induced by different concentrations of BMP-2 were statistically significant (P < 0.01), and the expression level of Smad1 protein was linearly correlated with p65 (P < 0.05).
ConclusionsBMP-2 can promote the fusion of mouse spinal surgery model.BMP-2 is involved in the process of osteoclast activation, and may mediate osteoclast activation via Smad1/p65 signaling pathway.

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