骨髓间充质干细胞旁分泌对成骨细胞生物学活性的影响

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摘要目的探讨在骨修复中骨髓间充质干细胞(bone mesenchymal stem cells，BMSCs)旁分泌对成骨细胞生物活性的作用与影响。方法研究通过transwell对BMSCs与类成骨细胞MG63行非接触性共培养方法，排除BMSCs成骨分化的影响，单纯观察BMSCs旁分泌对MG63细胞生物学特性的改变。应用迁移实验和划痕实验检测成骨细胞迁移运动能力，流式细胞仪检测细胞周期，茜素红染色和碱性磷酸酶染色分别检测成骨细胞的钙化结节和碱性磷酸酶水平，实时荧光定量聚合酶链式反应(real time quantitative polymerase chain reaction，RT-qPCR)检测成骨与迁移相关基因基质金属蛋白酶(matrix metalloproteinase，MMP)-2、MMP-9、骨钙素(osteocalcin，OCN)、骨桥素(osteopontin，OPN)、Osterix、Runt相关转录因子2(Runt related transcription factor 2，RUNX2)的转录水平。结果在共培养环境下，共培养组成骨细胞迁移、运动能力较对照组显著增高(P＜0.05)。共培养组成骨细胞增殖加快并且S期与G2/M期细胞比例较对照组明显增高(P＜0.05)。共培养组成骨细胞的钙化结节和碱性磷酸酶水平较对照组显著上升(P＜0.05)。共培养组成骨细胞MMP-2、MMP-9、OPN、Osterix、RUNX2基因转录水平显著增高(P＜0.05)。结论在非接触共培养条件下，BMSCs旁分泌促进了成骨细胞的运动迁移、增殖分裂、钙化、碱性磷酸酶活性、多种成骨基因表达。这些证据表明BMSCs除具有成骨分化能力外，还具有增强周围成骨细胞骨修复活性的旁分泌功能，这些证据为BMSCs移植在骨缺损及骨坏死中应用提供新的思路和依据。

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关键词 ：骨髓间充质干细胞, 成骨细胞, 骨修复, 旁分泌

Abstract：Objective To investigate the effect of paracrine of bone marrow mesenchymal stem cells (BMSCs) on the biological activity of osteoblasts in bone repair. Methods In order to exclude the influence of BMSCs osteogenic differentiation，we use non-contact co-culture of BMSCs and osteoblastic cells by transwell to observe the changes of BMSCs paracrine on the biological characteristics of MG63 cells.Migration test and scratch test were used to detect the migration and movement ability of osteoblasts.Flow cytometry was used to detect the changes of cell cycle.Alizarin red staining and alkaline phosphatase staining were used to detect the calcification nodule and ALP level of osteoblasts，and RT-qPCR was used to detect the MMP-2，MMP-9，OCN，OPN，Osterix，RUNX2 transcription level.Results In the co-culture environment，the migration and movement ability of osteocytes in the co-culture group were significantly higher than those in the control group (P＜0.05).The proliferation of osteocytes in the co-cultured group was accelerated and the proportion of cells in S phase and G2/M phase in the co-culture group was significantly higher than that in the control group （P＜0.05).The levels of calcified nodules and ALP in co-cultured osteocytes were significantly higher than those in the control group (P＜0.05).The transcriptional level of MMP-2，MMP-9，OPN，Osterix，RUNX2 in co-cultured osteocytes was significantly increased (P＜0.05).Conclusion Under the condition of non-contact co-culture，BMSCs paracrine promotes osteoblast migration，proliferation，division，calcification，ALP activity and multiple osteogenic gene expression.These evidences show that BMSCs not only have the ability of osteogenic differentiation，but also have the paracrine function of enhancing the bone repair activity of surrounding osteoblasts，which provides a new theoretical basis for the application of BMSCs transplantation in bone defect and osteonecrosis.

Key words： bone marrow mesenchymal stem cells osteoblast bone repair paracrine

基金资助:辽宁省教育厅科学研究经费项目(L2019605)
大连市科技创新基金项目(2018J11CY030)

通讯作者: 赵德伟

作者简介: 王广阔(1991－ )，男，研究生在读，大连大学附属中山医院骨科医学研究中心，116001。

引用本文:

王广阔 1，张秀智 2*，王韦丹 1，曹放 3，赵德伟 1*. 骨髓间充质干细胞旁分泌对成骨细胞生物学活性的影响[J]. 实用骨科杂志, 2021, 27(8): 713-718.
Wang Guangkuo 1，Zhang Xiuzhi 2*，Wang Weidan 1，et al. Effect of Paracrine of Bone Mesenchymal Stem Cells on Biological Activity of Osteoblasts. sygkzz, 2021, 27(8): 713-718.

链接本文:

http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2021/V27/I8/713

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