摘要： 目的：明确牙髓间充质干细胞对巨噬细胞极化的调节作用，探索调节作用分子机制。方法：使用酶消化法获取健康人的牙髓组织，差速贴壁法纯化培养DPSCs（dental pulp stem cells）。使用流式细胞术分析其表面蛋白表达；使用油红O、茜素红S染色，分析其成脂肪、成骨诱导的潜能。转录组测序分析DPSCs和BMSCs基因表达谱的差异，并使用KEGG、String数据库对可能参与初始免疫信号通路的相关基因进行富集分析和蛋白相互作用预测。使用免疫印迹定量分析MYD88及TLR3蛋白在DPSCs和BMSCs中表达水平。在Transwell共培养体系中，将DPSCs和经Poly（I:C）激活的DPSCs分别与巨噬细胞进行共培养，使用流式细胞术分析共培养之后的巨噬细胞CD206、Arg-1的表达水平。结果：纯化培养的DPSCs形态呈成纤维细胞样，表达CD90、CD105、CD73，不表达CD34、CD45，CD11b，经成骨、成脂肪诱导液诱导后，油红O、茜素红S染色均呈阳性反应。转录组测序富集分析发现DPSCs在初始免疫信号通路基因表达水平高于BMSCs，核心基因MYD88、TLR3的蛋白水平也高于BMSCs。使用TLR3通路的激动剂Poly（I:C）激活DPSCs后，发现能够使巨噬细胞表达CD206、Arg-1的水平增加。结论：DPSCs能够促进巨噬细胞向抗炎型的巨噬细胞极化，TLRs信号通路可能是重要的分子途径。

关键词: 牙髓干细胞, 巨噬细胞, 极化, Toll受体信号通路

Abstract: Objective: To investigate the regulatory effect and molecular mechanism of macrophage polarization on dental pulp mesenchymal stem cells. Methods: DPSCs were obtained from healthy human premolars treated with orthodontics by enzyme digestion method and expanded in long-term culture. DPSCs were identified by surface markers by flow cytometry and multipotentiality analysis. Transcriptome analyses were carried out on both of cell types. KEGG and String databases were used to perform enrichment analysis and protein interaction prediction of related genes that may participate in the initial immune signaling pathway. Western blot technique was used to detect the expression of MYD88/TLR3 protein in the sample. DPSCs were co-cultured with macrophages in transwell co-culture system. The CD206 and Arg-1 expression level in co-cultured macrophages was measured by flow cytometry. Results: Purified DPSCs showed the morphology, surface protein expression and differentiation of osteoblasts and adipocytes as typical MSCs. DPSCs compared with BMSCs showed that high expression of genes related to initial immune signaling pathway by transcriptome analyses and western blot. DPSCs had a stronger ability to induce the macrophage to develop into M2 polarization with agonist of TLR3 than control transwell co-culture system. Conclusion: DPSCs can promote macrophages to polarize anti-inflammatory macrophages, TLRs signal pathway may be an important molecular pathway.

Key words: dental pulp stem cells, macrophage, polarization, Toll like receptor signaling pathway

中图分类号: 

• R782