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目的为提高BMSCs的静脉移植效率,研究单核细胞趋化蛋白1(monocyte chemoattractant protein1,MCP-1)对经成肌诱导分化后的小鼠BMSCs的体外趋化迁移作用。方法以差速贴壁法与密度梯度离心法相结合分离培养C57/BL10小鼠BMSCs,并采用ALP Gomori改良钙钴法染色行成骨诱导鉴定。取第3代BMSCs以5氮杂胞苷(5-azacytidine,5-Aza)为诱导剂进行成肌细胞诱导分化。以不同浓度(25、50、100、200、400ng/mL)的MCP-1对经成肌诱导分化后的小鼠BMSCs进行体外趋化迁移,计数迁移细胞数,以单纯L-DMEM培养基为对照;应用流式细胞仪检测经成肌诱导分化后的小鼠BMSCs的C-C趋化因子受体(chemokine receptor2,CKR-2)的表达。结果第3代细胞能诱导分化为成骨细胞,提示所用细胞为BMSCs。经成肌诱导分化后的BMSCs可形成肌小管结构。MCP-1浓度为25、50、100、200、400ng/mL时,迁移至Millicell小室聚碳酸酯膜外层的细胞数分别为(35.0667±6.5842)、(43.2000±6.4608)、(44.4667±4.8235)、(45.6000±8.6503)、(50.7333±7.5825)个,与对照组(28.3333±8.9176)个比较差异均有统计学意义(P<0.05),25、50、400ng/mL组间两两比较差异均有统计学意义(P<0.05)。流式细胞仪检测显示,加入一抗和二抗的待测样本CKR-2表达率为48.0%,与空白对照(0.6%)和仅加入二抗的阴性对照(17.0%)比较差异均有统计学意义(P<0.001)。结论 MCP-1对经成肌诱导分化后的小鼠BMSCs有趋化迁移作用,MCP-1/CKR-2通路参与了小鼠BMSCs的迁移。
Objective To study the effect of monocyte chemoattractant protein 1 (MCP-1) on the migration of BMSCs induced by myoblast in vitro in order to improve the efficiency of vein grafting of BMSCs. Methods The BMSCs of C57 / BL10 mice were isolated and cultured by differential adhesion method and density gradient centrifugation. The osteogenic differentiation of C57 / BL10 mice was evaluated by ALP Gomori staining. The 3rd generation BMSCs were induced to differentiate into myoblasts with 5-azacytidine (5-Aza) as an inducer. Mice BMSCs induced by myoblast were treated with MCP-1 at different concentrations (25,50,100,200,400ng / mL) for chemotaxis migration. The numbers of migrating cells were counted and counted in L-DMEM medium alone The expression of chemokine receptor 2 (CKR-2) in mouse BMSCs induced by myoblast was detected by flow cytometry. The results of the third generation of cells can be induced to differentiate into osteoblasts, suggesting that the cells used for the BMSCs. Myogenic differentiation induced BMSCs can form myotube structure. The number of cells that migrated to the outer layer of Millicell cell polycarbonate membrane was (35.0667 ± 6.5842), (43.2000 ± 6.4608) and (44.4667 ± 4.8235) at MCP-1 concentrations of 25, 50, 100, 200 and 400 ng / , (45.6000 ± 8.6503) and (50.7333 ± 7.5825), respectively, which were significantly different from those in the control group (28.3333 ± 8.9176) (P <0.05) There was statistical significance (P <0.05). The results of flow cytometry showed that the positive rate of CKR-2 in primary antibody and secondary antibody was 48.0%, which was statistically different from that of blank control (0.6%) and negative control (17.0%) Significance (P <0.001). Conclusion MCP-1 can induce the chemotactic migration of BMSCs induced by myoblast differentiation. MCP-1 / CKR-2 pathway is involved in the migration of BMSCs.