摘要
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 investigate the effect of monocyte chemoattractant protein 1（MCP-1） on the migration of the induced and differentiated mouse bone marrow mesenchymal stem cells（BMSCs） for raising the efficacy ofintravenous transplantation of BMSCs.Methods The BMSCs were cultured with the method of differential adhesion and density gradient centrifugation of C57/BL10 mice,and were identified by alkaline phosphatase Gomori modified staining after osteogenic inducing.At the 3rd passage,the BMSCs were induced to the myoblasts with 5-azacytidine（5-Aza）.The chemotaxis of MCP-1 in the induced and differentiated BMSCs in vitro at concentrations of 25,50,100,200,and 400 ng/mL was observed through the migration test,by counting the number of the migrated cells.The expression of the chemokine receptor 2（CKR-2） in the induced and differentiated BMSCs was detected with the flow cytometry.Results The cells could be cultured with the methods of differential adhesion and density gradient centrifugation and still had higher proliferative and differentiative potency;the induced cells at the 3rd passage could differenciate to the osteoblasts,confirming that the cells were BMSCs;the myogenic induced BMSCs possesed the sarcotubule structure.The number of the migrating BMSCs at MCP-1 concentrations of 25-400 ng/mL were respectively 35.066 7 ± 6.584 2,43.200 0 ± 6.460 8,44.466 7 ± 4.823 5,45.600 0 ± 8.650 3,and 50.733 3 ± 7.582 5;showing significant difference when compared with control group（28.333 3 ± 8.917 6,P 0.05）,and presenting significant difference among 25,50,400 ng/mL groups compared with each other（P 0.05）.The expression of CKR-2 in the mouse BMSCs（48.0%） was significantly higher（P 0.001） than those of blank control（0.6%） and negative control（17.0%）.Conclusion The results indicate that the MCP-1 can induce the migration of mouse BMSCs by MCP-1/CKR-2 pathway.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第7期834-837,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30200085)~~
关键词
BMSCS
成肌诱导分化
单核细胞趋化蛋白1
趋化迁移作用
小鼠
Bone marrow mesenchymal stem cells Myogenic inducing and identification Monocyte chemoattractant protein 1 Chemotatic migration Mouse
