骨髓基质干细胞分化为神经元样细胞迁移能力变化的研究

摘要

Aim To study the methods of bone marrow mesenchymal stem cells ( BMSCs) differentiation into the neural-like cells and investigate the mechanism of the migration capacity changes of neuron-like cells derived from BMSCs. Methods BMSCs were cultured with 1 mmol · L-1 β-mercaptoethanol (BME) for 12 h or 24 h to induce the differentiation into neuron like cells in vitro. After 12 h or 24 h, differentiation was observed under phase contrast microscope. Reverse Transcription-Polymerase Chain Reaction ( RT-PCR) techniques was used to detect the expression of mRNA of specificity enolase of neuron ( NSE) , neurofilament and glial fibrillary acidic protein ( GFAP) , β-tubulin Ⅲ. Western-blot was applied to detect expression of CXCR4. Cell migration was measured using a modified Boyden chamber assay. Results Partly cytoplasm of differentiated BMSCs contracted with protruding after induction. RT-PCR demonstrated that the mRNA expression of NSE, neurofilament, β-tubulin Ⅲ of neuron-like cells induced from BMSCs was markedly enhanced. Western blot showed that the expression level of CXCR4 of neuron-like cells was obviously increased. SDF-1 significantly increased the migration of the differentiated BMSCs in a Boyden chamber assay. When the cells were treated with a specific antagonist for CXCR4, AMD3100,cell migration was significantly inhibited. Conclusion BME is an effective inducer for the differentiation of BMSCs into neuron-like cells in vitro, and it can increase the differentiated BMSCs migration via up-regulating SDF1/CXCR4 axis.%目的 研究体外定向诱导骨髓间充质干细胞(BMSCs)分化为神经元样细胞的方法,探讨其分化后迁移能力变化的分子机制.方法 体外培养BMSCs,以β-巯基乙醇(BME)1 mmol·L-1诱导分化,于诱导后12 h、24 h分别观察细胞形态变化,RT-PCR鉴定神经元特异性烯醇化酶(NSE) 、神经丝蛋白(Neurofilament) 、胶质纤维酸性蛋白(β-tubulin Ⅲ) mRNA的变化.Western blot检测CXCR4表达变化,细胞迁移实验检测BMSCs诱导前后迁移能力的变化.结果 诱导后,BMSCs胞体收缩,突起伸出;RT-PCR发现诱导出的神经元样细胞的NSE、Neurofilament 、β-tubulin Ⅲ mRNA明显增加;Western blot结果表明神经元样细胞表面CXCR4的表达明显增加,细胞迁移实验发现神经元样细胞向SDF-1α的迁移能力明显增加.用CXCR4特异的抑制剂AMD3100处理后细胞迁移被明显抑制.结论 在体外骨髓基质干细胞以BME为诱导剂可定向分化为神经元样细胞,且发现其迁移能力明显增加,其机制可能与其表面CXCR4表达上调有关.