甲钴胺体外诱导大鼠骨髓间充质干细胞向神经元样细胞分化

Methylcobalamin induces differentiation of rat bone mesenchymal stem cells into neuron-like cells in vitro

背景:目前骨髓间充质干细胞移植到脊髓损伤动物体内后对脊髓损伤的恢复效果非常有限。甲钴胺是治疗神经系统疾病及损伤的常见药物,其对骨髓间充质干细胞的影响尚不清楚。目的:探讨甲钴胺体外定向诱导骨髓间充质干细胞向神经元样细胞分化的可行性,观察分化后的细胞增殖和生长情况。方法:取大鼠胫腓骨骨髓,采用密度梯度离心贴壁细胞培养法分离、培养骨髓间充质干细胞,取第四五代骨髓间充质干细胞,分别以25,50,100mg/L的甲钴胺进行诱导分化24,48和72h。倒置相差显微镜下连续观察细胞形态学变化,MTT法检测细胞活性,RT-PCR和Western blot法检测特异性标志物Nestin和NSE的表达。结果与结论:甲钴胺诱导骨髓间充质干细胞后大部分细胞变成神经元样细胞。与对照组比较,甲钴胺诱导后细胞活性无明显变化。不同剂量甲钴胺诱导48h后,Nestin和NSE在mRNA和蛋白水平表达均上调,其中100mg/L组表达上调最明显;100mg/L甲钴胺诱导24,48和72h后,Nestin和NSE在mRNA和蛋白水平表达均上调,其中72h表达上调最明显。说明甲钴胺可定向诱导骨髓间充质干细胞向神经元样细胞分化,100mg/L为甲钴胺的较佳诱导浓度。

BACKGROUND： Currently, transplantation of bone marrow mesenchymal stem cells into the spinal cord is very limited to the recovery of animals following spinal cord injury. Methylcobalamin is a common drug for the treatment of neurological diseases and injuries, but its effects on bone marrow mesenchymal stem cells are unclear. OBJECTIVE： To study the feasibility of bone marrow mesenchymal stem cells differentiating into neuron-like cells induced by methylcobalamin in vitro and to observe the cell viability and proliferation of differentiated cells. Methods： Rat bone marrow mesenchymal stem cells were isolated, cultured and purified by density gradient centrifugation and adherent culture. The fourth to fifth generation of bone marrow mesenchymal stem cells were treated for 24, 48 and 72 hours with different concentrations （25, 50 and 100 mg/L） of methylcobalamin. The morphological changes and cell growth were continuously observed under an inverted phase constract microscope. The viability of induced cells was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. The expressions of Nestin and neuron-specific enolase were identified by reverse transcription PCR and western blot. RESULTS AND CONCLUSION： Most of bone marrow mesenchymal stem cells could differentiate into neuron-like cells after induction with methylcobalamin. The expressions of Nestin and neuron-specific enolase were up-regulated after 48 hours of methylcobalamin treatment at different concentrations, especially after treatment with 100 mg/L methylcobalamin. Similarly, the expressions of Nestin and neuron-specific enolase could be increased significantly after 100 mg/L methylcobalamin treatment for 24, 48 and 72 hours, especially for 72 hours. It is indicated that methylcobalamin can induce bone marrow mesenchymal stem cells differentiating into neuron-like cells, and the optimal concentration of methylcobalamin is 100 mg/L.