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☆

摘要

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25，50，100 mg/L的甲钴胺进行诱导分化24，48和72 h。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48 h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中100 mg/L组表达上调最明显；100 mg/L 甲钴胺诱导24，48和72 h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中72 h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，100 mg/L为甲钴胺的较佳诱导浓度。%BACKGROUND:Currently, transplantation of bone marrow mesenchymal stem cel s into the spinal cord is very limited to the recovery of animals fol owing spinal cord injury. Methylcobalamin is a common drug for the treatment of neurological diseases and injuries, but its effects on bone marrow mesenchymal stem cel s are unclear. OBJECTIVE:To study the feasibility of bone marrow mesenchymal stem cel s differentiating into neuron-like cel s induced by methylcobalamin in vitro and to observe the cel viability and proliferation of differentiated cel s. Methods:Rat bone marrow mesenchymal stem cel s were isolated, cultured and purified by density gradient centrifugation and adherent culture. The fourth to fifth generation of bone marrow mesenchymal stem cel s were treated for 24, 48 and 72 hours with different concentrations (25, 50 and 100 mg/L) of methylcobalamin. The morphological changes and cel growth were continuously observed under an inverted phase constract microscope. The viability of induced cel s 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 cel s could differentiate into neuron-like cel s after induction with methylcobalamin. The expressions of Nestin and neuron-specific enolase were up-regulated after 48 hours of methylcobalamin treatment at different concentrations, especial y 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, especial y for 72 hours. It is indicated that methylcobalamin can induce bone marrow mesenchymal stem cel s differentiating into neuron-like cel s, and the optimal concentration of methylcobalamin is 100 mg/L.