诱导骨髓基质细胞表达神经细胞标记物Nestin,NSE和GFAP:脑源性神经节苷脂定向诱导细胞分化作用的研究

目的：研究脑源性神经节苷脂诱导成年大鼠骨髓基质细胞定向分化为神经前体细胞的作用。方法：成年大鼠骨髓基质细胞传代纯化后，分别在无血清、无外源性生长因子、含2％t327的Neurobasal-A培养基及含20％胎牛血清的DMEM培养基中添加脑源性神经节苷脂作为诱导物，观察细胞的形态变化。结果：在无血清、无外源性生长因子、含2?7的Neurobasal-A培养基中，脑源性神经节苷脂能诱导骨髓基质细胞定向分化为神经前体细胞，其形态发生显著变化，胞体逐渐变小、折光性增强，突起逐渐增多、延长，部分细胞间可见突起连接、细胞核和核仁；诱导第5d免疫细胞荧光染色显示，神经前体细胞标记物Nestin染色阳性，同时部分细胞可见神经元特异性标记物NSE及星形胶质细胞标记物GFAP染色阳性，而少突胶质细胞标记物Nogo-A染色阴性。在含20％胎牛血清的DMEM（高糖）培养基中，脑源性神经节苷脂的这种诱导、分化作用明显被抑制，表现为细胞形态变化不明显，Nestin、NSE及GFAP染色均为阴性。两组对照组细胞形态无变化、上述染色均为阴性。结论：脑源性神经节苷脂具有诱导骨髓基质细胞成为神经前体细胞的作用，这种作用不需要外源性生长因子存在，血清可抑制这种作用，脑源性神经节苷脂在成体干细胞可塑性研究中有重要价值。

Induction of Neural Markers Nestin, NSE and GFAP Expression in Cultured Bone Marrow Stromal Cells: Differential Effects of Brain Gangliosides

Objective:To study the effects of brain gangliosides on inducing marrow stromal cells (MSCs) into neural progenitor cells. Methods:Adult rat MSCs were purified by passaging.Purified MSCs were induced by brain gangliosides (in process of applying the pa- tent) respectively in Neurobasal-A medium containing 2% B27 and DMEM (high glucose) containing 20% fetal bovine serum(FBS).Cell morphology was observed after being induced 1,3,5 day (s).Nestin,and neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence after 5 days of induction.Results:Cell morphological changes were remarkable after being induced by brain gangliosides in Neurobasal-A medium,such as the cell body getting small,the refraction of the cell body getting strong, the processes getting more abundant and elongated.The connection of the processes and the nuclei (nucleoli) among part of the cells were seen.Cell immunofluorescence staining showed a large number of cells were positive for Nestin and part of the ceils were positive for NSE and GFAP.Nogo-A immunofluorescence staining for oligodendrocyte was negative.However,No change of the cell morphology was seen after being induced by brain gangliosides in DMEM medium with 20% FBS.Cell immunofluorescence staining for Nestin,NSE,and GFAP were negative.Conclusion:Brain gangliosides can induce MSCs into neural progenitor cells and it is independent of extraneous growth fac- tors.The role was inhibited by serum.Brain gangliosides may play an important role in the plasticity studies of adult stem cells.