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目的:探讨单唾液酸四己糖神经节苷脂(monosialoteterahexosyl ganglioside,GM1)联合生长因子(bFGF+EGF)能否更为高效地促进骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化为神经元样细胞,从而为细胞替代治疗缺血性脑卒中提供实验依据。方法:采用密度梯度离心法获取骨髓单个核细胞,行原代、传代培养,取第3代BMSCs,用流式细胞术鉴定其表面标志物;同时鉴定其多分化潜能。将第3代BMSCs分四组向神经元样细胞诱导分化,A组:单用(bFGF+EGF);B组:单用GM1;C组:GM1联合(bFGF+EGF);D组:对照组。分别在正式诱导前后行免疫细胞化学法检测各组细胞Nestin、β-TubulinⅢ、GFAP的阳性表达率并行数据统计分析。结果:流式细胞术鉴定BMSCs表面标志物呈CD29(+)、CD105(+)、CD34(-)、CD45(-);同时其具分化为成骨细胞和成脂细胞潜能。BMSCs诱导为神经元样细胞之前,四组细胞Nestin、β-Tubulin、GFAP表达均呈阴性;诱导2~4 d时,A、B、C三组Nestin阳性细胞表达率均呈上升趋势,且C组高于A、B、D组;诱导6~8 d时,A、B、C三组Nestin阳性细胞表达率均有所降低,而β-TubulinⅢ、GFAP阳性细胞表达率则呈较明显上升趋势,且C组较A、B组升高明显。综合实验数据,C组Nestin、β-TubulinⅢ、GFAP阳性细胞表达率均高于A、B、D组。结论:GM1联合生长因子(bFGF+EGF)相比单独的生长因子(bFGF+EGF)或GM1,可更为高效地促进BMSCs诱导分化为神经元样细胞。
Objective: To investigate whether monosialoteterahexosyl ganglioside (GM1) combined with growth factor (bFGF + EGF) can promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into Neuron-like cells, so as to provide experimental evidence for alternative cell therapy for ischemic stroke. Methods: Bone marrow mononuclear cells (BMMNC) were obtained by density gradient centrifugation. Primary passage and subculture were performed. The third generation BMSCs were obtained and their surface markers were identified by flow cytometry. Their differentiation potential was also identified. Group B: GM1 alone; Group C: GM1 combined with (bFGF + EGF); Group D: control group . The positive expression rates of Nestin, β-Tubulin Ⅲ and GFAP in each group were detected by parallel immunocytochemistry before and after formal induction. Results: The surface markers of BMSCs were CD29 (+), CD105 (+), CD34 (-) and CD45 (-). Flow cytometry showed that BMSCs differentiated into osteoblasts and adipocytes. The expression of Nestin, β-Tubulin and GFAP in all four groups of cells were negative before BMSCs were induced into neuron-like cells. At 2 and 4 days after induction, the expression of Nestin positive cells in groups A, B and C showed an upward trend, and C Group A, B and D were higher than those in group A, B and D. When induced for 6-8 days, the expression of Nestin positive cells in groups A, B and C were decreased, while the expression of β-TubulinⅢ and GFAP positive cells was significantly increased , And C group than A, B group increased significantly. Comprehensive experimental data, C group Nestin, β-Tubulin Ⅲ, GFAP positive cells were higher than the A, B, D group. CONCLUSION: GM1 combined with bFGF + EGF can promote the differentiation of BMSCs into neuron-like cells more efficiently than bFGF + EGF alone or GM1.