GM1联合生长因子诱导骨髓间充质干细胞分化为神经元样细胞的研究

Study on the differentiation of bone marrow mesenchymal stem cells into neuron-like cells by GM1 and growth factors

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 approach a way to more efficiently induce BMSCs（bone marrow mesenchymal stem cells） into neuron-like cells by GM1（monosialoteterahexosyl ganglioside） combined with growth factors（bFGF ＋ EGF） differentiatly and supply experimental evidence for cell replacement therapy to ischemia stroke.Methods：Bone marrow mononuclear cells were obtained by density gradient centrifugation,then primary culture and subculture was proceed,and surface markers of the third passage BMSCs were identified by flow cytometry.Meanwhile,pluripotency of BMSCs were identified.Subsequently,BMSCs were divided into four groups in order to induce into neuron-like cells with different inducers.Group A：（bFGF ＋ EGF） alone,group B：GM1 alone,group C：GM1 and growth factors（bFGF ＋ EGF） and group D：control group.Respectively,the positive rate of Nestin,β-Tubulin Ⅲ and GFAP were detected by using immunocytochemistry before and after induction and the data was statistically analyzed.Results：The positive rate of surface markers of BMSCs were CD29（＋）,CD105（＋）,CD34（-）,CD45（-） by flow cytometry.Meanwhile,the BMSCs induced into osteoblasts and adipocytes were detected.Before BMSCs induced into neuron-like cells,the rate of Nestin,β-Tubulin Ⅲand GFAP were negative in four group.On the 2nd to 4th day for induction,the Nestin positive rate of group A,B and C showed an upward trend,and that group C was much higher than group A,B and D.On the 6th to 8 th day for induction,the Nestin positive rate of group A,B and C decreased,however,the β-Tubulin Ⅲ and GFAP positive rate was detected significantly upward trend,and that group C was higher than group A and B.Comprehensive experimental data,the Nestin,β-Tubulin Ⅲ and GFAP positive rate of group C were higher than group A,B and D.Conclusion：GM1 combined with growth factors（bFGF ＋ EGF） group can more effectively induce BMSCs into neuron-like cells in comparison with growth factors（bFGF ＋ EGF） group or GM1 group.