GM1体外诱导人脐带血间充质干细胞分化为神经样细胞研究

目的 研究单唾液酸四己糖神经节苷脂通过体外诱导人脐带间充质干细胞分化为神经样细胞并探索其最佳诱导浓度.方法 取足月妊娠剖宫产健康胎儿的脐带,剔除脐动脉和脐静脉,用酶消化法获得MSCs,贴壁培养,采用流式细胞技术行细胞表型检测.扩增后,取第4代细胞,分为A、B、C、D、E5组,前4组为实验组,E组为对照组,实验组各组GM1浓度分别为:A组50μg/mL、B组100μg/mL、C组150μg/mL、D组200μg/mL,诱导液用L-DMEM培养基配制,对照组仅使用L-DMEM培养基.观察5组诱导人脐带血间质干细胞向神经样细胞分化的作用,每60min观察细胞诱导前后的形态,在第360min时采用免疫细胞染色法检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经丝蛋白(neurofilaments protein H,NF-H)以及神经元特异性标记物微管结合蛋白-2(microtubule associated protein 2,MAP-2)的表达情况,并计算阳性细胞率.结果 ①大部分原代细胞在接种9h后贴壁,呈多边形、菱形,之后变为长梭形,细胞开始以漩涡、放射状生长.②P3、P5和P10代细胞表面的分子标记经流式细胞术检测发现,均表达CD105、CD90和CD73,却不表达HLA-DR、CD19、CD11b、CD45以及CD34.③诱导至第360分钟后,实验组各组细胞均有神经样细胞出现,表现为细胞胞体收缩成椭圆形,伸出较长突起,以双极细胞居多.免疫细胞化学检测显示实验组各组NF-H、MAP-2表达阳性,胶质纤维酸性蛋白GFAP表达为阴性,C组(150μg/mL)细胞NF-H、MAP-2阳性表达率最高.对照组细胞诱导前后变化不明显.结论 GM1可以诱人脐带间充质干细胞向神经样细胞分化,150μg/mL为最合适诱导剂量.

Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells into Neuron Like Cells in Vitro Induced by GM1

Objective To study the monosialo four hexose ganglioside (GM1) induced human umbilical cord mesenchymal stem cells into neuron like cells and to explore the optimal concentration in vitro.Methods The umbilical cord,umbilical artery and umbilical vein were collected from the healthy fetus in full term pregnancy.The MSCs was obtained by enzyme digestion method and the cell phenotype was determined by flow cytometry.After amplification,the fourth generation cells were divided into A,B,C,D,E five groups.The first 4 groups were the experimental groups,E as control group.The experimental group cells were treated with different concentrations of GM1,group A 50μg/mL,B 100μg/mL group and C group 150μg/mL group D 200μg/mL.Inducing medium was diluted by L-DMEM,the control group was only treated with L-DMEM medium.The morphology of 5 groups was observed and compared every 60 min.At 360 min,the expression of glial fibrillary acidic protein (GFAP) and neurofilament protein (neurofilaments protein H,NF-H) and neuron specific markers of microtubule-associated protein 2 (MAP-2) was examined by immunohistochemistry,and calculate the rate of positive cells.Results Most of the primary cells were adherent at 9h after inoculation.The cells evolved from polygonal and diamond shapes to spindle shape,and then into spiral and radial shape.Expression of CD105 and CD73,but not HLA-DR,CD90,CD19,CD11b,CD45,CD34,and P3 were detected by flow cytometry.The molecular markers of P5 and P10 on cell surface were detected by flow cytometry.At 360th minutes after induction,the cells of the experimental group were found to be neuron like cells,which showed that the cell bodies were contracted into oval shape,extending out longer processes,and the bipolar cells were the majority.The expression of NF-H and MAP-2 were detected in the experimental group,whereas GFAP was negative.NF-H and MAP-2 in C group (150μg/mL) was at highest rate.Conclusion monosialo four hexose ganglioside,GM1 can induce umbilical cord mesenchymal stem cells to differentiate into neuron like cells.The concentration of 150μg/mL was the most appropriate induction dose.