人重组表皮生长因子和牛磺酸体外诱导人脐血干细胞分化为神经元样细胞

目的 探讨人重组表皮生长因子(rhEGF)和牛磺酸体外诱导人脐血间充质干细胞(MSC)分化为神经元样细胞的实验条件和可能机制.方法 用含有105U/L青霉素、100 mg/L链霉素、10%胎牛血清(FBS)、5%自体血浆、4 mmol/L-谷氨酰胺、30 ng/ml rhEGF的Dulbeceo改良Eagle培养基与F12以1:1混合(DMEM/F-12)培养液对人脐血MSC进行原代培养,传至第3代后改用含牛磺酸的培养基进行诱导.流式细胞仪检测细胞表面抗原CD90、CD29、CD34、CD44及CD45的表达.免疫细胞化学方法观察神经元特异性烯醇化酶(NSE)、视紫红质、巢蛋白的阳性细胞表达情况.统计学方法采用两组资料的卡方检验分析NSE、视察红质、巢蛋白的表达.结果 脐血MSC首次培养5～7 d,有间充质样细胞贴壁,3～4周后细胞达80%～90%融合,传代后生长加速;形态学类似骨髓源性MSC.脐血MSC均一稳定地表达MSC的相关抗原CD29、CD44、CD90,不表达CD34、CD45.经牛磺酸体外诱导后的脐血MSC中,3120个细胞中有2515个细胞表达神经元特异性烯醇化酶(NSE),3050个细胞中有903个细胞表达巢蛋白,3175个细胞中有1168个细胞表达视紫红质;未诱导组的2965个脐血MSC中仅有234个细胞表达NSE,其他抗原未见表达.两组间NSE、巢蛋白和视紫红质的表达差异具有统计学意义(χ2=3 242.9,1 073.0,1 444.5;P值均<0.01).结论 人脐血MSC经rhEGF和牛磺酸诱导后能使部分MSC向神经元样细胞或视紫红质阳性细胞分化.

Recombined human epidermal growth factor and taurine induce human umbilical cord blood derived mesenchymal stem cells differentiating into neuron-like cells

Objective To investigate the experimental condition and mechanism of differentiation of human umbilical cord blood derived mesenchymal stem cells(hUCB-MSC)into neuron-like cells induced by recombined human epidermal growth factor (rhEGF) and taurine in vitro.Methods hUCB-MSC were primary cultured in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12)which supplemented with 105U/L penicillin G,100 mg/L streptomycin sulfate,10% fetal bovine serum (FBS),5% autologous plasma,4 mmol L-glutamine,30 ng/ml rhEGF.The DMEM/F-12 medium was replaced by taurine medium after 3 passages.The expression of surface antigen CD90.CD29.CD34.CD44 and CD45 were detected by flow cytometry;the expression of neuron specific enolase,rhodopsin and nestin were investigated by immunoeytochemistry.The statistical method was chi square test.Results Morphologically similar to bonemarrow MSC,hUCB-MSC became attached cells after the first 5 to 7 days in culture,and reached 80% to 90% confluent after 3 to 4 weeks.Growth accelerated after passage,hUCB-MSC were positive for CD29,CD44 and CD90 but negative for CD34 and CD45.After taurine induction,2515/3120 cells expressed NSE,1168/3175 cells expressed rhodopsin and 903/3050 cells expressed nestin while only 234/2965 cells expressed NSE in the control group (P<0.01).Conclusion rhEGF and taurine can induce hUCB-MSC differentiating into neuron-like or rhodopsin positive cells.