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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.

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.