人脐带间充质干细胞定向诱导分化成类神经元的实验研究

Differentiation of mesenchymal stem cells derived from human umbilical cord

目的:采用不同细胞因子定向诱导人脐带间充质干细胞分化成类神经元,为脐带间充质干细胞移植治疗脊髓损伤提供理论依据。方法:采用Ⅱ型胶原酶消化法分离培养人脐带间充质干细胞,采用流式细胞术鉴定细胞。取第5代细胞随机分成A、B、C、D 4组,在A组基础培养基中加入b FGF,B组中加入b FGF和BDNF,C组中加入b FGF、BDNF和BHA诱导培养,D组为对照组,使用含有10%胎牛血清的DMEM-F12培养基培养。诱导2周后采用实时荧光定量PCR检测细胞的nestin、NEFH和GFAP mRNA表达情况,并通过原子力显微镜观察细胞超微结构的的变化。结果:Ⅱ型胶原酶消化法能成功分离人脐带间充质干细胞。通过流式细胞术检测第3代细胞发现,细胞均强表达CD29、CD44和CD105,而CD34、CD45和HLA-DR均未见表达。经定向诱导分化成类神经元后,原子力显微镜观察细胞表面突起增多,实时荧光定量PCR检测显示nestin在A、B、C组均呈阳性表达,NEFH在A、B组呈阳性表达,而GFAP在A、B、C、D 4组均不表达。A、B组n EFH和nestin表达具有显著差异(P﹤0.05)。结论:本实验成功分离培养人脐带间充质干细胞,所培养的细胞扩增迅速,生物学性状稳定;与b FGF单独处理相比,b FGF联合BDNF更能有效诱导人脐带间充质干细胞分化成类神经元。

AIM： To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchymal stem cells（ h UCMSCs） into neuron-like cells and to provide some evidence for the transplantation of h UCMSCs for spinal cord injury. METHODS： The h UCMSCs were isolated from human umbilical cord digested with collagenase Ⅱ. The h UCMSCs was verified by flow cytometry analysis. The passage 5 cells were randomly divided into 4 groups. The differentiation of h UCMSCs was induced by b FGF in group A,b FGF and BDNF in group B,or BHA,b FGF and BDNF in group C,while the cells in group D served as a control group cultured with DMEM-F12 and 10% FBS. Two weeks later,the expression of nestin,neurofilament protein H（ NEFH） and glial fibrillary acidic protein（ GFAP） was detected by realtime PCR and immunocytochemistry. The morphological changes of cells were observed under an atomic force microscope.RESULTS： Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion. h UCMSCs expressed CD29,CD44 and CD105,but no CD34,CD45 or HLA-DR. After cultured with inducing medium for 2 weeks,the cells were successfully induced into neuron-like cells. The appearance of the cells had great change. The induced h UCMSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope. The result of real-time PCR showed that nestin was positive in A,B and C groups,and NEFH was positive in A and B groups,but GFAP was negative in 4 groups. The difference of nestin and NEFH expression among the induced groups was significant（ P〈0. 05）. CONCLUSION： Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion in vitro,and all the h UCMACs presented stable biological properties. Moreover,h UCMSCs were induced to differentiate into neuron-like cells in vitro via b FGF combined with BDNF.