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

摘要

AIM:To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchy-mal stem cells (hUCMSCs) into neuron-like cells and to provide some evidence for the transplantation of hUCMSCs for spi-nal cord injury .METHODS:The hUCMSCs were isolated from human umbilical cord digested with collagenase Ⅱ.The hUCMSCs was verified by flow cytometry analysis .The passage 5 cells were randomly divided into 4 groups.The differentiation of hUCMSCs was induced by bFGF in group A , bFGF and BDNF in group B, or BHA, bFGF 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 real-time 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 .hUCMSCs 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 hUC-MSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope .The re-sult 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 signifi -cant (P<0.05).CONCLUSION:Mesenchymal stem cells were isolated and cultured from human umbilical cord by en-zyme digestion in vitro, and all the hUCMACs presented stable biological properties .Moreover, hUCMSCs were induced to differentiate into neuron-like cells in vitro via bFGF combined with BDNF .%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bFGF，B组中加入bFGF和BDNF，C组中加入bFGF、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nEFH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bFGF单独处理相比，bFGF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。