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目的:探讨通过化学诱导的方法,诱导脐带间充质干细胞分化为类许旺细胞,为组织工程神经寻找一种新的种子细胞来源。方法:通过酶消化法,分离获得原代脐带间充质干细胞。体外培养至第3代(P3),以1×103/cm2接种于培养瓶中,待细胞长至亚融合状态,吸弃培养液,加入含β-巯基乙醇的培养液预诱导24 h。然后,加入含有全反式维甲酸的培养液进一步诱导72 h。最后,加入含有胶质细胞生长因子的培养液,作用两周。对诱导后的脐带间充质干细胞用免疫荧光及RT-PCR法,进行形态观察和表型鉴定。结果:经过上述诱导过程,脐带间充质干细胞的形态,由开始时的扁平、片状、多极,类似成纤维细胞状,逐渐变为长梭形,双极或三极,类似许旺细胞。同时,表达许旺细胞特异性标志S-100、P75;而且S100,P75的m RNA水平也明显上调,并且可以观测到GFAP的m RNA条带。结论:化学诱导法,可以将脐带间充质干细胞诱导分化为类许旺细胞,有望为组织工程神经提供一种新的许旺细胞来源。
OBJECTIVE: To explore a method for inducing umbilical cord mesenchymal stem cells to differentiate into Schwann cells through chemical induction and find a new source of seed cells for tissue engineering nerves. Methods: Primary umbilical cord mesenchymal stem cells were isolated by enzyme digestion. The cells were in vitro cultured to the third generation (P3) and inoculated into culture flasks at 1 × 10 3 / cm 2. After the cells were grown to the sub-fusion state, the medium was aspirated and the medium was pre-induced with β-mercaptoethanol for 24 h. Then, the medium containing all-trans retinoic acid was further induced for 72 h. Finally, adding glial cell growth factor-containing medium for two weeks. The induced umbilical cord mesenchymal stem cells were observed by immunofluorescence and RT-PCR method for morphological observation and phenotypic identification. Results: After the above induction process, the morphology of umbilical cord mesenchymal stem cells changed from flat, flake, multipolar and fibroblast-like at the beginning to fusiform, bipolar or tripolar, similar to Schwann cells . At the same time, the Schwann cell specific markers S-100 and P75 were expressed. Moreover, the m RNA level of S100 and P75 was also significantly up-regulated, and the m RNA band of GFAP was observed. Conclusion: The chemical induction method can differentiate umbilical cord mesenchymal stem cells into Schwann cells, which is expected to provide a new source of Schwann cells for tissue engineering nerves.