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背景:人脐带Wharton’s Jelly源间充质干细胞避免了伦理的限制,来源丰富,可以作为种子细胞进行组织修复。目的:观察体外诱导脐带Wharton’s Jelly中间充质干细胞向许旺细胞分化的可行性。方法:分离、培养脐带Wharton’s Jelly中间充质干细胞,流式细胞术鉴定细胞表面标志。利用神经细胞培养基、碱性成纤维生长因子、表皮生长因子、维甲酸、血小板源性生长因子等采用两步法将脐带间充质干细胞诱导分化为许旺细胞,倒置显微镜下观察细胞形态变化。利用免疫细胞化学染色法检测巢蛋白、S-100、纤维酸性蛋白的表达,反转录-聚合酶链反应、免疫印记技术检测许旺细胞特异性蛋白产物表达。结果与结论:脐带细胞培养第7天形态发生变化,部分细胞变成梭形。原代细胞培养10d左右可达80%～90%融合,细胞呈梭形。分离培养的细胞表达具有间充质干细胞表面特有标志:CD44(91.4%),CD29(91.3%),CD105(99.2%),不表达CD34(0.2%),CD45(0.9%),CD14(0.6%)。脐带Wharton’s Jelly中间充质干细胞经第一阶段诱导后,细胞由短梭形变成长梭形或纺锤形,并出现聚集现象,由形状规则、表面圆滑的球形细胞团形成。第二阶段诱导后,有长梭形细胞从球形细胞团爬出,96h后细胞形态多为长梭形,伴有多极现象。免疫细胞化学染色结果示:长梭形多极细胞具有许旺细胞特异的纤维酸性蛋白、S100蛋白染色。结果表明脐带Wharton’s Jelly中间充质干细胞可在体外诱导分化为许旺细胞。
Background: Human umbilical cord Wharton’s Jelly source mesenchymal stem cells avoid ethical limitations and are rich in sources that can be used as seed cells for tissue repair. OBJECTIVE: To observe the feasibility of differentiation of Wharton’s Jelly mesenchymal stem cells into Schwann cells in vitro. Methods: Wharton’s Jelly mesenchymal stem cells were isolated and cultured, and cell surface markers were identified by flow cytometry. Umbilical cord mesenchymal stem cells were induced to differentiate into Schwann cells by using neural cell culture medium, basic fibroblast growth factor, epidermal growth factor, retinoic acid and platelet-derived growth factor by two-step method. The morphological changes of cells were observed under an inverted microscope . Immunocytochemical staining was used to detect the expression of Nestin, S-100, fibrillary acidic protein, reverse transcription-polymerase chain reaction and Western blotting to detect the expression of Schwann cell specific protein. RESULTS AND CONCLUSION: The morphology of umbilical cord cells was changed on the 7th day and part of cells became fusiform. Primary cell culture up to about 10d about 80% to 90% fusion, spindle-shaped cells. The cells cultured in isolation expressed CD44 (91.4%), CD29 (91.3%), CD105 (99.2%), no CD34 (0.2%), CD45 (0.9%), CD14 ). Umbilical cord Wharton’s Jelly mesenchymal stem cells after induction by the first phase, the cells from the short spindle deformation spindle fusiform or spindle shape, and the phenomenon of aggregation, the shape of regular, smooth surface spherical cell mass formed. After induction in the second phase, there were long spindle cells that climbed out from the globular cell mass. After 96 hours, the cells were mostly fusiform with multipolarity. Immunocytochemical staining results show: long spindle-shaped multipolar cells with Schwann cell specific fibrillary acidic protein, S100 protein staining. The results show that the umbilical cord Wharton’s Jelly mesenchymal stem cells can be induced to differentiate into Schwann cells in vitro.