无血清培养条件诱导人脐血间充质干细胞向神经细胞的分化

Differentiation of human umbilical cord blood mesenchymal stem cells into neural cells under serum-free culture conditions

于2005-06/2007-09在南昌大学第二附属医院江西省分子医学重点实验室拟建立一种在无血清培养条件下诱导脐血间充质干细胞向神经细胞分化的方法。所用无血清培养基血清替代物是将纯化人转铁蛋白直接溶于DMEM培养基中,再将去毒后的BSA、超声波粉碎后的胆固醇、胰岛素及过氧化氢酶直接加入DMEM培养基中稀释至所需浓度。体外分离的脐血单个核细胞以1×109L-1接种,加入含氢化可的松、纤维连接蛋白的无血清培养基,7d后消化传代。取传至2,5,8代的脐血间充质干细胞,达60%～70%融合时以含β-巯基乙醇、丁化羟基苯甲醚的无血清培养基诱导其向神经细胞分化。结果在无血清条件下能培养扩增出大量脐血间充质干细胞,融合后可继续传代扩增;不表达CD34、CD13和CD45,强表达CD166、CD29、CD105,较强表达CD54,80%以上细胞处于G0/G1期;脐血间充质干细胞经诱导后,能形成具有典型神经元形态的神经细胞,神经元特异性烯醇化酶、神经丝蛋白、神经胶质元纤维酸性蛋白均呈阳性表达。提示在自行研制的无血清培养条件下可成功诱导脐血间充质干细胞向神经细胞分化。

A method of inducing mesenchymal stem cells （MSCs） derived from umbilical cord blood to differentiate into neural cells under serum-free conditions in vitro was established at the Molecular Medical Key Laboratory, Second Affiliated Hospital, Nanchang University from June 2005 to September 2007. The serum-free medium is obtained by： the purified human transferring was directly dissolved in the DMEM. Following detoxicating, BSA, cholesterol, insulin and hydrogen peroxidase were directly added in the DMEM, and diluted to a needed concentration. In vitro harvested mononuclear cells at 1×10^9/L were incubated in serum-free medium, supplemented with hydrocortisone and fibronectin. Seven days later, cells were passaged. At the second, fifth and eighth passages, 60%-70% confluent cells were induced to differentiate into neural cells by serum-free medium, supplemented with β -mercaptoethanol and butyl-hydroxyanisol. It is possible to culture, passage and harvest large quantities of umbilical MSCs under sreum-free conditions. Umbilical MSCs were negative for CD34, CD13 and CD45, but strongly positive for specific surface markers such as CD166, CD29, CD106 and CD54. The percentage of umbilical MSCs at G0-G1 cell cycle was 80%. After being induced, umbilical MSCs cultured under serum-free conditions showed a relatively high commitment to neuronal fate and were strongly positive for neural cells specific surface markers such as neuron specific enolase, neurofilament protein and glial fibrillary acidic protein. The above results showed that umbilical MSCs could be induced to differentiate to neural cells under self-made serum-free culture conditions.