背景：以往相关报道大多为成人或者动物骨髓间质干细胞，关于儿童骨髓间质干细胞体外培养及定向分化的研究相对较少。 目的：探讨儿童骨髓间质干细胞诱导分化为神经干细胞及神经元细胞的能力。 方法：取传12代儿童骨髓间质干细胞，加入预诱导液(含体积分数为10%胎牛血清及1mmol/L β-巯基乙醇的DMEM培养液)、诱导液(含2%二甲基亚砜及150μmol/L丁基羟基茴香醚的DMEM培养液)进行定向诱导分化。诱导后30 min，7 d，免疫细胞化学检测神经干细胞特异性抗原nestin、神经丝蛋白β-tublinⅢ的表达，诱导后0，5.5，6 d，RT-PCR检测nestin mRNA的表达。 结果与结论：①经二甲基亚砜、丁基羟基茴香醚联合诱导之后，细胞胞体逐渐收缩为锥形和多角形、类圆形，胞体伸出突起并逐渐延长为细丝状。联合诱导至第6天，相邻细胞突起发生交互，形成网状；②诱导30 min后，nestin抗原表达呈阳性，诱导7 d后，β-tublinⅢ表达呈阳性；③诱导开始时(0 h)未出现nestin mRNA表达，诱导5.5 h时nestin mRNA表达呈阳性，诱导6 d时不再表达；④结果表明，小儿骨髓间质干细胞经二甲基亚砜、丁基羟基茴香醚体外联合诱导可分化为神经干细胞与神经元细胞。%BACKGROUND:In the past, the culture and differentiation of bone marrow mesenchymal stem celsin vitrowere mostly reported in the adult or animal rather than in children. OBJECTIVE: To explore the ability of bone marrow mesenchymal stem cels from children differentiating into neural stem cels and nerve cels. METHODS: Bone marrow mesenchymal stem cels from children were isolated and cultured, and passage 12 cels were cultured in the pre-induction medium (DMEM culture medium containing 10% fetal bovine serum and 1 mmol/L β-mercapto ethanol) and induction medium (DMEM containing 2% dimethyl sulfoxide and 150 μmol/L butylated hydroxyanisole). Expression of nestin and β-tublin III was detected using immunocytochemistry method at 30 minutes and 7 days after induction, while RT-PCR was used to detect nestin mRNA expression at 0, 5.5, 6 days after induction. RESULTS AND CONCLUSION: After combined induction, the cels shrank from round shape to tapered, polygonal or oval shape, and cel processes extended gradualy and became filament-like shape. Interconnected cels formed a network at 6 days after combined induction. The expression of nestin antigen was positive at 30 minutes after induction, while the expression of β-tublin was positive at 7 days. RT-PCR findings showed that positive expression of nestin mRNA was detected at 5.5 hours of induction, and then disappeared at 6 days. These findings show that the combined use of dimethyl sulfoxide and butylated hydroxyanisole can induce bone marrow mesenchymal stem cels from children to differentiate into neural stem cels and nerve cels in vitro.
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