Notch-1在骨髓间质干细胞分化为神经细胞中的作用

目的观察Notch-1信号在骨髓间质干细胞(MSC)分化为神经细胞中的作用。方法采用RNA干扰技术,构建小鼠Notch-1shRNA,转染小鼠MSC后诱导其分化为神经细胞;同时以未转染MSC和转染甘油醛-3-磷酸脱氢酶(GAPDH)shRNA为对照。采用免疫细胞化学染色和Western印迹检测巢蛋白、神经元特异性烯醇化酶(NSE)、神经微丝蛋白200(NF200)、胶质纤维酸性蛋白(GFAP)和Notch-1蛋白表达;原位末端标记染色评价细胞凋亡率。结果诱导6d后,转染Notch-1shRNA诱导后细胞形成较典型的神经细胞形态,同时高效表达巢蛋白、NSE和NF200等,不表达GFAP,对照组无此现象。但是转染mNotch-1shRNA后细胞凋亡率(13·3%±2·3%)显著高于对照组。结论MSC的横向分化过程可能与神经干细胞类似,阻断Notch信号通路,会增加MSC向神经细胞分化的效果。

Effects of Notch-1 signaling in bone marrow mesenchymal stem cells differentiating into neurons: experiment in vitro

OBJECTIVE: To investigate the role of Notch-1 signaling in bone marrow mesenchymal stem cells (MSCs) differentiating into neurons. METHODS: Mice Notch-1 small hairpin RNA (mNotch-1 shRNA) was constructed and transfected into the MSCs obtained from the tibiae of BALB/c mice. MSCs transfected with glyceraldehyde-3-phosphate hydoxygenase (GADPH) shRNA and untransfected MSCs were used as controls. The cell survival rate was detected by ELISA. The MSCs of different groups were cultured in Neurobasal-A medium so as to be induced to differentiate into neurons. Apoptosis of the MSCs was detected by TUNEL. RESULTS: After induction of 6 days the MSCs transfected with mNotch-1 shRNA displayed typical neuronal morphology and high expression of neuron-specific markers: nestin, neuron-specific enolase (NSE), neurofilament 200 (NF 200), and Notch-1 protein, however, gilal fibrillary acidic protein (GFAP), the glia-specific marker, was not detected. The percentage of apoptotic cells in the MSCs transfected with mNotch-1 shRNA was 13.3% +/- 2.3%, significantly higher than those of the MSCs transfected with mGAPH shRNA and untransfected MSCs (4.7% +/- 0.5% and 4.5% +/- 0.4%, both P < 0.01). CONCLUSION: Block of the Notch signal pathway may increase the differentiation of MSCs into neurons.