NeuroD1基因过表达对大鼠神经干细胞增殖和分化的影响

The influence of overexpression of NeuroD1 on proliferation and differentiation of rat neural stem cells

【目的】神经干细胞(neural stem cells,NSCs)是一群能自我更新并具有多种分化潜能的细胞,可分化成神经元、少突胶质细胞和星形细胞。NSCs分化受多种转录因子调控网络的影响,本实验旨在研究神经分化因子1(neuronal differentiation1,Neuro D1)过表达对NSCs增殖和分化的影响。【方法】分离培养原代大鼠NSCs,免疫荧光染色鉴定NSCs。将携带Neuro D1基因的逆转录病毒转染NSCs,QPCR检测Neuro D1过表达神经干细胞系的构建。QPCR和MTT检测过表达Neuro D1对NSCs增殖和分化的影响;光镜下观察分化细胞形态变化。【结果】第3代NSCs经免疫荧光染色证实所培养细胞为NSCs,可分化为神经元和星形胶质细胞。QPCR结果显示Neuro D1 m RNA在NSCs-Neuro D1组表达最高,与其他两组有统计学差异(P<0.05)。QPCR结果显示MAP2在NSCs-Neuro D1组表达最高,与其他两组有统计学差异(P<0.05)。MTT结果显示NSCs-Neuro D1组细胞增殖水平最高,与其他两组有统计学差异(P<0.05)。光镜下可见NSCs-Neuro D1组细胞多分化为神经元细胞。【结论】成功构建Neuro D1过表达神经干细胞系。Neuro D1可促进NSCs增殖和向神经元方向分化。

[Objective] Neural stem ceils （NSCs） is a group of cells with self-renewing and muhiple differentiation potential, and it can differentiate into neurons, oligodendrocytes and astrocytes. The differentiation of NSCs is affected by multiple transcription factors. This experiment aims to study the role of NeuroD1 overexpression on proliferation and differentiation of NSCs. [Methods]The rat NSCs were isolated and cultured. NSCs were identified with immunofluorescence staining. NeuroD 1 was overexpressed in NSCs via retroviral transfection technique, NeuroD1 overexpression NSCs were confirmed by QPCR. The influence of NeuroDl on the proliferation and differentiation of NSCs was detected with QPCR and MTT. Morphological changes and neuronal axon length were observed under light microscope. [Results] The cultured cells were identified to be NSCs and it can differentiate into neuron and astrocyte cells. The group NSCs-NeuroD1 is the highest on NeuroD1 mRNA expression by QPCR, and it had significant difference with the other two groups （P〈 0.05）. The group NSCs-NeuroD1 is the highest on MAP2 mRNA expression by QPCR, and it had significant difference with the other two groups （P〈0.05） MTT results showed that the proliferation of NSCs-NeuroD 1 group was increased, and it had significant difference with the other two groups （P〈0.05）. [ Conclusion ] NeuroD1 overexpression NSCs line were constructed successfully. Overexpression of NeuroD1 can promote the proliferation and differentiation of neural stem cells into neuron.