脊髓神经管神经上皮干细胞的分离培养和诱导分化

ISOLATION AND INDUCED DIFFERENTIATION OF NEUROEPITHELIAL STEM CELLS FROM SPINAL CORD NEURAL TUBE

目的 从胚胎大鼠脊髓神经管中分离神经上皮干细胞并诱导其向多巴胺能神经元方向分化。 方法 利用无血清悬浮培养、单细胞克隆技术分离神经上皮干细胞 ;采用 5 溴 2 脱氧尿苷 (BrdU)标记新生细胞 ,免疫细胞化学单标或双标染色技术 ,检测神经上皮干细胞蛋白 (nestin)和分化后特异性神经细胞抗原的表达 ;用纹状体组织提取液 ,诱导神经上皮干细胞向多巴胺能神经元方向分化。 结果 从胚胎大鼠脊髓神经管中分离的细胞可以连续传代 ,表达nestin ,它们分化后可以表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原 ;与对照组 3%相比 ,纹状体组织提取液可以诱导这些细胞中的 12 %分化成为多巴胺能神经元。 结论 分离自脊髓神经管的细胞具有自我更新能力和多分化潜能 (multipotent) ,是增殖能力很强的神经干细胞 ;这些干细胞在一定的体外环境中能被诱导成为特定的神经元 。

Objective To isolate neuroepithelial stem cells from the spinal cord neural tube of the embryonic rat and induce them to differentiate into dopaminergic neurons. Methods Serum free cells suspension culture and single cell cloning technique were used to isolate neuroepithelial stem cells. 5 bromo 2 deoxyuridine(BrdU) to label new cells combined with single or double immunocytochemistry staining to detect nestin antigen before differentiation and neural cell specific antigens after differentiation, such as neurofilament (NFM 160?kD), glial fibrillary acidic protein(GFAP), galactocerebroside(GalC) and tyrosine hydroxylase(TH). Striatal extracts were used to induce neuroepithelial stem cells to differentiate into dopaminergic neurons. Results The cells isolated from the spinal cord neural tube of the embryonic rat expressed nestin antigen. They had the potential to serially passage and differentiate into neurons, astrocytes and oligodendrocytes. Striatal extracts could induce 12% of them to differentiate into dopaminergic neurons compared with 3% in controls.Conclusion The cells, which express nestin antigen, isolated from neural tube are multipotent and have the ability to self renew, therefore, they are neural stem cells. These stem cells can be induced to differentiate into specific neurons in vitro. Which can provide materials for neural transplantation.