目的 探索新生SD大鼠神经干细胞体外分离培养基鉴定的方法。方法 分离新生SD大鼠海马组织, 无血清培养技术培养神经干细胞, 免疫组织化学技术检测其巢蛋白的表达;并用溴脱氧尿嘧啶核苷掺入试验, 免疫荧光双标技术观测神经干细胞的增殖状况;诱导神经干细胞分化, 分别用神经元特异性烯醇化酶抗体和胶质纤维酸性蛋白抗体进行免疫组织化学技术鉴定分化细胞。结果 获得大量悬浮生长的单克隆神经球, 并可以分化为神经元和星形胶质细胞。结论 成功培养的新生SD大鼠神经干细胞具有增殖、自我更新和多分化潜能, 为体外基础和临床研究奠定基础。
Abstract
Objective To investigate the cultivation, identification and identification cultivation of the neural stem cells (NSCs) from neonatal rats. Methods Hippocampus was isolated from neonatal SD rats and NSCs were cultured in serum free medium.Nestin expression in NSCs was detected by immunohistochemical fluorescence.The proliferation of NSCs was assessed by both of 5-bromodeoxyuridine (5-BrdU) incorporation assay and double-marked immunofluorescence.The differentiation of NSCs was induced.To identify the differentiated NSCs, antibodies of neuron-specific enolase (NSE) and antibody of glial fibrillary acidic protein (GFAP) were employed respectively with immunohistochemical fluorescence staining. Results A mass of neurospheres were obtained.Those NSCs could differentiate into neurons and astrocytes. Conclusions The fetal rat NSCs are successfully obtained, which have the capacities of proliferation, self-renew and pluripotentiality.They could differentiate into neurons and astrocytes, lay the foundation for the basic and clinical research in vitro.
关键词
新生鼠 /
神经干细胞 /
细胞培养 /
免疫细胞化学
Key words
neonatal rat /
neural stem cells /
cell culture /
immunohistochemistry
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