摘要
为建立成年活体嗅粘膜神经干细胞的简便、实用的体外培养方法 ,进而为神经干细胞自体移植治疗中枢神经系统损伤的研究提供更为安全的候选细胞 ,本实验自成年大鼠活体分离剪取部分嗅粘膜经胰酶消化制成细胞悬液 ,接种于玻璃培养皿 ,用含 10 %胎牛血清的 F12培养基培养 ,动态观察神经干细胞克隆球的形成及分化过程 ,并用 nestin、NSE、GFAP、vim entin及laminin等抗体作免疫细胞化学染色 ,对阳性细胞进行形态学鉴定。结果显示 ,成年大鼠经嗅粘膜活体取材后 ,可长期存活。成体嗅粘膜细胞混合体外培养时不同类型细胞的贴壁时间存在差异 ,神经干细胞呈球形 ,不直接贴附于培养皿 ,仅粘着在首先贴壁的扁平基底细胞上分裂形成克隆球 ,球内细胞 nestin免疫细胞化学染色阳性。原代培养 14 d后克隆球不再生长 ,球周细胞逐渐向外迁移分化为嗅细胞和嗅鞘细胞。提示 ,成体嗅粘膜神经干细胞可在普通培养基中形成干细胞克隆球 ,嗅粘膜中的其它细胞作为神经干细胞的基底饲养细胞有助于干细胞克隆球的形成 ;活体分离成体嗅粘膜作混合细胞体外培养获取神经干细胞的方法简便、安全、可行 ;本结果为嗅粘膜神经干细胞自体移植治疗中枢神经系统损伤的可行性研究提供了动物实验资料。
To offer the new donation of neural stem cells (NSCs) from the adult alive nasal olfactory mucous membrane for the study of NSCs transplantation reparing injured central nervous system(CNS), the simple and applicable culture method of the NSCs was established. The nasal olfactory mucous membrane was alive removed from the anaesthetic adult SD rats, the cells of the mucous membrane were then cultured in F12 medium containing 10% fetal bovin serum. The growth and morphologic characteristics of cultured NSCs and other cells were observed by nestin, NSE, GFAP, p75NGFR, vimentin and laminin immunocytochemistry. The animals suffering from operations could live for a long time. The cells of olfactory mucous membrane grew well in vitro. There was difference in attachements between the different cultured cell types from the nasal olfactory mucous membrane. The cells attached to the bottom of culture vessels first were flat and positive for vimentin and laminin immunocytochemistry. The NSCs were spherical and adhered to the flat subtrate cells then formed clonal spheres by mitosis. The NSCs and clonal spheres were positive for nestin immunocytochemistry. Cultured for 7 days, the clonal spheres ceased growing and the NSCs in clonal spheres migrated out then differentiated into olfactory neurones(positive for NSE)and olfactory ensheathing cells(positive for GFAP and p75NGFR). The above results demonstrate that the NSCs from the adult rat alive nasal olfactory mucosa can be cultured in common medium, and the other type cells as substrate feeder cells may promote the NSCs to grow and differentiate. The NSCs harvested by means of the foregoing culture method can be used as donative cells for autologous transplantation to repare the injured CNS.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2004年第4期371-376,共6页
Chinese Journal of Neuroanatomy
基金
江苏省教育厅自然科学基金 (0 2 kzd3 2 0 0 0 4)资助项目
