大鼠胚胎神经干细胞定位及干细胞克隆实验研究  

Experimental stating study on local cloning of embryinic neural stem cell of rat

作　　者：马以骝[1] 杨志敏[1] 王秦秦 余化霖[1] 金丽昆[1] 王义[1] 李玉[1] 刘文春[1] 

机构地区：[1]昆明医学院第一附属医院神经外科,650032 [2]云南省肿瘤研究所

出　　处：《立体定向和功能性神经外科杂志》2001年第2期63-69,共7页Chinese Journal of Stereotactic and Functional Neurosurgery

基　　金：云南省自然科研基金资助课题

摘　　要：目的 :探索大鼠神经干细胞在胚胎脑内的分布特点 ,并用三种不同的方法对干细胞进行克隆化培养。方法 :对胚龄 16天左右的大鼠胚胎脑组织做连续冠状位冰冻切片 ,特异的抗nestin免疫组化染色 ,以显示干细胞在胚胎的分布特点。我们还把胚龄 16天左右的胎鼠室管膜下脑进行原代及传代培养 ,传代的神经干细胞用三种方法有限稀释法 ,饲细胞克隆培养法 ,软琼脂克隆培养法进行克隆培养。结果 :1 神经干细胞在胚胎的分布主要集中在胚胎发育的中轴部位如 :脑室、脑室下区 ,中脑导水管周围。神经干细胞多表现为复层上皮样排列 ,细胞突起呈放射状伸向脑的表层并之与保持密切联系。有的干细胞在离室管膜较远处呈簇状分布好似群状迁移的干细胞群也好似干细胞的发生中心。 2 干细胞的原代培养及传代培养显示 :神经细胞易呈聚集状生长的特点及神经细胞的迁移性的生长特点 ,各细胞群之间有密切的纤维联系。 3 神经细胞生长的细胞密度依赖效应与细胞之间的接触在生长发育中的重要作用。结论 :神经干细胞在胚胎的分布主要集中在胚胎发育的中轴部位 ,软琼脂克隆法是一种成功的干细胞克隆方法。Objectiue:To study the method of the cloning of embryonic neural stem cell.Methods:The embryinic brain at E16 in rat was sectioned at coronal section,at 30 μm thick.The charcter of disrtribution of embryonic neural progenitor cell was revealed by special anti-nestin immunocytochemistry.Embryonic rat beain tissue at E16 from subventral zone was used in primary culture and subculture;furthemore.There methods of cell cloning (limited-dilute, feeder cell,agar-culture system)were applied in subculture cell.Result:① The distribution of progenitors was mainly aggregated on intermediate sturcture of developing embryonic brain,such as ventricle,subventral zone and ambient tissue of mesocephalomic dust.Neural progenitor cells were organized as striatified epithelail arrangement on subventral zone (SZ). Cell processed radially extended form SZ to embryinic brain tissue surface and kept close relation to brain tissue.Clusters of nueral progenitor cells were observed in the brain parenchyma,as if they settled down on the migration way,or they were cell generative centres;②Primary culture and subculture of progenitors showde that noncolonal aggregates rather than colonies were formed.Clusters of progenitors closely cintactes by their processes.Migrations of neural cell occured along neural fibre; ③Experimental study on neural stem cell revealed that in vitro cell density effects were observed in these cultrue systems.Conclusion:The cell colonic method of agar-medium culture stsyem was the best among the three methods.

关 键 词：大鼠 胚胎 神经干细胞 定位 克隆 培养 

分 类 号：R329.1[医药卫生—人体解剖和组织胚胎学]