人胎脑神经干细胞体外培养及分化研究

目的研究人胚胎神经干细胞体外长期培养的条件、不同部位脑组织神经干细胞数量及其分化能力和特点。方法分离人胚胎海马、皮质、室周、纹状体脑组织，培养在含碱性成纤维细胞生长因子（b-FGF）、表皮生长因子（EGF）、B27、N2掭加剂的无血清培养基中，形成神经球，用有限稀释法进行克隆、传代。计算神经球数量，用溴脱氧尿苷（BrdU）标记神经球，使用免疫细胞化学法鉴定神经干细胞自主分化能力。结粜人胚胎海马、皮质、室周、纹状体部位脑组织均能培养出具有自我增殖能力的神经干细胞，其中海马所含神经干细胞最丰富。室周次之。BrdU检测有正在分裂、增殖的细胞。液氮冻存6个月的细胞复苏后仍具增殖分化能力。细胞贴壁分化后可形成Nestin、胶质原性纤维酸性蛋白（GFAP）、微管相关蛋白2（MAP2）表达阳性细胞。结论胚胎脑组织具有丰富的神经干细胞。具有自我更新和增殖能力，可长期培养。含b-FOF、EGF的无血清培养基能促进神经干细胞连续稳定增殖，保持神经干细胞特性。分离培养的神经干细胞可向神经元、胶质细胞分化。

Study on in vitro Culture and Differentiation of Neural Stem Cells from Human Embryonic Brain

Objective To study the conditions of invitro culture,quantities of neural stem cells(NSCs) derived from different location brain,and abilities of differentiation of NSCs from human embryonic hippocampus.Methods NSCs were isolated from human embryonic hippocampus,cortex,sub-ependymal layer,corpus striatum.The cells were cultured and formed neuro-spheres in serum-free medium with basic fibroblast growth factor(b-FGF),epidermal growth factor(EGF),B27 and N2 supplement.Then neuro-spheres were cloned and transferred proliferated by limited dilution.The neuro-spheres were labeled with BrdU.Differentiation happened while neuro-spheres adhered to dish wall.These cells were for observation of cell mitosis and proliferation.The NSCs and default differentiated cells were distinguished by immunocytochemisty.Results NSCs from human embryonic brain had ability of proliferation.Hippocampus was most abundant of neural stem cells;the following was sub-ependymal layer.They could be cultured for passage and differentiate toward multiple directions.The cells labeled with BrdU had ability of mitosis and proliferation;these cells stored in liquid nitrogen had ability,too.Nestin, glid fiboillary acidic protein(GFAP),and microtubule associated protein 2(MAP2) expressing cells were identified after adhesion and differentiation via immunocytochemisty.Conclusions Embryonic brain is rich of neural stem cells which have ability of self-renewal and proliferation,and remained ability when they are cultured in serum-free medium with EGF,b-FGF in vitro lasting a long time.They are able to differentiate into neurons and astrocytes.