人神经干细胞在宿主鼠脑内分化成神经元呈脑位点特征

目的研究人胎脑神经干细胞植入年幼大鼠脑后的成神经元分化作用，探讨神经干细胞替代治疗小儿脑病的可行性。方法自孕16周的胎脑组织分离培养出神经干细胞球，在小儿脑脊液中培养诱导分化以证明其分化潜能。将培养14d的神经干细胞球移植10d龄鼠的侧脑室内，于移植后第4、7、14天杀鼠取脑，切片，行人神经丝特异性标志的免疫荧光分析，显示神经元的分布和细胞形态。结果培养获得典型神经干细胞球，呈漂浮生长，在小儿脑脊液中能分化为神经元、星形胶质细胞和少突胶质细胞。用抗人神经丝混合单抗检测移植物的成神经元分化，移植后的第4天，观察到阳性反应细胞在颗粒层表现为颗粒性细胞，锥体细胞层则出现长突起的锥体细胞，还有连接神经元样中间神经元，小脑内有单层排列的浦肯野细胞。对比各时间点的观察结果，阳性细胞分布位置未变。随着移植后天数的后延，阳性细胞数量呈减少趋势，但锥体细胞的突起明显加长。结论体外培养获得的人神经干细胞脑室途径移植年幼大鼠，在脑内发生迁移，并分化成形态上与其临近宿主细胞一致的神经元。提示宿主脑组织局部微环境在引导移植物分化成神经元中发挥了重要作用。

Site Specific Neuronal Differentiation of Human Neural Stem Cells in Host Rat Brains

Objective To investigate the neuronal differentiation of the neural stem cells derived from a human fetal brain after transplanted into young rats' brains.To study the possibility of cell-replacement therapy for children's brain disorders with neural stem cells.Methods The neural stem cell spheres were cultured out from the fetal brain tissues of 16 week gestation.The differentiation multipotency of the neurosphere was identified when cultured in children cerebrospinal fluid(CSF).The neurospheres cultured in vitro for fourteen days were injected into the lateral ventricles of young rats of 10 days old.The rats were killed at 4d,7d,and 14d post-transplantation.The brains were sectioned,and the special immuno-fluorescent assays were performed using anti-human neurofilament(anti-hNF) to show the location and morphology of graft neurons.Results Typical floating neurospheres were obtained,with the potency to differentiate into neurons,astrocytes and oligodendrocytes.The neuronal differentiation of grafts was detected with the mixture of three monoclonal antibodies against human neurofilament.Four days after transplantation,the positive staining cells within the granule cell layer of cerebral cortex were shown in the shape of granule cells,or within the pyramid cell layer in the shape of pyramid cells with long processes,and the interneuron-like cells also were watched.The Purkinje cells arranging in a monolayer row were detected in the cerebellum.At the different time points of 4d,7d,14d after injection,the graft cells were less and the processes were longer with the time extending while the location of grafts were the same.Conclusion The in vitro cultured neurosphere cells can migrated into brain tissues and differentiated into site-specific neurons in shape after transplanted into the lateral ventricles of young rats,suggesting that the environment of host brain tissue plays an important role in guiding the graft differentiation into neurons.