人胚脑神经干细胞的分离培养、克隆和分化

探讨从人胚脑分离培养的神经干细胞在增殖和分化方面的生物学特点。用无血清培养技术从 4月龄人胎中脑组织中分离培养出神经干细胞 ,用有限稀释法获得单细胞克隆球 ,消化后用含 Brd U的培养液培养 ,待形成神经干细胞球后 ,进行 Brd U和nestin免疫荧光检测。取第 4代神经干细胞球用含 10 % FBS的培养液诱导分化 ,3周后分别进行神经元、星形胶质细胞和少突胶质细胞特异性标记物 MAP-2、GFAP和 CNP免疫荧光检测。结果显示 ,单细胞悬液培养 2周后可形成神经干细胞球 ;神经球 Br-d U和 nestin免疫荧光检测均呈阳性 ;神经干细胞分化后呈 MAP-2、GFAP和 CNP阳性的三种类型细胞 ,但分化的神经元数量较少、胞体较小、突起较少。提示从人胚中脑组织中分离得到的神经干细胞具有增殖和自我更新能力 ,并具有分化为神经元、星形胶质细胞和少突胶质细胞的多分化潜能 ;与啮齿类动物相比 ,人神经干细胞增殖速度较慢 ,分化的神经元较少且稍欠成熟

ISOLATION,CLONING AND DIFFERENTIATION OF NEURAL STEM CELLS FROM HUMAN FETAL BRAIN

To study the biological characteristics in proliferation and differentiation of human neural stem cells(hNSCs), the population of hNSCs was isolated from 4 month-old human fetal midbrain using serum-free medium. The single-cell clone sphere obtained by limiting dilution method was dissociated into single cells. These cells proliferated in the serum-free medium containing BrdU. After the neurospheres formed, immunofluorescence detection of BrdU and nestin were employed. The 4th passage of neurospheres were induced to differentiate in the medium containing 10% FBS. In order to determine the phenotype of the differentiated cells, the expressions of MAP-2, GFAP and CNP(the distinctive marker for neuron, astrocyte and oligodendrocyte respectively) were detected by immunofluorescence labeling technique after 3 weeks. The results showed that single cell suspension became neurospheres after 2 weeks' culturing; The neurospheres exhibited both nestin and BrdU positive; The differentiated cells expressed MAP-2, GFAP and CNP, respectively, but the number of MAP-2 positive neurons was less, the size of cell body was smaller and the processes were scarce. These results suggest that neural stem cells isolated from human fetal mescencephalon have the strong ability in proliferating and self-renewing, and have multipotential ability to differentiate into neurons,astrocytes and oligodendrocytes. Comparing with the rodent counterpart, the velocity of hNSCs proliferation is slower, and the differentiated neurons are less and slightly immature.