人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。

THE GROWTH CHARACTERISTIC OF HUMAN NEURAL STEM CELLS IN VITRO

To assess the human neural stem cell (NSCs) induced by mitogens in vitro and investigate their growth and differentiation character istic. The cells from the human fetal cerebral hemispheres at 10 - 22 weeks of gestation were placed into growth medium treated wiih epidermal growth factor (EOF, 20ng/ml) and basic fibroblast growth factor (bFGF, 20ng/ml) alone or in combination. The cell types were identified by immunochemistry method. The cell clone, passaging and telomerase activity were also analyzed and detected. NSCs induced by EGF and/or bFGF grew in culture as free - floating spheres (neurospheres) and were immunoreactive for the intermediate filament nestin. NSC can proliferate and be passaged without losing their proliferative and multilineage potential. After removing EGF and bFGF, the cells cease mitosis and can be induced to differentiate into neurons, astrocytes, and oligodendrocytes. But EGF- induced NSC were more restricted to a glia lineage fate and FGF2 - induced NSC had a greater capacity for neuronal differentiation. Clonal growth was density dependent (no growth below 1000 cell/ml) and thus single cell cloning was not accomplished. Human NSC express very low levels of telomerase activity and was decreased with the prolongation of culture time. The growth and differentiation of NSC can be regulated by extrinsic and intrinsic fators, related mechanism remain to be elucidated.