PROLIFERATION, DIFFERENTIATION, AND LONG-TERM CULTURE OF PRIMARY HIPPOCAMPAL-NEURONS
PROLIFERATION, DIFFERENTIATION, AND LONG-TERM CULTURE OF PRIMARY HIPPOCAMPAL-NEURONS
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DOI：
10.1073/pnas.90.8.3602
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
GAGE, FH
中科院分区：
文献类型：
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作者：
RAY, J;PETERSON, DA;GAGE, FH
初级胚胎海马神经元在培养中可以在形态和功能上发育，但存活时间不超过几周。据报道，碱性成纤维细胞生长因子（bFGF）促进胎儿海马神经元的存活和神经突伸长。我们报道bFGF以剂量依赖性的方式诱导胚胎海马祖神经元体外存活（50 pg至1 ng/ml）和增殖（10-20 ng/ml）。在含高浓度bFGF的无血清培养基中，神经元不仅增殖（4 d倍增时间）、形态分化，而且可以传代、连续生长。通过几种不同的神经元特异性标记物的免疫染色和详细的超微结构分析，证实了增殖细胞的神经元性质。利用bFGF的增殖作用产生几乎纯的神经细胞培养物，可以传代、冷冻、解冻和再次培养。神经元在培养中维持了50个月。建立长期原代神经元培养的能力提供了从中枢神经系统的特定区域分离不同神经元细胞类型的克隆系的可能性。这种长期的神经元培养在研究单个细胞水平的神经元以及探索体外神经元之间的相互作用方面应该证明是有价值的。观察到的剂量依赖性提出了不同水平bFGF可能影响体内细胞存活和增殖的可能性。
Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained >5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.