Kinase-dependent differentiation of a retinal ganglion cell precursor
Kinase-dependent differentiation of a retinal ganglion cell precursor
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DOI：
10.1167/iovs.05-0340
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Levin, LA
中科院分区：
文献类型：
--
作者：
Frassetto, LJ;Schlieve, CR;Levin, LA
目的.细胞系常用于阐明疾病的病理生理机制。然而，将细胞系的结果外推到神经退行性疾病是困难的，因为它们是有丝分裂的，并且通常具有其他非神经元性质。RGC-5细胞系具有视网膜神经节细胞（RGC）的许多特征。尽管其表达Thy-1和NMDA受体，如在原发性RGC中发现的，但该细胞系的增殖能力和非神经元外观将其与其他中枢神经元区分开来，使其用于神经元存活、电生理学或神经突延伸的研究变得复杂。鉴定了使用非特异性蛋白激酶抑制剂星形孢菌素分化RGC-5细胞的方法。用100 nM至3.16 μ M星形孢菌素处理培养物，并评估各种分化标志物。分化的RGC-5细胞表达了许多神经元特性，包括在不诱导凋亡的情况下抑制增殖、诱导神经元形态、上调神经元标志物和建立外向整流通道。分化不依赖于单一的激酶依赖性途径，基于分析多个激酶磷酸化靶点，并尝试用多个特异性激酶底物复制分化。这种从增殖细胞系中产生RGC样细胞的方法有利于以下以前不切实际的技术：高通量筛选神经保护剂或影响离子通道的试剂;通过非病毒转染技术直接转导中枢神经元中的基因表达，包括产生稳定的转染子;在没有基于细胞表面抗原或解剖位置纯化的情况下，对纯的RGC样细胞进行生物化学和其它测定。
PURPOSE. Cell lines are frequently used to elucidate mechanisms of disease pathophysiology. Yet extrapolation of results with cell lines to neurodegenerative disorders is difficult because they are mitotic and usually have other non-neuronal properties. The RGC-5 cell line has many features of retinal ganglion cells (RGCs). Despite its expression of Thy-1 and NMDA receptors, as found in primary RGCs, this line's ability to proliferate and non-neuronal appearance differentiate it from other central neurons, complicating its use for the study of neuronal survival, electrophysiology, or neurite extension.METHODS. A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 mu M staurosporine and assessed for a variety of differentiation markers.RESULTS. Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors.CONCLUSIONS. This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.