Frassetto Laura J, Schlieve Christopher R, Lieven Christopher J, Utter Amy A, Jones Mathew V, Agarwal Neeraj, Levin Leonard A
Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, WI 53792, USA.
Invest Ophthalmol Vis Sci. 2006 Jan;47(1):427-38. doi: 10.1167/iovs.05-0340.
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.
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 muM staurosporine and assessed for a variety of differentiation markers.
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.
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.
细胞系常用于阐明疾病病理生理学机制。然而，将细胞系的研究结果外推至神经退行性疾病却很困难，因为它们具有有丝分裂能力且通常具有其他非神经元特性。RGC - 5细胞系具有视网膜神经节细胞（RGCs）的许多特征。尽管它如原代RGCs一样表达Thy - 1和NMDA受体，但该细胞系的增殖能力和非神经元外观使其有别于其他中枢神经元，这使其在用于神经元存活、电生理学或神经突延伸研究时变得复杂。
确定了一种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 μM的星形孢菌素处理培养物，并评估多种分化标志物。
分化后的RGC - 5细胞表达了众多神经元特性，包括增殖停止而不诱导凋亡、诱导神经元形态、上调神经元标志物以及建立外向整流通道。基于对多种激酶磷酸化靶点的分析以及尝试用多种特异性激酶抑制剂复制分化过程，发现分化并不依赖于单一的激酶依赖性途径。
这种从增殖细胞系产生类RGC细胞的方法有助于开展以下以前不切实际的技术：对具有神经保护作用或影响离子通道的药物进行高通量筛选；通过非病毒转染技术在中枢神经元中直接进行基因表达转导，包括产生稳定转染子；对纯类RGC细胞进行生化及其他检测，而无需基于细胞表面抗原或解剖位置进行纯化。
