Recharacterization of the RGC-5 Retinal Ganglion Cell Line
Recharacterization of the RGC-5 Retinal Ganglion Cell Line
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DOI：
10.1167/iovs.09-3484
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发表时间：
2009-09-01
影响因子：
4.4
通讯作者：
Crowston, Jonathan G.
中科院分区：
文献类型：
--
作者：
Van Bergen, Nicole J.;Wood, John P. M.;Crowston, Jonathan G.
目的。转化后的RGC-5视网膜神经节细胞系广泛应用于青光眼研究。在已发表的文献中，对谷氨酸的抗性增加导致了对RGC-5细胞系的重新鉴定。方法：通过对核Thy1基因的一个区域进行测序，以及对d-loop和tRNAPhe基因的一个区域进行线粒体DNA测序，对RGC-5细胞系进行了表征。检测未分化细胞标志物的表达，用50mU g/mL琥珀酰刀豆球蛋白A(S刀豆蛋白A)诱导分化3d。结果：3-(4，5-Dimethylthiazol-2-yl)-2，5-diphenyltetrazolium-5细胞经线粒体和细胞核DNA分析发现，RGC5细胞来源于小鼠(Mus Musculus)而不是大鼠(Rattus Norveicus)。在第二个实验室测得的RGC-5 DNA序列随后被发现来自肌肉分枝杆菌。细胞对神经元标志物β-微管蛋白和PGP9.5以及微管相关蛋白tau呈阳性染色，但对神经节细胞的已知标志物如神经丝或Thy1.2则不阳性，这表明它们可能代表了小鼠神经元前体细胞的谱系。与S刀豆蛋白A诱导分化不会增加RGC-5对谷氨酸兴奋毒性的敏感性，也不会增加视网膜或神经节细胞标志物蛋白的表达。结论：使用RGC-5细胞的研究者在考虑将其用作视网膜神经节样细胞之前，应确认其为大鼠来源和视网膜特异性标志物的表达。(投资眼科VS科学。2009年；50：42674272)doi：10.1167/iovs.09-3484
PURPOSE. The transformed RGC-5 retinal ganglion cell line is used widely in glaucoma research. Increased resistance to glutamate was noted in published literature and led to the recharacterization of the RGC-5 cell line.METHODS. Characterization of the RGC-5 cell line was performed by sequencing of a region of the nuclear Thy1 gene and mitochondrial DNA sequencing of a region of the d-loop and tRNAPhe gene. Marker expression was examined in undifferentiated cells, and cells differentiated with 50 mu g/mL succinyl concanavalin A (S Con A) for 3 days. Glutamate sensitivity was examined in undifferentiated and S Con A differentiated cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24-hours of glutamate treatment.RESULTS. RGC-5 cells were found to be of mouse (Mus musculus), not rat (Rattus norvegicus), origin by mitochondrial and nuclear DNA analyses. RGC-5 DNA sequenced in a second laboratory was subsequently found to be of M. musculus origin. Cells stained positively for the neuronal markers beta-tubulin and PGP9.5 and for the microtubule-associated protein tau, but not for known markers of ganglion cells such as neurofilaments or Thy1.2, suggesting that they likely represented a lineage of mouse neuronal precursor cells. Differentiation with S Con A did not increase RGC-5 sensitivity to glutamate excitotoxicity or increase the expression of retinal or ganglion cell marker proteins.CONCLUSIONS. Investigators using cells designated as RGC-5 should confirm the species to be of rat origin and retinal-specific marker expression before considering their use as retinal ganglion-like cells. (Invest Ophthalmol Vis Sci. 2009; 50: 4267-4272) DOI: 10.1167/iovs.09-3484