Characterization of a transformed rat retinal ganglion cell line (Retracted article. See vol. 1544, pg. 62, 2014)
Characterization of a transformed rat retinal ganglion cell line (Retracted article. See vol. 1544, pg. 62, 2014)
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DOI：
10.1016/s0169-328x(00)00224-2
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发表时间：
2001-01-31
期刊：
影响因子：
--
通讯作者：
Agarwal, N
中科院分区：
文献类型：
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作者：
Krishnamoorthy, RR;Agarwal, P;Agarwal, N
本研究的目的是通过转化大鼠视网膜细胞建立大鼠视网膜神经节细胞系。在这项研究中，从出生后第 1 天 (PN1) 大鼠中分离出视网膜细胞，并用 psi2 E1A 病毒进行转化。为了分离视网膜神经节细胞（RGC），从转化细胞中随机选择单细胞克隆。使用逆转录酶聚合酶链式反应 (RT-PCR)、免疫印迹和免疫细胞化学研究了 Thy-1（RGC 标记）、胶质纤维酸性蛋白（GFAP，Muller 细胞阳性标记）、HPC-1/syntaxin（无长突细胞标记）、8A1（水平细胞和神经节细胞标记）和神经营养因子的表达。其中一个视网膜细胞克隆，称为 RGC-5，Thy-1、Brn-3C、Neuritin、NMDA 受体、GABA-B 受体和突触素表达呈阳性，而 GFAP、HPC-1 和 8A1 呈阴性，表明它代表了假定的 RGC 克隆。 RT-PCR 分析的结果通过 Thy-1 和 GFAP 的免疫细胞化学得到证实。通过免疫印迹进一步表征，RGC-5 克隆 Thy-1 呈阳性，GFAP、8A1 和突触融合蛋白呈阴性。 RGC 5 细胞神经营养因子及其同源受体的表达也呈阳性。为了确定 RGC-5 的生理相关性，分析了血清/营养因子剥夺和谷氨酸毒性的影响，以确定这些细胞是否会发生凋亡。还研究了血清剥夺后神经营养因子对 RGC-5 的保护作用。通过末端脱氧核苷酸转移酶介导的荧光素化 dUTP 缺口末端标记 (TUNEL) 研究细胞凋亡。血清剥夺导致细胞凋亡，在生长培养基中补充 BDNF 和 NT-4，可以保护 RGC-5 细胞免于凋亡。在用琥珀酰刀豆球蛋白 A (sConA) 进行分化时，RGC-5 细胞对谷氨酸毒性变得敏感，而这种毒性可以通过包含 ciplizone (MK801) 来逆转。总之，基于 Thy-1 和 Brn-3C 的表达及其对谷氨酸兴奋毒性和神经营养素撤退的敏感性，转化的大鼠视网膜细胞系 RGC-5 具有视网膜神经节细胞的某些特征。这些细胞对于理解视网膜神经节细胞生物学和生理学（包括青光眼实验模型的体外操作）可能有价值。 (C) 2001 Elsevier Science B.V. 保留所有权利。
The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavaIin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma. (C) 2001 Elsevier Science B.V. All rights reserved.