Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways
Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways
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DOI：
10.1167/iovs.04-0363
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发表时间：
2005-04-01
影响因子：
4.4
通讯作者：
Agarwal, N
中科院分区：
文献类型：
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作者：
Charles, I;Khalyfa, A;Agarwal, N
目的。在从培养基中去除血清后，在缺乏生长因子的培养的大鼠视网膜神经节细胞（RGC-5）系中研究了细胞凋亡相关的信号通路。方法。将RGC-5细胞进行血清剥夺2至6天，并与在含有10%胎牛血清的生长培养基中培养的RGC-5细胞进行比较。通过中性红染料摄取测定法测定细胞活力。 RGC-5细胞的凋亡通过DNA梯状法建立。通过免疫印迹分析和/或逆转录聚合酶链反应（RT-PCR）分析研究各种凋亡相关基因的表达。通过生化方法测定细胞的氧化还原状态，包括通过电泳迁移率凝胶位移测定 (EMSA) 测定 NF-kappa B 结合活性，并使用活细胞共聚焦显微镜通过 JC-1（5,5', 6,6'-四氯 1,1', 3,3'-四乙基苯并咪唑基-碘化碳青）染色测定线粒体损伤 和细胞色素 c 的胞质释放。结果。中性红染料摄取测定表明，血清剥夺 2 天后，明显有 50% 的细胞损失。根据 DNA 梯形图确定，这种细胞损失是由于细胞凋亡所致。细胞裂解物中丙二醛 (MDA) 的增加和还原型谷胱甘肽 (GSH) 水平的降低表明，去血清的 RGC-5 细胞的氧化状态受到干扰。 RGC-5细胞的凋亡与caspase-3、-8和-9的激活以及Bax水平升高相关，同时Bcl-2水平和NF-κB（NF-κB）结合活性相应降低。细胞色素 c 的胞质释放和垂死 RGC-5 细胞线粒体的 JC-1 染色显示，血清剥夺也与线粒体功能丧失相关。结论。综上所述，这些结果表明血清撤除通过线粒体途径诱导 RGC-5 细胞凋亡。这些研究导致推测，神经营养素逆行转运阻断引起的生长因子剥夺可能涉及青光眼中视网膜神经节细胞死亡的类似机制。
PURPOSE. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium.METHODS. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappa B binding activity by electrophoretic mobility gel shift assays ( EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1', 3,3'-tetraethylbenzimidazolyl- carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c.RESULTS. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, - 8, and - 9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappa B (NF-kappa B) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells.CONCLUSIONS. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.