Charles Irma, Khalyfa Abdelnaby, Kumar D Maneesh, Krishnamoorthy Raghu R, Roque Rouel S, Cooper Nigel, Agarwal Neeraj
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas, USA.
Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1330-8. doi: 10.1167/iovs.04-0363.
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.
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-kappaB 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.
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-kappaB (NF-kappaB) 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.
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.
在从培养基中撤出血清后剥夺生长因子的培养大鼠视网膜神经节细胞（RGC-5）系中研究凋亡相关信号通路。
将RGC-5细胞进行2至6天的血清剥夺，并与在含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试验测定细胞活力。通过DNA梯状条带分析确定RGC-5细胞的凋亡。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。通过生化方法确定细胞的氧化还原状态，包括通过电泳迁移率凝胶迁移试验（EMSA）测定NF-κB结合活性以及使用活细胞共聚焦显微镜和细胞色素c的胞质释放通过JC-1（5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基-羰花青碘化物）染色测定线粒体损伤。
中性红染料摄取试验表明，血清剥夺2天后明显有50%的细胞丢失。如DNA梯状条带分析所确定的，这种细胞丢失是由于凋亡性细胞死亡。细胞裂解物中丙二醛（MDA）增加和还原型谷胱甘肽（GSH）水平降低表明，血清剥夺的RGC-5细胞的氧化状态受到干扰。RGC-5细胞的凋亡与半胱天冬酶-3、-8和-9的激活相关，并且Bax水平升高，同时Bcl-2水平和NF-κB（NF-κB）结合活性相应降低。如垂死的RGC-5细胞的细胞色素c胞质释放和线粒体的JC-1染色所揭示的，血清剥夺还与线粒体功能丧失相关。
综上所述，这些结果表明血清撤出通过线粒体途径诱导RGC-5细胞发生凋亡性细胞死亡。这些研究引发了这样的推测，即神经营养因子逆行运输受阻导致的生长因子剥夺可能涉及青光眼视网膜神经节细胞死亡的类似机制。
