Department of Ophthalmology, Duke University Eye Center, Durham, North Carolina 27710, USA.
Invest Ophthalmol Vis Sci. 2011 Aug 17;52(9):6545-51. doi: 10.1167/iovs.10-6772.
PURPOSE. Retinal pigment epithelial (RPE) cell survival plays a critical role in normal physiology and in retinal diseases, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). We have previously demonstrated that Bcl-x(L) is an important cell survival protein in human RPE (hRPE) cells. Herein, we determined the role of Bcl-x(L) as a survival protein in mouse RPE (mRPE) cells. METHODS. Survival factor gene expression and Bcl-x(L) protein distribution were determined using qRT-PCR and immunohistochemistry, respectively. Cultured mRPE cells were transfected with two modified 2'-O-methoxyethoxy antisense oligonucleotides (ASOs): Bcl-x(L)-mismatched control and Bcl-x(L)-specific. Bcl-x(L) protein levels were analyzed using Western blot. To determine the effects of survival factor regulation in mRPE cells, cultured cells were treated for 24 hours with mouse TNF-α, human IL-1β, and human TNF-α. RESULTS. Bcl-x(L) was the most highly expressed survival factor in both mouse eyecup and cultured mRPE cells, whereas Bax was the most highly expressed antisurvival factor. Bcl-x(L) was expressed in the RPE layer, and the distribution among the retinal layers was similar to that observed in human eyecups. IL-1β and TNF-α had minimal effect on Bcl-x(L) and Bax expression and strongly upregulated Traf-1. Transfection with Bcl-x(L)-specific ASO resulted in markedly diminished Bcl-x(L) gene expression, Bcl-x(L) protein levels, and cell number. CONCLUSIONS. Bcl-x(L) is the most highly expressed survival gene in mRPE cells and is essential for mRPE cell survival. Our data suggest that mouse tissue is an appropriate model for investigations of RPE survival factor genes.
目的。视网膜色素上皮 (RPE) 细胞的存活在正常生理和视网膜疾病中起着关键作用，如年龄相关性黄斑变性 (AMD) 和增生性玻璃体视网膜病变 (PVR)。我们之前已经证明 Bcl-x(L) 是人类 RPE (hRPE) 细胞中重要的细胞存活蛋白。在此，我们确定了 Bcl-x(L) 在小鼠 RPE (mRPE) 细胞中作为存活蛋白的作用。方法。使用 qRT-PCR 和免疫组织化学分别确定存活因子基因表达和 Bcl-x(L) 蛋白分布。用两种修饰的 2'-O-甲氧基乙氧基反义寡核苷酸 (ASO) 转染培养的 mRPE 细胞：Bcl-x(L)-错配对照和 Bcl-x(L)-特异性。使用 Western blot 分析 Bcl-x(L) 蛋白水平。为了确定存活因子调节对 mRPE 细胞的影响，用小鼠 TNF-α、人 IL-1β 和人 TNF-α 处理培养的细胞 24 小时。结果。Bcl-x(L) 是小鼠眼杯中和培养的 mRPE 细胞中表达最高的存活因子，而 Bax 是表达最高的抗存活因子。Bcl-x(L) 在 RPE 层中表达，在视网膜层中的分布与在人眼杯中观察到的分布相似。IL-1β 和 TNF-α 对 Bcl-x(L) 和 Bax 表达的影响很小，但强烈地上调了 Traf-1。用 Bcl-x(L)-特异性 ASO 转染导致 Bcl-x(L) 基因表达、Bcl-x(L) 蛋白水平和细胞数量明显减少。结论。Bcl-x(L) 是 mRPE 细胞中表达最高的存活基因，对 mRPE 细胞存活至关重要。我们的数据表明，小鼠组织是研究 RPE 存活因子基因的合适模型。
