Effect of oxidative stress on RPE and its pertinence to the pathogenesis of AMD

实验目的和方法： 将从另外三个供体获得的培养的hfRPE细胞用于测定。将细胞与叔丁基氢过氧化物（TBH）（Sigma）在含有MEM-α、改良培养基（Sigma-Aldrich）、谷氨酰胺-青霉素-链霉素（Sigma-Aldrich）1：100 mL/mL和非必需氨基酸溶液（Sigma-Aldrich）1：100 mL/mL的无血清培养基中孵育不同时间点。在TBH治疗前或治疗后立即测量跨上皮电位（TER）。使用活-死测定来确定细胞死亡的水平，并且用配备有高分辨率AxioCam照相机（Axioplan 2，具有Axiovision 3.4软件AxioCam-MRm，Carl Zeiss Meditec，Inc.，都柏林，CA）。在细胞死亡定量的情况下，通过MosaiX Axiovision模块对插入物的整个过滤器进行成像;通过配备ITCN（基于图像的细胞核计数工具）的NIH Image J 13.6对阳性细胞核的数量进行计数。对于慢性损伤过程，将非致死浓度的TBH应用于培养物长达一周，将测定TBH对TER的影响，并使用荧光染料-JC-1（分子探针，Invitrogen）监测线粒体电位。用Western blot和免疫细胞化学方法检测连接蛋白表达和分布的变化。TBH和细胞因子（其他免疫调节剂）在RPE损伤中的潜在相互作用也将被研究。 结果如下： TBH在hfRPE中诱导剂量依赖性细胞死亡和TER降低。当应用超过6小时时，致死浓度的TBH处理产生了强烈的细胞死亡。毒性接触率的降低似乎是渐进的。细胞死亡水平和TER降低似乎相关。 结论：虽然致死浓度的氧化刺激物（例如TBH）可以引起hfRPE中的细胞死亡，但急性和强烈的过程不可能模仿年龄相关性黄斑变性（AMD）中发生的过程。用低水平的氧化剂或/和炎症刺激（与AMD相关的两种类型的风险因素）治疗hfRPE将采用相关的损伤过程。

Experimental purpose and approaches: Cultured hfRPE cells obtained from three more donors were used for the assay. The cells were incubated with Tert-butyl-hydroperoxide (TBH) (Sigma) in serum free culture media containing MEM-alpha; modified medium (Sigma-Aldrich), glutamine-penicillin-streptomycin (Sigma-Aldrich) 1:100 mL/mL, and nonessential amino acid solution (Sigma-Aldrich) 1:100 mL/mL for various time points. Trans epithelial potential (TER) was measured prior to or immediately after the treatment of TBH. Live-dead assay was used to determine the level of cell death and fluorescent images were captured with a microscope equipped with high resolution AxioCam camera (Axioplan 2 with Axiovision 3.4 software AxioCam-MRm, Carl Zeiss Meditec, Inc., Dublin, CA). In case of cell death quantification, the whole filter from insert was imaged through MosaiX Axiovision module; the numbers of positive nuclei were counted via NIH Image J 13.6 equipped with ITCN (image based tool for counting nuclei). For chronic injury process, nonlethal concentrations of TBH will be applied to the culture for up to a week, the impact of TBH on TER will be assayed and mitochondrial potential will be monitored using the fluorescent dye-JC-1 (molecular probes, invitrogen). Changes of expression and distribution o f junction proteins will be examined by Western blot analysis and immunocytochemistry. Potential interaction of TBH and cytokines(other immunoregulators)in RPE injury will be studied as well. Results: TBH induced a dose dependent cell death and TER reduction in hfRPE. Lethal concentrations of TBH treatment produced a robust cell death when applied for more than 6 hours. TER reduction appears to be gradual events. The level of cell death and TER reduction appear to be associated. Conclusion: Although lethal concentrations of oxidative stimulant, such as TBH can elicit cell death in hfRPE, the acute and robust process doesn't possibly mimics the process as occurred in age-related -macular degeneration (AMD). Treating hfRPE with low levels of oxidant or/and inflammatory stimulations, two types of risk factors that are associated AMD, will employ an relevant injury process.