方法：人视网膜色素上皮细胞(human retinal pigment epithelium，hRPE)体外常规培养和传代，取3～6代细胞用于实验。将培养的人RPE细胞分为常氧对照组和缺氧处理组，常氧对照组用DMEM完全培养液培养细胞，缺氧处理组培养液中含有终质量浓度为200μmol/L CoCl₂，分别观察缺氧后4、6、8、12、24h后终止。采用细胞免疫荧光染色技术及Western-blot方法鉴定人RPE细胞中NOX4的表达以及缺氧不同时间对NOX4表达的影响。

结果：常氧对照组及缺氧处理组人RPE细胞生长良好，形态呈梭形。缺氧处理组细胞的形态及排列方式与空白对照组相比无明显改变。免疫荧光染色显示，常氧对照组RPE细胞内有少量的NOX4表达，细胞质呈绿色荧光，细胞核呈蓝色荧光； 缺氧处理后NOX4的表达量增加，一直持续到缺氧后期； Western-bolt显示缺氧条件下人RPE细胞中NOX4的表达明显增加，与缺氧时间呈正比，与常氧对照组相比，差异具有统计学意义(P<0.05)；

结论：NADPH氧化酶在人RPE细胞中有表达，缺氧条件下NOX4的表达显著提高，且与时间呈正比。提示NADPH氧化酶可能在脉络膜新生血管形成中发挥重要调节作用。

METHODS:Human RPE cells were cultured and passaged, and 3-6 generation cells were used in the experiment. The experiment was divided into normoxia control group and hypoxia group. The human RPE cells in normoxia control group were routinely cultured. The culture medium containing 200μmol/L CoCl₂ was used to establish the hypoxia model of human RPE cells cultured in vitro for 0,4,6,8,12 and 24h, and the RPE cells cultured normoxia were as controls. The effects of different time of hypoxia on the expression of NOX4 were identified by Immunofluorescence staining and Western-blot assay.

RESULTS:RPE cells grew well in the control group and hypoxia group. The morphology was spindle. The morphology and arrangement of cells in hypoxia group had no significant change compared with the control group. Immunofluorescence staining showed that there was a small amount of NOX4 expression in the RPE cells of the normoxia control group. Cytoplasm showed green fluorescence while the nucleus was blue. The expression of NOX4 increased after hypoxia, and continued until the late stage of hypoxia. Western-bolt results showed that the expression level of NOX4 under hypoxic conditions in RPE cells increased significantly, and is proportional to the hypoxia time, the difference was statistically significant(P<0.05).

CONCLUSION: NADPH oxidase 4 in normal human RPE cells have a small amount of expression, hypoxia can significantly increase the expression of NOX4, and is proportional to the time. It suggests that NADPH oxidase may play an important role in the formation of choroidal neovascularization.