NADPH氧化酶参与兔晶状体上皮细胞中超氧阴离子的生成

The Role of NADPH Oxidase in Superoxide Anion Generation in Rabbit Lens Epithelial Cells

目的:观察兔晶状体上皮细胞内是否存在内源性超氧阴离子生成机制。方法:用不同浓度的花生四烯酸作用于兔晶状体上皮细胞N/N1003A,利用化学发光检测仪测定超氧阴离子的浓度变化,观察超氧阴离子歧化酶(Superoxide anion dismutase,SOD)和NADPH氧化酶抑制剂DPI对超氧阴离子的影响;用RT-PCR方法检测NADPH氧化酶(NADPH oxidase)亚单位NOX家族在N/N1003A细胞中的表达。结果:花生四烯酸可以刺激兔晶状体上皮细胞N/N1003A迅速生成超氧阴离子,该活性氧能被SOD有效清除,黄素蛋白抑制剂DPI能显著抑制N/N1003A细胞生成超氧阴离子。RT-PCR结果表明,NOX蛋白的5种同源异构体[包括NOX1、NOX2(gp91phox)、NOX3、NOX4、NOX5]的mRNA在兔晶状体上皮细胞中都有表达。结论:NADPH氧化酶参与兔晶状体上皮细胞中超氧阴离子的产生,NOX蛋白5种同源异构体的mRNA都在N/N1003A细胞中表达。眼科学报2007;23:199-204.

Purpose： To identify the presence of endogenous superoxide anion-generating system in rabbit lens epithelial cells. Methods： The generation of superoxide anion was quantified by using lucigenin- amplyfied chemiluminescence （LUCL） in intact rabbit lens epithelial cells （N/N 1003A）. The LUCL readings were recorded with a luminometer （LumiStar BMG） immediately upon arachidonic acid （AA） addition. To test the effects of superoxide dismutase （SOD） and DPI （non-specific inhibitor to NADPH oxidase）, cells were preincubated for 30 minutes with indicated concentrations of inhibitors before stimulation with AA. The expression of NOX family, including NOX1, NOX2 or gp91^phox, NOX3, NOX4 and NOXS, was detected by RT-PCR. Results： AA at dosage of 30 -90 μM proportionally induced luminescence in N/N 1003A cells. Production of superoxide occurred quickly within 30 seconds of AA addition, reached the highest level after 200 -260 seconds and dissipated after 600 seconds. The amount of superoxide anions, expressed as relative light unit of luminescence （RLU）, was proportional to the concentration of AA used. Dosedependent effect could be seen. Superoxide generation was inhibited in N/N1003A cells preloaded with SOD. DPI eliminated AA-induced superoxide anion generation. RT-PCR using primers specific for mRNAs of the five isoforms of the NOX proteins documented that mRNA encoding NOX1 through NOX5 were constitutively present in N/N1003A cells. The expression of NOX1 was much weaker than that of the other four NOX isoforms. Conclusions： NADPH oxidase complex is involved in superoxide anion generation in rabbit lens epithelial cells. N/N 1003A cells constitutively produce mRNA encoding five isoforms of NOX proteins.