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目的以花生四烯酸为刺激因子,研究人晶状体上皮细胞中是否存在超氧阴离子生成体系。设计实验研究。研究对象人晶状体上皮细胞B3(HLE B3)。方法用花生四烯酸及其衍生物刺激HLE B3细胞,超氧阴离子用化学发光法定量检测。用超氧阴离子歧化酶和山梨醇与细胞预培养作为阴性对照;以3%乙醇(花生四烯酸及其衍生物的溶剂)刺激细胞作基线;测定超氧阴离子前,所有抑制剂先与细胞预培养30分钟。花生四烯酸对丝裂原激活蛋白激酶(MAPK)磷酸化影响的时间依赖实验和剂量依赖实验用蛋白质印迹杂交进行检测。主要指标超氧阴离子的含量,MAPK磷酸化的变化。结果30～150mM花生四烯酸能刺激HLE B3细胞迅速产生超氧阴离子,超氧阴离子歧化酶(SOD)和山梨醇抑制了该反应,尼克酰胺二磷酸腺苷(NADPH)氧化酶抑制剂DPI也抑制了超氧阴离子的产生。花生四烯酸的衍生物二十碳二烯酸、二十碳三烯酸以及硬脂酸、亚油酸不能引起超氧阴离子的产生。环氧合酶、细胞色素p450的抑制剂对超氧阴离子的产生无影响,提示花生四烯酸这两条代谢途径不参与超氧阴离子的生成。脂氧合酶,尤其是5-脂氧合酶的抑制剂完全阻断了该反应。30～150mM花生四烯酸能在2.5～30分钟瞬间激活ERK和JNK,并有剂量和时间依赖性。结论抑制超氧离子的生成可能是抑制品状体上皮细胞增殖、预防后发发障的新途径。
Aim To study the existence of superoxide anions in human lens epithelial cells with arachidonic acid as stimulating factor. Design experiment research. Study object Human lens epithelial cells B3 (HLE B3). Methods HLE B3 cells were stimulated with arachidonic acid and its derivatives, and the superoxide anion was quantified by chemiluminescence. Superoxide anion dismutase and sorbitol were used as negative controls with cells pre-culture; cells were stimulated with 3% ethanol (arachidonic acid and its derivatives) as baseline; before all superoxide anion was measured, all inhibitors were incubated with cells Pre-incubate for 30 minutes. Time-dependent and aa-dependent experiments of arachidonic acid on the phosphorylation of mitogen-activated protein kinase (MAPK) were performed by Western blotting. The main indicators of superoxide anion content, MAPK phosphorylation changes. Results 30 ~ 150mM arachidonic acid can stimulate the rapid generation of HLE B3 cells superoxide anion, superoxide anion dismutase (SOD) and sorbitol inhibition of the reaction, the NIH DPPH inhibitor of adenosine diphosphate (NADPH) oxidase Inhibit the production of superoxide anion. Arachidonic acid derivatives eicosadienoic acid, eicosatrienoic acid and stearic acid, linoleic acid can not cause superoxide anion generation. Cyclooxygenase and cytochrome p450 inhibitors had no effect on the production of superoxide anion, suggesting that the two metabolic pathways of arachidonic acid do not participate in the generation of superoxide anion. Inhibitors of lipoxygenase, especially 5-lipoxygenase, completely blocked this reaction. 30 ~ 150mM arachidonic acid can instantly activate ERK and JNK in 2.5-30 minutes in a dose-and time-dependent manner. Conclusion Inhibition of superoxide generation may be a new way to inhibit the proliferation of ECs and prevent the development of post-emergence syndrome.