EPO对氧化损伤的人视网膜色素上皮细胞bcl-2表达的影响

Effect of erythropoietin on the expression of bcl-2 in human RPE induced by oxidative injuries

目的研究实验性氧化损伤的人视网膜色素上皮(RPE)细胞中凋亡相关基因(bcl-2)表达的变化及促红细胞生成素(EPO)对bcl-2基因表达的影响。方法制备RPE细胞氧化损伤模型,加入40U/ml的重组人促红细胞生成素(rhEPO)共同孵育,采用免疫细胞化学法检测bcl-2蛋白的表达,RT-PCR测定bcl-2mRNA的表达量。结果bcl-2在正常RPE细胞有弱表达,在氧化损伤2、8、24h其表达水平依次降低,氧化损伤组在各时间点与正常组比较差异均有统计学意义(P<0.05)。EPO组bcl-2表达变化规律与氧化损伤组一致,但较氧化损伤组明显增加(P<0.05)。bcl-2mRNA表达量与bcl-2蛋白表达的变化趋势一致。结论bcl-2参与视网膜氧化损伤的机制,EPO促进bcl-2在氧化损伤的RPE细胞中的表达。

Objective Retinal pigment epithelium （RPE） situate in the outer layer of retina and is easily injured by oxidation. This research was to investigate the effect of erythropoietin （EPO） on the expression of apoptosis-inhibiting gene, bcl- 2,in human RPE cells induced by experimental oxidative injuries. Methods The RPE cells strain was take out from -70 ℃ refrigetator and put into water bath of 55 ℃ - 65 ℃. After resuscitation, the RPE cell was inoculated into culture flask and cultured in 5% CO2 incubator. H202 of 600 μmol/L was added into RPE cell culture medium to establish the model of oxidative injuries in human RPE. Meanwhile, EPO of 40 U/ml was also added into the culture medium and continued to culture for 2,8 and 24 hours. The expression of bcl-2 protein was studied by cellular immunochemical technique. The bcl-2 mRNA was studied by RT- PCR. Results The brown particles in cytoplasm of normal cultured RPE cell were displayed,demonstrating the expression of bcl-2, bcl-2 expression at 2, 8,24 hours after oxidative injuries was 161.83 ±4. 83, 156.32 ± 6.40 and 142. 10 ± 5, 52 respectively,indicating gradually decrease with time（P 〈 0. 05 ）, The changes of bcl-2mRNA paralleled to the changes of its protein. The aherative trend of bcl-2 in EPO treatment groups was in coincidence with oxidative injury groups, Conclusion The expression of bcl-2 is downregulated in human RPE cells of experimental oxidative injuries. Bcl-2 may have a pivotal role in apoptotic pathway of oxidative-damaged RPE cells. EPO can rescue RPE cells from retinal oxidative injuries through upregulation of bcl-2 expression and may represent an important mechanism for therapeutic method of retinal disease related to RPE cells.