氧化应激下视网膜色素上皮细胞外泌体对视网膜色素上皮细胞血管内皮生长因子A及丝氨酸／苏氨酸激酶表达的影响

Influence of oxidative stress-induced exosomes on Akt and vascular endothelial growth faetor-A of retinal pigment epithelium cells

目的 观察氧化应激下视网膜色素上皮细胞（RPE）外泌体对RPE细胞血管内皮生长因子（VEGF）-A 及丝氨酸/苏氨酸激酶（Akt）表达的影响。 方法 培养人RPE（ARPE-19）细胞，细胞培养基中加入浓度为2.5 μmol/L鱼藤酮诱导ARPE-19细胞发生氧化应激损伤。收集上清液，采取梯度超速离心法分离纯化外泌体。应用透射电子显微镜观察外泌体形态；蛋白免疫印迹法（Western blot）检测 ARPE-19 细胞外泌体表面特异性标志蛋白CD63的表达。将氧化应激下ARPE-19细胞外泌体与ARPE-19细胞共培养作为实验组，等量正常RPE细胞外泌体与ARPE-19细胞共培养作为对照组。采用噻唑蓝比色法检测ARPE-19细胞的细胞活力，以酶联免疫检测仪测量波长570 nm处各孔的吸光度[A，旧称光密度（OD）]值表示；免疫荧光染色法和Western blot检测ARPE-19细胞VEGF-A、Akt 蛋白表达；实时定量聚合酶链反应（RT-PCR）检测ARPE-19细胞VEGF-A、Akt mRNA表达。 结果 透射电子显微镜观察发现，ARPE-19细胞外泌体呈圆形或椭圆形膜性小囊泡，直径50～150 nm。Western blot检测结果显示，ARPE-19细胞外泌体表面表达特异性标志蛋白CD63。噻唑蓝比色法结果显示，实验组、对照组ARPE-19细胞在波长570 nm处的A 值分别为0.582±0.015、0.787±0.032。实验组ARPE-19细胞在波长570 nm处的A 值较对照组明显降低，差异有统计学意义（t=20.886，P＜0.05）。免疫荧光染色法检测结果显示，与对照组比较，实验组ARPE-19细胞VEGF-A蛋白表达明显增强，Akt蛋白表达明显减弱。Western blot 检测结果显示，与对照组比较，实验组ARPE-19细胞VEGF-A蛋白相对表达量明显升高，Akt蛋白相对表达量明显降低，差异均有统计学意义（t=3.822、6.527，P＜0.05）。RT-PCR 检测结果显示，与对照组比较，实验组ARPE-19细胞VEGF-A mRNA相对表达量明显升高，Akt mRNA 相对表达量明显降低，差异均有统计学意义（t=8.805、？7.823，P＜0.05）。 结论 氧化应激下ARPE-19细胞外泌体可抑制正常AREP-19细胞增生，上调VEGF-A表达，下调 Akt 表达。

Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium （ARPE-19） cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A （VEGF-A） and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes＇ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction （RT-PCR） was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 （affected by rotenone）-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant （t=3.822, 6.527;P〈0.05）. RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant （t=8.805, ？7.823;P〈0.05）. Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.