中华眼底病杂志

中华眼底病杂志

聚嘧啶束结合蛋白相关剪接因子对过氧化氢诱导下视网膜色素上皮细胞凋亡的影响

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目的 观察聚嘧啶束结合蛋白相关剪接因子(PSF)高表达对过氧化氢(H2O2)诱导下视网膜色素上皮(RPE)细胞凋亡的影响。 方法 体外培养的人RPE细胞分为正常细胞组、损伤组(N+H2O2组)、空载体对照组(Vec+H2O2组)、PSF高表达组(PSF+H2O2组)。应用脂质体2000将pEGFP空载体、pEGFP-PSF真核表达质粒分别导入Vec+H2O2组、PSF+H2O2组;N+H2O2组仅作转染处理;正常细胞组为正常培养细胞。细胞转染24 h后,以200 μmol/L H2O2刺激细胞2 h。苏木精-伊红染色观察各组细胞形态;噻唑蓝比色法检测N+H2O2组、Vec+H2O2组、PSF+H2O2细胞活性,以酶联免疫检测仪测量波长490 nm处各组细胞的吸光度[A,旧称光密度(OD)]值表示;LIVE/DEAD®细胞活性/细胞毒性试剂盒检测各组细胞生存力;细胞凋亡检测试剂盒检测N+ H2O2组、Vec+H2O2组、PSF+H2O2组细胞凋亡;二氯荧光素二乙酸酯检测各组细胞内活性氧(ROS)水平。 结果 N+H2O2组、Vec+H2O2组细胞体积缩小,胞质致密浓缩、嗜酸性染色增强;PSF+H2O2组细胞形态尚饱满,胞质染色较均匀,偶见体积缩小。与PSF+H2O2组比较,N+H2O2组、Vec+H2O2组细胞活性下降,差异有统计学意义(F=46.98,P=0.000)。正常细胞组细胞呈绿色,偶见红色荧光;N+H2O2组、Vec+H2O2组细胞中呈红色荧光的死亡细胞数量明显增多,呈绿色荧光的活细胞数量显著减少;PSF+H2O2组活细胞数量明显增多,死亡细胞数量显著减少。与N+H2O2组、Vec+H2O2组细胞凋亡比较,PSF+ H2O2组细胞凋亡下降,差异有统计学意义(F=62.24,P=0.000)。与N+H2O2组、Vec+H2O2组比较,PSF+H2O2组细胞中ROS表达量下降,差异有统计学意义(F=295.235,P=0.000)。 结论 高表达PSF通过抑制ROS的产生缓解H2O2诱导的人RPE细胞凋亡。

Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P<0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.

关键词: 视网膜色素上皮; 聚嘧啶区结合蛋白质; 细胞凋亡; 过氧化氢

Key words: Retinal pigment epithelium; Polypyrimidine tract-binding protein; Apoptosis; Hydrogen peroxide

引用本文: 田芳, 李文博, 黄亮瑜, 高美子, 赵今稚, 胡博杰, 张晓敏, 李筱荣, 东莉洁. 聚嘧啶束结合蛋白相关剪接因子对过氧化氢诱导下视网膜色素上皮细胞凋亡的影响. 中华眼底病杂志, 2018, 34(2): 159-163. doi: 10.3760/cma.j.issn.1005-1015.2018.02.012 复制

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