摘要
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.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2018年第2期159-163,共5页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金（81570872、81460089）
国家自然科学基金青年基金（81400425）
天津市应用基础与前沿技术研究计划（15JCYBJC24900）
天津医科大学自然科学基金（2016KYZM14）
天津医科大学眼科研究所基金（15YKYJS002）
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
Retinal pigment epithelium
Polypydmidine tract-binding protein
Apoptosis
Hydrogen peroxide
