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目的探讨miR-155、miR-125b-5p、miR-210及其靶基因蛋白在氧化应激血管内皮细胞中的表达和作用。方法分离人脐静脉内皮细胞(HUVEC)并通过免疫荧光染色法检测内皮细胞Ⅷ因子相关抗原、流式细胞术检测CD31的表达进行鉴定;将分离的HUVEC进行原代培养,采用子痫前期患者血清和H2O2建立氧化应激的血管内皮细胞的模型,实验分为正常妊娠血清组、子痫前期血清组、0μmol/L H2O2组、100μmol/L H2O2组;实时定量PCR检测造模后24 h细胞内miR-155、miR-125b-5p、miR-210的水平;Western blot法检测细胞内miR-125b-5p靶基因Smad4蛋白的水平;免疫荧光染色法检测细胞内Smad4蛋白的表达;异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染色结合流式细胞术检测细胞凋亡情况。结果与0μmol/L H2O2组相比,100μmol/L H2O2处理的内皮细胞miR-125b-5p显著降低、Smad4蛋白显著增加、细胞凋亡率和坏死率显著增加。与正常孕妇血清组相比,子痫前期血清处理的内皮细胞凋亡率和坏死率显著增加,但miR-125b-5p及Smad4蛋白变化不明显。结论 H2O2处理的HUVEC miR-125b-5p降低,Smad4蛋白增加。
Objective To investigate the expression of miR-155, miR-125b-5p, miR-210 and their target genes in oxidative stress vascular endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) were isolated and the Ⅷ factor related antigens of endothelial cells were detected by immunofluorescence staining. The expression of CD31 was detected by flow cytometry. The isolated HUVECs were cultured in primary culture. The serum of preeclampsia patients And H2O2 to establish oxidative stress vascular endothelial cells model, the experiment is divided into normal pregnancy serum group, preeclampsia serum group, 0μmol / L H2O2 group, 100μmol / L H2O2 group; real-time quantitative PCR 24 hours after modeling intracellular miR-125, miR-125b-5p and miR-210 were detected by immunohistochemistry. The level of Smad4 protein was detected by Western blot. The expression of Smad4 protein was detected by immunofluorescence staining. Fluorescein labeled annexin Ⅴ / propidium iodide (annexin Ⅴ-FITC / PI) double staining and flow cytometry were used to detect apoptosis. Results Compared with 0μmol / L H2O2 group, the expression of miR-125b-5p in the endothelial cells treated with 100μmol / L H2O2 was significantly decreased, the Smad4 protein was significantly increased, and the apoptosis rate and necrosis rate were significantly increased. The apoptosis rate and necrosis rate of endothelial cells treated with serum of preeclampsia increased significantly compared with that of normal pregnant women, but the changes of miR-125b-5p and Smad4 proteins were not obvious. Conclusion HUVEC treated with H2O2 decreased miR-125b-5p and Smad4 protein increased.