摘要
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 explore the expressions and functions of miR-155, miR-125b-5p, miR-210, and their target proteins, in vascular endothelial cells in oxidative stress. Methods Human umbilical vein endothelial cells （HUVECs） were isolated and cultured; the expression of endothelial cell factor related antigen was detected by immunofluorescence staining; and the expression of CD31 was detected and identified by flow cytometry. The oxidative stress model of vascular endothelial cells was established by preeclampsia serum and hydrogen peroxide （H=O2） treatment. The treated HUVECs were divided into 4 groups： normal pregnancy serum group, preeclampsia serum group, 0 μmoVL H202 group and 100 μmoVL H202 group. The levels of miR-155, miR-125b-5p, and miR-210 were tested by real-time quantitative PCR within 24 hours after modeling. The expression of Smad4 protein was detected by Western blotting and immunofluorescence staining. Cell apoptosis was detected by flow cytometry combined with annexin V-FITC/PI staining. Results Compared with 0 tamoVL H202 group, miR-125b-5p significantly decreased in 100 μmOl/L H202 group, while Smad4 protein significantly increased. In addition, 100 μmoVL H202 treatment promoted cell apoptosis and necrosis. Compared with normal pregnancy serum group, the apoptosis and necrosis were significantly enhanced in preeclampsia serum group, but there were no significant differences in miR-125b-5p and Smad4 between the groups. Conclusion High level of H202 up-regulates Smad4 protein expression and inhibits miR-125b-5p expression.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第8期1088-1093,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
陕西省自然科学基础研究计划(2014JQ2-8055
2016JM8046)
陕西省卫生科研项目-重点支持项目(2014A)
