高葡萄糖刺激血管内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调、活性氧增加及细胞凋亡

High Glucose Induce Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Expression,Reactive Oxygen Species Generation and Cell Apoptosis to Human Umbilical Endothelial Cells

目的研究在高葡萄糖刺激的条件下,人脐静脉内皮细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的表达、细胞内活性氧以及细胞凋亡的变化。方法倒置显微镜下观察人脐静脉内皮细胞形态;免疫荧光法检测人脐静脉内皮细胞Ⅷ因子相关抗原的表达;高葡萄糖刺激人脐静脉内皮细胞,用逆转录聚合酶链反应检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA水平,Western blotting检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4蛋白表达的变化,DCFH-DA检测细胞内活性氧生成量,流式细胞仪和Hoechst染色检测细胞凋亡。结果高葡萄糖刺激人脐静脉内皮细胞时,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA及蛋白表达上调,细胞内活性氧生成增加,细胞凋亡增加。结论高葡萄糖处理促进人脐静脉内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调并引起细胞内活性氧生成和凋亡增加。

Aim To investigative nicotinamide adenine dinucleotide phosphate oxidase（NOX4） expression,intracellular reactive oxygen species（ROS） level and cell apoptosis in human umbilical vein endothelial cells（HUVEC） when stimulated with high concentration of glucose.Methods Morphology of HUVEC was observed with inverted microscope,expression of factor Ⅷ related antigen in HUVEC was detected by immunofluorescence.NOX expression in HUVEC was convicted by reverse transcription polymerase chain reaction（RT-PCR） with positive control.Intracellular ROS generation was measured by DCFH-DA,cell apoptosis were measured by PI and Hoechet 33342 staining respectively.Results With high glucose in media,expression of NOX4,intracellular ROS generation and cell apoptosis were all increased.Conclusions High glucose could upregulate NOX4 expression,intracellular ROS production and then lead to cell apotosis.