CAJ | 학술논문

目的研究硫氧还蛋白(Trx)在动脉粥样硬化中对血管内皮细胞保护作用的分子机制. 方法应用腺病毒感染的方法在原代人脐静脉内皮细胞(HUVECs)中建立过表达硫氧还蛋白及其对照的细胞模型.以致动脉粥样硬化重要危险因子氧化型低密度脂蛋白(oxLDL)为刺激剂.应用免疫印迹及间接免疫荧光法检测Trx,黏附分子(ICAM-1,VCAM-1)及其上游信号分子(Smad3,AP-1)的蛋白表达及细胞定位.应用胰岛素还原法检测Trx的活性,应用荧光探针DCFHDA进行细胞内活性氧检测. 结果和对照组相比过表达Trx组Trx表达量明显提高,活性检测显示Ad-Trx的活性上调率为(26.2±3.3)％,细胞内活性氧(ROS)检测提示过表达Trx显著抑制细胞内ROS的产生.和对照组相比在基础及氧化型低密度脂蛋白(ox-LDL)刺激下过表达Trx组明显下调了内皮细胞黏附分子的表达(P＜0.05),显著提高了内皮细胞中Smad3的磷酸化(P＜0.05).而应用Smad3磷酸化特异性的抑制剂SIS3预处理细胞反转了Trx对黏附蛋白的抑制作用.SIS3预处理细胞进一步上调了oxLDL刺激下AP-1亚基c-Fos的核蛋白表达. 结论 Trx抑制内皮细胞黏附分子表达的作用可能是通过上调Smad3蛋白的磷酸化及抑制核转录因子AP-1亚基c-Fos的核表达来调节的.
목적 연구류양환단백(Trx)재동맥죽양경화중대혈관내피세포보호작용적분자궤제. 방법 응용선병독감염적방법재원대인제정맥내피세포(HUVECs)중건립과표체류양환단백급기대조적세포모형.이치동맥죽양경화중요위험인자양화형저밀도지단백(oxLDL)위자격제.응용면역인적급간접면역형광법검측Trx,점부분자(ICAM-1,VCAM-1)급기상유신호분자(Smad3,AP-1)적단백표체급세포정위.응용이도소환원법검측Trx적활성,응용형광탐침DCFHDA진행세포내활성양검측. 결과 화대조조상비과표체Trx조Trx표체량명현제고,활성검측현시Ad-Trx적활성상조솔위(26.2±3.3)％,세포내활성양(ROS)검측제시과표체Trx현저억제세포내ROS적산생.화대조조상비재기출급양화형저밀도지단백(ox-LDL)자격하과표체Trx조명현하조료내피세포점부분자적표체(P＜0.05),현저제고료내피세포중Smad3적린산화(P＜0.05).이응용Smad3린산화특이성적억제제SIS3예처리세포반전료Trx대점부단백적억제작용.SIS3예처리세포진일보상조료oxLDL자격하AP-1아기c-Fos적핵단백표체. 결론 Trx억제내피세포점부분자표체적작용가능시통과상조Smad3단백적린산화급억제핵전록인자AP-1아기c-Fos적핵표체래조절적.
Objective To investigate the molecular mechanisms of protective effects of thioredoxin (Trx) on human vascular endothelial cells in atherosclerosis.Methods The cell models of Trx-overexpressing cells (Ad Trx) and the control cells (Ad-con) were established by adenovirus vector gene transfer technology in human umbilical vein endothelial cells (HUVECs).The oxidized low density lipoprotein,a risk factor of atherosclerosis,was used as a stimulator.Western blot and indirect immunofluorescence were used to detect the protein expression levels and the cellular localization of Trx,adhesion molecules (ICAM-1,VCAM-1) and the upstream signal pathways.Trx activity was detected by insulin disulfide reduction assay,and cellular reactive oxygen species (ROS)production was detected by fluorescent probe DCFH-DA.Results As compared with control group,Trx protein expression level was enhanced in Ad-trx group and the Trx activity in Ad-Trx group was upregulated by (26.2 ±3.3)％.The result of ROS detection showed that overexpression of Trx significantly inhibited the cellular ROS generation.As compared with control group,overexpression of Trx obviously inhibited the adhesion molecules expression but markedly promoted the phosphorylation of Smad3 in endothelial cells with or without oxLDL stimulation (P＜0.05).Pretreatment of cells with SIS3,a specific inhibitor of Smad3 phosphorylation,reversed Trx-induced inhibition of adhesion molecules expression.Further studies showed that pretreatment of cells with SIS3 enhanced oxLDL-induced AP-1 subunit c-fos nuclear expression.Conclusions The enhancement of Smad3 phosphorylation and c-Fos nuclear expression are mainly responsible for the Trx-induced downregulation of adhesion molecules.